Reproducibility of 18F-FDG microPET studies in mouse tumor xenografts.

(18)F-FDG has been used to image mouse xenograft models with small-animal PET for therapy response. However, the reproducibility of serial scans has not been determined. The purpose of this study was to determine the reproducibility of (18)F-FDG small-animal PET studies. METHODS: Mouse tumor xenografts were formed with B16F10 murine melanoma cells. A 7-min small-animal PET scan was performed 1 h after a 3.7- to 7.4-MBq (18)F-FDG injection via the tail vein. A second small-animal PET scan was performed 6 h later after reinjection of (18)F-FDG. Twenty-five sets of studies were performed. Mean injected dose per gram (%ID/g) values were calculated from tumor regions of interest. The coefficient of variation (COV) from studies performed on the same day was calculated to determine the reproducibility. Activity from the second scans performed after 6 h were adjusted by subtracting the estimated residual activity from the first (18)F-FDG injection. For 7 datasets, an additional scan immediately before the second injection was performed, and residual activity from this additional delayed scan was subtracted from the activity of the second injection. COVs of both subtraction methods were compared. Blood glucose values were measured at the time of injection and used to correct the %ID/g values. RESULTS: The COV for the mean %ID/g between (18)F-FDG small-animal PET scans performed on the same day 6 h apart was 15.4% +/- 12.6%. The delayed scan subtraction method did not produce any significant change in the COV. Blood glucose correction increased the COV. The injected dose, tumor size, and body weight did not appear to contribute to the variability of the scans. CONCLUSION: (18)F-FDG small-animal PET mouse xenograft studies were reproducible with moderately low variability. Therefore, serial small-animal PET studies may be performed with reasonable accuracy to measure tumor response to therapy.     

Maturation of immature oocytes by coculture with granulosa cells

To increase the number of embryos available for transfer, immature human oocytes were cocultured with granulosa cells from preovulatory follicles. Greater numbers of immature oocytes incubated with granulosa cells had dispersion of the cumulus and corona cells compared with immature oocytes cultured in media alone. Fifty-four percent of immature oocytes were fertilized after coculture with granulosa cells compared with 20% fertilization of immature oocytes cultured without granulosa cells. There were no cases in which only embryos developed from immature oocytes were transferred, and thus we could not determine if the immature oocytes could contribute to a pregnancy.     

Metastatic mammary carcinoma of the middle ear

Malignancies of the middle ear and mastoid are rare and secondaries in the ear are extremely rare. A rare case of metastic adenocarcinoma from the breast is presented herewith.     

Localization of simian immunodeficiency virus in the central nervous system of rhesus monkeys.

Simian immunodeficiency virus (SIV), like the human immunodeficiency virus (HIV), is a lentivirus that is both immunosuppressive and neurovirulent. Rhesus macaques (Macaca mulatta) inoculated with SIV often develop a giant cell encephalitis similar to that seen in humans infected with HIV. The authors examined SIV expression by immunohistochemistry and RNA in situ hybridization in the cerebrum, cerebellum, choroid plexus, and spinal cord from five macaques with and two macaques without giant cell encephalitis. Selected portions of the central nervous system (CNS) also were examined by electron microscopy. Simian immunodeficiency virus was detected in the CNS of all seven monkeys whether or not they had giant cell encephalitis. Both SIV antigen and RNA were present in all levels of the CNS examined. Macrophage/giant cell lesions always contained viral RNA and antigen and were the only sites where viral particles were detected by electron microscopy. However, SIV antigen and RNA also were commonly associated with small vessels, the choroid plexus, and meninges; these were the only locations where virus was detected in animals without giant cell encephalitis. Immunophenotyping showed that the cellular infiltrates consisted primarily of monocyte/macrophages and occasional CD8-positive T cells. Macrophages and T cells also were present in the stroma of the choroid plexus and were intimately associated with vessels in the CNS of SIV-infected but not uninfected macaques. Simian immunodeficiency virus infection of the macaque CNS provides an excellent model for studying the pathogenesis, treatment, and prevention of HIV-1-encephalitis.     

