Protein changes in macrophages induced by plasma from rats exposed to 35 GHz millimeter waves.

A macrophage assay and proteomic screening were used to investigate the biological activity of soluble factors in the plasma of millimeter wave-exposed rats. NR8383 rat macrophages were incubated for 24 h with 10% plasma from male Sprague-Dawley rats that had been exposed to sham conditions, or exposed to 42 °C environmental heat or 35 GHz millimeter waves at 75 mW/cm² until core temperature reached 41.0 °C. Two-dimensional polyacrylamide gel electrophoresis, image analysis, and Western blotting were used to analyze approximately 600 protein spots in the cell lysates for changes in protein abundance and levels of 3-nitrotyrosine, a marker of macrophage stimulation. Proteins of interest were identified using peptide mass fingerprinting. Compared to plasma from sham-exposed rats, plasma from environmental heat- or millimeter wave-exposed rats increased the expression of 11 proteins, and levels of 3-nitrotyrosine in seven proteins, in the NR8383 cells. These altered proteins are associated with inflammation, oxidative stress, and energy metabolism. Findings of this study indicate both environmental heat and 35 GHz millimeter wave exposure elicit the release of macrophage-activating mediators into the plasma of rats.

Gene expression changes in the skin of rats induced by prolonged 35 GHz millimeter-wave exposure.

To better understand the cellular and molecular responses to overexposure to millimeter waves, alterations in the gene expression profile and histology of skin after exposure to 35 GHz radiofrequency radiation were investigated. Rats were subjected to sham exposure, to 42 degrees C environmental heat, or to 35 GHz millimeter waves at 75 mW/cm(2). Skin samples were collected at 6 and 24 h after exposure for Affymetrix GeneChip analysis. The skin was harvested from a separate group of rats at 3-6 h or 24-48 h after exposure for histopathology analysis. Microscopic findings observed in the dermis of rats exposed to 35 GHz millimeter waves included aggregation of neutrophils in vessels, degeneration of stromal cells, and breakdown of collagen. Changes were detected in 56 genes at 6 h and 58 genes at 24 h in the millimeter-wave-exposed rats. Genes associated with regulation of transcription, protein folding, oxidative stress, immune response, and tissue matrix turnover were affected at both times. At 24 h, more genes related to extracellular matrix structure and chemokine activity were altered. Up-regulation of Hspa1a, Timp1, S100a9, Ccl2 and Angptl4 at 24 h by 35 GHz millimeter-wave exposure was confirmed by real-time RT-PCR. These results obtained from histopathology, microarrays and RT-PCR indicate that prolonged exposure to 35 GHz millimeter waves causes thermally related stress and injury in skin while triggering repair processes involving inflammation and tissue matrix recovery.

Field collection and genetic classification of tick-borne Rickettsiae and Rickettsiae-like pathogens from South Texas: Coxiella burnetii isolated from field-collected Amblyomma cajennense.

We are reporting the first known isolation of the Q-fever agent Coxiella burnetii from field-collected cayenne ticks Amblyomma cajennense in North America. Q-fever affects a number of domestic ungulates where it can lead to abortion in sheep and goats. There is far less known about the disease's effects on wild species, primarily because of the tendency of the disease to self resolve and to provide long-term immunity to subsequent infections. The first recovery of C. burnetii in North America was from the tick species Dermacentor andersoni. Since the original isolation C. burnetii has been recovered from five other North American tick species. The currently accepted mode for the majority of human infections is inhalation. The Centers for Disease Control and Prevention, Division of Viral and Rickettsial Diseases, Rickettsial Zoonoses Branch asserts the Q-fever agent as requiring as few as one organism to cause disease via inhalation in susceptible humans. However, with more and more isolations from ticks, evidence linking C. burnetii and ticks is mounting. The true role of tick species as competent vectors is still unconfirmed. Preemptive field collections of possible vector arthropods, hosts, and reservoirs can provide invaluable baseline environmental data that will prove supportive in follow-up studies and abatement efforts.

Viral association with the elusive rickettsia of viper plague from Ghana, West Africa.

