The biological effects of syngeneic and allogeneic cytokine-expressing prophylactic whole cell vaccines and the influence of irradiation in a murine melanoma model

Allogeneic whole tumour cell vaccines are inherently practical compared with autologous vaccines. Cell lines are derived from allogeneic tumour, grown in bulk and then administered as a vaccine to the patient, following irradiation, which not only prevents any replication but also enhances antigen presentation. Protection is believed to occur through the presentation of antigens shared between the syngeneic and allogeneic tumours. Although cytokine-transfected tumour whole cell vaccines have been used clinically, little data is available comparing the effects of immunomodulatory cytokine-transfection directly on the same cells when used as both an allogeneic and autologous vaccine. To address this, weakly immunogenic B16-F10 (H-2^b) murine melanoma was transfected to secrete either GM-CSF, IL-4 or IL-7. Prophylactic vaccination of both syngeneic C57/BL6 (H-2^b) (B6) and allogeneic C3H/Hej (H-2^k) (C3H) mice showed the effects of transfected cytokine varied between models. Both GM-CSF and IL-7 significantly (P<0.05) increased the levels of protection within syngeneic B6 mice, but had a diminished effect (P>0.05) within C3H allogeneic mice. Allogeneic B16-F10 cells and syngeneic K1735 cells generated CTL against K1735 suggesting cross-reactive immunity. Using cells labeled with fluorescent dye we demonstrate that irradiated vaccines, of either syngeneic or allogeneic origin, appear to generate potent immune responses and fragments of either vaccine remain at the injection site for up to 9 days. This study shows that protection can be enhanced in vivo by using transfected cytokine, but suggests that irradiated whole cell vaccines, of either tissue-type, are rapidly processed. This leads to the conclusion that the cytokine effects are transient and thus transfection with cytokine may be of limited long-term use in situ.     

Heat stress reduces cerebral blood velocity and markedly impairs orthostatic tolerance in humans

Orthostatic tolerance is reduced in the heat-stressed human. This study tested the following hypotheses: 1) whole body heat stress reduces cerebral blood velocity (CBV) and increases cerebral vascular resistance (CVR); and 2) reductions in CBV and increases in CVR in response to an orthostatic challenge will be greater while subjects are heat stressed. Fifteen subjects were instrumented for measurements of CBV (transcranial ultrasonography), mean arterial blood pressure (MAP), heart rate, and internal temperature. Whole body heating increased both internal temperature (36.4+/-0.1 to 37.3+/-0.1 degrees C) and heart rate (59+/-3 to 90+/-3 beats/min); P<0.001. Whole body heating also reduced CBV (62+/-3 to 53+/-2 cm/s) primarily via an elevation in CVR (1.35+/-0.06 to 1.63+/-0.07 mmHg.cm-1.s; P<0.001. A subset of subjects (n=8) were exposed to lower-body negative pressure (LBNP 10, 20, 30, 40 mmHg) in both normothermic and heat-stressed conditions. During normothermia, LBNP of 30 mmHg (highest level of LBNP achieved by the majority of subjects in both thermal conditions) did not significantly alter CBV, CVR, or MAP. During whole body heating, this LBNP decreased MAP (81+/-2 to 75+/-3 mmHg), decreased CBV (50+/-4 to 39+/-1 cm/s), and increased CVR (1.67+/-0.17 to 1.92+/-0.12 mmHg.cm-1.s); P<0.05. These data indicate that heat stress decreases CBV, and the reduction in CBV for a given orthostatic challenge is greater during heat stress. These outcomes reduce the reserve to buffer further decreases in cerebral perfusion before presyncope. Increases in CVR during whole body heating, coupled with even greater increases in CVR during orthostasis and heat stress, likely contribute to orthostatic intolerance.     

Flow cytometric detection of tandem repeat mutations induced by various chemical classes