Role of imprint cytology in the diagnosis of gastrointestinal tract malignancies

Endoscopic biopsies obtained from 275 patients (180 from the upper gastrointestinal tract and 95 from the lower gastrointestinal tract) were studied to compare the accuracy of biopsy imprint cytology and histology in the diagnosis of gastrointestinal lesions, and also to establish the degree of reliability of imprint cytology alone for an early diagnosis of malignancy. Biopsy histology results were found to be correct in 100% cases. Imprint cytology had an overall accuracy of 100%, 96.7%, 95.8% and 95.8% for the diagnosis of malignancies of the oesophagus, stomach, duodenum and colorectum respectively. False negative results were obtained with lymphomas. Regenerative cellular atypia was an important cause for false positive results. It was concluded that imprint cytology can serve as a useful and simple tool for an immediate diagnosis of malignancy. This should be subsequently correlated with histopathology which facilitates exact tumour typing and assessment of tumour invasion.     

Phenotypic and functional properties of murine {gamma}{delta} T cell clones derived from malaria immunized, {alpha}{beta} T cell-deficient mice

Six murine T cell clones expressing TCR were generated frommalaria immunized, ß T celldeficient mice. Phenotypiccharacterization of these clones has revealed that, in contrastto conventional ß T cells, there is a considerabledegree of heterogeneity among these clones with regard to theirsurface markers and their lymphokine profile. One clone wasfound to display significant anti-parasite activity in vivoupon adoptive transfer. We attempted to determine whether theprotective clone differs in one or more key characteristicsfrom the non-protective clones. Although no obvious patternpeculiar to the protective clone was observed, it appears thatmore than one parameter may, in combination, define a distinctprotective phenotype, and thus explain the functional differencebetween the protective and non-protective clones.     

Bystander CD8 T-cell-mediated demyelination is interferon-gamma-dependent in a coronavirus model of multiple sclerosis

Mice infected with the coronavirus mouse hepatitis virus, strain JHM (JHM) develop a disease that shares many histological characteristics with multiple sclerosis. We previously demonstrated that JHM-infected mice that only have CD8 T cells specific for an epitope not in the virus develop demyelination on specific activation of these cells. Herein we show that this process of bystander T-cell-mediated demyelination is interferon-gamma (IFN-gamma)-dependent. The absence of IFN-gamma abrogated demyelination but did not change T-cell infiltration or expression levels of inflammatory cytokines or chemokines in the spinal cord. These results are consistent with models in which IFN-gamma contributes to CD8 T-cell-mediated demyelination by activation of macrophages/microglia, the final effector cells in the disease process.     

When genetic distance matters: measuring genetic differentiation at microsatellite loci in whole-genome scans of recent and incipient mosquito species

Genetic distance measurements are an important tool to differentiate field populations of disease vectors such as the mosquito vectors of malaria. Here, we have measured the genetic differentiation between Anopheles arabiensis and Anopheles gambiae, as well as between proposed emerging species of the latter taxon, in whole genome scans by using 23-25 microsatellite loci. In doing so, we have reviewed and evaluated the advantages and disadvantages of standard parameters of genetic distance, F(ST), R(ST), (delta mu)(2), and D. Further, we have introduced new parameters, D' and D(K), which have well defined statistical significance tests and complement the standard parameters to advantage. D' is a modification of D, whereas D(K) is a measure of covariance based on Pearson's correlation coefficient. We find that A. gambiae and A. arabiensis are closely related at most autosomal loci but appear to be distantly related on the basis of X-linked chromosomal loci within the chromosomal Xag inversion. The M and S molecular forms of A. gambiae are practically indistinguishable but differ significantly at two microsatellite loci from the proximal region of the X, outside the Xag inversion. At one of these loci, both M and S molecular forms differ significantly from A. arabiensis, but remarkably, at the other locus, A. arabiensis is indistinguishable from the M molecular form of A. gambiae. These data support the recent proposal of genetically differentiated M and S molecular forms of A. gambiae.     

Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination,accessory, and epigenetic profiles

Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity.Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a serious infectious disease of humans and animals and a leading cause of community-acquired sepsis and pneumonia in endemic regions (Currie et al. 2010). Initially thought to be confined to Southeast Asia and Northern Australia, the prevalence of Bp appears to be spreading (Wiersinga et al. 2012), and Bp has been designated a biothreat select agent in the United States. Bp can persist in extreme environmental conditions and can infect several plant and animal hosts, including birds, dolphins, and humans (Wuthiekanun et al. 1995; Howard and Inglis 2003; Sprague and Neubauer 2004; Larsen et al. 2013). Treatment of clinical melioidosis is challenging because the bacterium is inherently resistant to many antibiotics, and Bp infections can persist in humans for more than a decade (Hayden et al. 2012; Wiersinga et al. 2012).The Bp genome comprises one of the largest and most complex bacterial genomes sequenced to date. Consisting of two large circular replicons (chromosomes) with a combined 7.2-Mb genome size (Holden et al. 2004), it contains a rich arsenal of genes related to virulence (e.g., Type III and Type VI secretion systems, polysaccharide biosynthesis clusters), metabolic pathways, and environmental adaptation (Wiersinga et al. 2012). Besides conserved regions, accessory genes on mobile elements and genomic islands may also contribute to phenotypic and clinical differences in microbial behavior (Currie et al. 2000; Sim et al. 2008). Analysis of the Bp genome has revealed previously unknown toxins and mechanisms of antibiotic resistance (Chantratita et al. 2011; Cruz-Migoni et al. 2011).Most large-scale studies of Bp genetic diversity to date have analyzed strains using multilocus sequence typing (MLST). These studies have suggested a high degree of genetic variability between Bp strains and related Burkholderia species (Cheng et al. 2008), and have shown that Bp strains belonging to different sequence types (STs) can often coexist in the same locale and sometimes even within the same sample (Pitt et al. 2007; Wuthiekanun et al. 2009). However, due to the limited number of genes analyzed by MLST, these studies cannot comment on the global proportion of genetic material shared between strains of different STs nor on the relative contribution of recombination, mutation, and horizontal gene transfer on intraspecies genetic diversity. Moreover, although previous studies have applied whole-genome sequencing (WGS) to study global patterns of Bp genetic heterogeneity and evolution, earlier Bp WGS reports have been confined to a limited number of isolates (10–12) derived from diverse geographical regions (Nandi et al. 2010), where geophysical barriers likely limit the propensity of the analyzed strains to exchange genetic material. To achieve a comprehensive understanding of genetic variation among closely related Bp strains, WGS analysis of much larger strain panels, ideally performed on strains isolated from a common region and belonging to the same (or closely related) ST groups, is required.In this study, we attempted to fill this important knowledge gap by performing WGS on 106 Bp strains drawn from a restricted Asian locale (Singapore and Malaysia). The WGS data, exceeding previous Bp WGS studies by 10-fold, enabled us to identify specific genomic clades of Bp, molecular features of Bp recombination at the whole-genome level, and accessory genome features contributing to recombination and horizontal gene transfer. We found a consistent pattern of genetic separation correlating with MLST, recombination haplotypes, shared accessory genes, and restriction modification (RM) systems. We provide evidence that restriction modification, beyond its role as defense against foreign DNA invasion, may have also partitioned the Bp species by restricting gene flow, resulting in the other observed correlations. Because RM systems are widely dispersed through the bacterial kingdom, it is possible that similar principles may apply to other bacterial species, implicating a potential role for epigenetic barriers as a driver of early incipient speciation.