We previously reported a rickettsial heartwater-like disease in vipers from Ghana that resembled heartwater in its gross lesions, was apparently transmitted by ticks (Aponomma and Amblyomma), and responded clinically favorably to early treatment with tetracycline. Cell culture showed consistent cytopathic effects in bovine endothelial cells, viper cells, and mouse cells, and inhibition of cytopathic effect by tetracycline in vitro. A type D retrovirus was observed in vacuoles in all infected cells. The virus and rickettsia infection was associated with transfer of cytopathic effect, regardless of cell species. Close association of virus and rickettsia may indicate a dual infection etiology of viper plague.

Aptamer selection express: a novel method for rapid single-step selection and sensing of aptamers.

Here we describe a new DNA capture element (DCE) sensing system, based on the quenching and dequenching of a double-stranded aptamer. This system shows very good sensitivity and thermal stability. While quenching, dequenching, and separating the DCE systems made from different aptamers (all selected by SELEX), an alternative method to rapidly select aptamers was developed-the Aptamer Selection Express (ASExp). This process has been used to select aptamers against different types of targets (Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2 bacteriophage, ovalbumin, and botulinum neurotoxin). The DCE systems made from botulinum neurotoxin aptamers selected by ASExp have been investigated. The results of this investigation indicate that ASExp can be used to rapidly select aptamers for the DCE sensing system.

Fluorescence assay based on aptamer-quantum dot binding to Bacillus thuringiensis spores.

A novel assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer-quantum dot conjugate to BT spores. The in vitro selection and amplification technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used in order to identify the DNA aptamer sequence specific for BT. The 60 base aptamer was then coupled to fluorescent zinc sulfide-capped, cadmium selenide quantum dots (QD). The assay is semi-quantitative, specific and can detect BT at concentrations of about 1,000 colony forming units/ml.

Emerging tick-borne disease in African vipers caused by a Cowdria-like organism.

Heartwater is a tick-borne infectious disease caused by the rickettsial organism Cowdria ruminantium, currently Ehrlichia ruminantium. It poses an imminent threat to the Western Hemisphere, where it could cause mortality in cattle and other ruminant livestock in excess of 70%. It has been reported in the Caribbean; and its vector, Amblyomma sparsum, has been found on imported African spurred tortoises (Geochelone sulcata) and leopard tortoises (Geochelone pardalis) in southern Florida in the United States, leading to an importation ban on these reptiles. Symptoms have not been previously reported in reptiles. Here, we report peracute and acute deaths in African vipers imported from Africa through Florida. Signs included vomiting mucoid fluid, diarrhea, emaciation, convulsions, and death. Postmortem showed few gross lesions. The most consistent peracute and acute lesions were the pulmonary lesions and pericarditis with considerable bloody fluid in the pericardial sac (hydropericardium). These lesions strongly resembled the lesions of heartwater and a coccobacillus of less than 1-micron diameter was isolated in viper cell culture. The outbreak was brought to a halt by tick control and treatment of all exposed snakes with tetracycline. This isolation, tetracycline sensitivity, clinical signs, preliminary results with polymerase chain reaction of pCS20 ORF, and the viper preference of the disease may indicate a Cowdria-related attenuated species that has adapted to infect reptiles or an emerging new form of this group of microbes.

Comparison of blood pressure and thermal responses in rats exposed to millimeter wave energy or environmental heat.

Electromagnetic fields at millimeter wave lengths are being developed for commercial and military use at power levels that can cause temperature increases in the skin. Previous work suggests that sustained exposure to millimeter waves causes greater heating of skin, leading to faster induction of circulatory failure than exposure to environmental heat (EH). We tested this hypothesis in three separate experiments by comparing temperature changes in skin, subcutis, and colon, and the time to reach circulatory collapse (mean arterial blood pressure, 20 mmHg) in male Sprague-Dawley rats exposed to the following conditions that produced similar rates of body core heating within each experiment: (1) EH at 42 degrees C, 35 GHz at 75 mW/cm, or 94 GHz at 75 mW/cm under ketamine and xylazine anesthesia; (2) EH at 43 degrees C, 35 GHz at 90 mW/cm, or 94 GHz at 90 mW/cm under ketamine and xylazine anesthesia; and (3) EH at 42 degrees C, 35 GHz at 90 mW/cm, or 94 GHz at 75 mW/cm under isoflurane anesthesia. In all three experiments, the rate and amount of temperature increase at the subcutis and skin surface differed significantly in the rank order of 94 GHz more than 35 GHz more than EH. The time to reach circulatory collapse was significantly less only for rats exposed to 94 GHz at 90 mW/cm, the group with the greatest rate of skin and subcutis heating of all groups in this study, compared with both the 35 GHz at 90 mW/cm and the EH at 43 degrees C groups. These data indicate that body core heating is the major determinant of induction of hemodynamic collapse, and the influence of heating of the skin and subcutis becomes significant only when a certain threshold rate of heating of these tissues is exceeded.