To facilitate detection of genotoxicity from environmental mutagen exposure, we generated an in vitro enhanced green fluorescence protein (EGFP) reactivation assay that quickly and effectively detects frameshift mutations in tandem repeat sequences (TRS). Two murine cell lines, C3H10T1/2 and mismatch repair deficient MC2a, were stably transfected with EGFP reporter plasmids in which the EGFP constructs contain TRS that put the EGFP sequence out of frame. These included several 2, 3, 4, 5 and 6 bp repeat sequences, a control non-repetitive sequence and a human gene sequence containing a 4 bp repeat motif. Transfected cultures were exposed to five model mutagens and carcinogens: hydrogen peroxide (H(2)O(2)), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), benzo-a-pyrene-diol-epoxide (BPDE), ethyl nitrosourea (ENU), 9-aminoacridine (9AA) and two controls: acetone and ethanol. Frameshift mutations resulted in green fluorescent revertants, as determined by flow cytometry, and were confirmed, for 9AA treatments, by sequencing. All five treatments with model agents induced statistically significant sequence- and exposure-dependent responses in MC2a cells and a negative response with the two negative control treatments, acetone and ethanol. Similar responses were seen in a smaller panel of treatments and plasmids in C3H10T1/2 cells. The mutation frequencies were higher in cells transfected with the plasmids containing TRS than those harbouring the control construct lacking repeats. The highest mutation frequencies were observed with H(2)O(2) and 9AA treatments, yielding up to a 50-fold difference between vehicle and highest concentration treatment. ENU, BPDE, and to a lesser extent TPA treatments, also showed a statistically significant exposure response. Results from these experiments reveal that the assay responds robustly to various classes of mutagenic substances, as well as to rodent carcinogens that are inactive in conventional mutation assays, and that responses are not linked to cytotoxicity. This assay is a promising approach for detecting chemically induced frameshifts within certain DNA sequences of interest, but further characterization and validation are required prior to general use in genotoxicity screening.     

Triumphalone, a diketone from the volatile oil of the leaves of Melaleuca triumphalis, and its spontaneous conversion into isotriumphalone

The major component (35-65%) of the volatile oil obtained by steam distillation of the leaves of Melaleuca triumphalis has been identified as (rel)-1beta-pentyl-1alpha,6alpha-dihydroxy-3,3,5,5-tetramethylcyclohexa-2,4-dione (trivial name triumphalone). Relative stereochemistry was established by nuclear Overhauser experiments and X-ray studies on the 2-(3,5-dinitrobenzoic acid) derivative. The remainder of the oil was composed of mono- and sesquiterpene hydrocarbons and alcohols. On prolonged standing the presence of a rearrangement product of triumphalone was observed which was characterized as (rel)-1beta-pentyl-1alpha,3alpha-dihydroxy-4,4,6,6-tetramethylcyclohexa-2,5-dione (trivial name isotriumphalone), presumably arising from an acid catalyzed shift of the pentyl group from C-1 to C-2.     

The behavioral ecology of sympatric African apes: implications for understanding fossil hominoid ecology

The behavioral ecology of the great apes is key evidence used in the reconstruction of the behavior of extinct ape and hominid taxa. Chimpanzees and gorillas have been studied in detail in the wild, and some studies of their behavioral ecology in sympatry have also been been carried out. Although the two ape species have divergent behavior and ecology in important respects, recent studies have shown that the interspecific differences are not as stark as previously thought and subsequently urge new consideration of how they share forest resources when sympatric. These new data require re-examination of assumptions about key aspects of chimpanzee-gorilla ecological divergence, such as diet, ranging and nesting patterns, and the mating system. Diet is a key component of the species adaptive complexes that facilitates avoidance of direct competition from the other. While the nutritional basis for chimpanzee food choice remains unclear and no doubt varies from site to site, this species is a ripe fruit specialist and ranges farther during periods of ripe fruit scarcity. Gorillas in the same habitat also feed on ripe fruit when widely available, but fall back onto fibrous plant foods during lean periods. The inclusion of animal protein in the diet of the chimpanzees and its absence in that of the gorillas also distinguish the species ecologically. It may also offer clues to aspects of ecological divergence among early members of the hominid phylogeny. The paper concludes by suggesting likely characteristics of sympatric associations of Pliocene hominids, based on field data from extant sympatric apes.     

Mitochondrial DNA mutations in individuals occupationally exposed to ionizing radiation

Mutations in a 443-bp amplicon of the hypervariable region HVR1 of the D-loop of mitochondrial DNA (mtDNA) were quantified in DNA extracted from peripheral blood samples of 10 retired radiation workers who had accumulated external radiation doses of >0.9 Sv over the course of their working life and were compared to the levels of mutations in 10 control individuals matched for age and smoking status. The mutation rate in the 10 exposed individuals was 9.92 x 10(-5) mutations/ nucleotide, and for the controls it was 8.65 x 10(-5) mutations/ nucleotide, with a procedural error rate of 2.65 x 10(-5) mutations/nucleotide. No increase in mtDNA mutations due to radiation exposure was detectable (P = 0.640). In contrast, chromosomal translocation frequencies, a validated radiobiological technique for retrospective dosimetric purposes, were significantly elevated in the exposed individuals. This suggests that mutations identified through sequencing of mtDNA in peripheral blood lymphocytes do not represent a promising genetic marker of DNA damage after low-dose or low-dose-rate exposures to ionizing radiation. There was an increase in singleton mutations above that attributable to procedural error in both exposed and control groups that is likely to reflect age-related somatic mutation.     

A novel gammaherpesvirus associated with genital lesions in a Blainville's beaked whale (Mesoplodon densirostris)

An adult male Blainville's beaked whale (Mesoplodon densirostris) was found stranded on the Atlantic coast of the USA on 28 January 2004. Necropsy revealed a focal papilloma-like penile lesion, the cells from which revealed single 4-6 microm basophilic intranuclear inclusions. Total DNA extracted from lesion material was tested using a pan-herpes-virus PCR assay that targets the DNA polymerase gene and found to be positive. When the amplified DNA fragment was cloned, sequenced, and compared to GenBank-deposited herpesvirus DNA polymerase sequences, the detected virus was determined to be a distinct member of the Gammaherpesvirinae subfamily of herpesviruses. This new virus, tentatively named Ziphiid herpesvirus type 1, was associated with but not determined to be the cause of genital disease in the Blainville's beaked whale.     

An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana

ARGONAUTE4 (AGO4) and RNA polymerase IV (Pol IV) are required for DNA methylation guided by 24 nucleotide small interfering RNAs (siRNAs) in Arabidopsis thaliana. Here we show that AGO4 localizes to nucleolus-associated bodies along with the Pol IV subunit NRPD1b; the small nuclear RNA (snRNA) binding protein SmD3; and two markers of Cajal bodies, trimethylguanosine-capped snRNAs and the U2 snRNA binding protein U2B'. AGO4 interacts with the C-terminal domain of NRPD1b, and AGO4 protein stability depends on upstream factors that synthesize siRNAs. AGO4 is also found, along with the DNA methyltransferase DRM2, throughout the nucleus at presumed DNA methylation target sites. Cajal bodies are conserved sites for the maturation of ribonucleoprotein complexes. Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci.     

Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. nodule bacteria on two Mimosa spp. in Costa Rica

rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two Mimosa species at three sites in Costa Rica belonged to the genera Burkholderia, Cupriavidus, and Rhizobium. This is the first report of Cupriavidus sp. nodule symbionts for Mimosa populations within their native geographic range in the neotropics. Burkholderia spp. predominated among samples from Mimosa pigra (86% of isolates), while there was a more even distribution of Cupriavidus, Burkholderia, and Rhizobium spp. on Mimosa pudica (38, 37, and 25% of isolates, respectively). All Cupriavidus and Burkholderia genotypes tested formed root nodules and fixed nitrogen on both M. pigra and M. pudica, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both Cupriavidus and Burkholderia spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of Rhizobium. Given the prevalence of Burkholderia and Cupriavidus spp. on these Mimosa legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both beta-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed.     

Identification, typing, and insecticidal activity of Xenorhabdus isolates from entomopathogenic nematodes in United Kingdom soil and characterization of the xpt toxin loci

Xenorhabdus strains from entomopathogenic nematodes isolated from United Kingdom soils by using the insect bait entrapment method were characterized by partial sequencing of the 16S rRNA gene, four housekeeping genes (asd, ompR, recA, and serC) and the flagellin gene (fliC). Most strains (191/197) were found to have genes with greatest similarity to those of Xenorhabdus bovienii, and the remaining six strains had genes most similar to those of Xenorhabdus nematophila. Generally, 16S rRNA sequences and the sequence types based on housekeeping genes were in agreement, with a few notable exceptions. Statistical analysis implied that recombination had occurred at the serC locus and that moderate amounts of interallele recombination had also taken place. Surprisingly, the fliC locus contained a highly variable central region, even though insects lack an adaptive immune response, which is thought to drive flagellar variation in pathogens of higher organisms. All the X. nematophila strains exhibited a consistent pattern of insecticidal activity, and all contained the insecticidal toxin genes xptA1A2B1C1, which were present on a pathogenicity island (PAI). The PAIs were similar among the X. nematophila strains, except for partial deletions of a peptide synthetase gene and the presence of insertion sequences. Comparison of the PAI locus with that of X. bovienii suggested that the PAI integrated into the genome first and then acquired the xpt genes. The independent mobility of xpt genes was further supported by the presence of xpt genes in X. bovienii strain I73 on a type 2 transposon structure and by the variable patterns of insecticidal activity in X. bovienii isolates, even among closely related strains.