Anti-Francisella tularensis DNA aptamers detect tularemia antigen from different subspecies by Aptamer-Linked Immobilized Sorbent Assay.

Aptamers are powerful candidates for molecular detection of targets due to their unique recognition properties. These affinity probes can be used to recognize and bind to their targets in the various types of assays that are currently used to detect and capture molecules of interest. They are short single-stranded (ss) oligonucleotides composed of DNA or RNA sequences that are selected in vitro based on their affinity and specificity for the target. Using combinatorial oligonucleotide libraries, we have selected ssDNA aptamers that bind to Francisella tularensis subspecies (subsp) japonica bacterial antigen. F. tularensis is an intracellular, nonmotile, nonsporulating, Gram-negative bacterial pathogen that causes tularemia in man and animals. Just as antibodies have been used to detect specific targets in varying formats, it is possible that nucleic acid-binding species or aptamers could be used to specifically detect biomolecules. Aptamers offer advantages over antibody-based affinity molecules in production, regeneration and stability due to their unique chemical properties. We have successfully isolated a set of 25 unique DNA sequences that specifically bind to F. tularensis subspecies japonica. When tested in a sandwich Aptamer-Linked Immobilized Sorbent Assay (ALISA) and dot blot analysis, the aptamer cocktail exhibited specificity in its ability to bind only to tularemia bacterial antigen from subspecies japonica, holarctica (also known as palaearctica) and tularensis but not to Bartonella henselae. Moreover, there is no binding observed either to pure chicken albumin or chicken lysozyme. Thus, it appears that this novel antitularemia aptamer cocktail may find application as a detection reagent for a potential biological warfare agent like F. tularensis.

Ultra-wideband pulses increase nitric oxide production by RAW 264.7 macrophages incubated in nitrate.

The possible effects of ultra-wideband (UWB) pulses on cellular nitric oxide production were tested by measuring nitrite in the medium bathing UWB exposed RAW 264.7 macrophages. A 30 min exposure to 1 ns UWB pulses, repeated at 600 Hz with an estimated SAR of 0.106 W/kg, did not change nitric oxide production by RAW 264.7 cells, with or without stimulation by gamma interferon and lipopolysaccharide. However, when nitrate was added to the medium of stimulated cells, nitric oxide production increased after UWB exposure, indicating a possible action of UWB pulses on induced nitric oxide synthase under certain conditions.

Use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods.

Systematic evolution of ligands by exponential enrichment (SELEX) was used to develop DNA ligands (aptamers) to cholera whole toxin and staphylococcal enterotoxin B (SEB). Affinity selection of aptamers was accomplished by conjugating the biotoxins to tosyl-activated magnetic beads. The use of magnetic beads reduces the volumes needed to perform aptamer selection, thus obviating alcohol precipitation and allowing direct PCR amplification from the bead surface. Following five rounds of SELEX, 5'-biotinylated aptamers were bound to streptavidin-coated magnetic beads and used for the detection of ruthenium trisbypyridine [Ru(bpy)3(2+)]-labeled cholera toxin and SEB by an electrochemiluminescence methodology. A comparison of control (double-stranded) aptamer binding was made with aptamers that were heat denatured at 96 degrees C (single-stranded) and allowed to cool (conform) in the presence of biotoxin-conjugated magnetic beads. Results suggest that control aptamers performed equally well when compared to heat-denatured DNA aptamers in the cholera toxin electrochemiluminescence assay and a colorimetric microplate assay employing peroxidase-labeled cholera toxin and 5'-amino terminated aptamers conjugated to N-oxysuccinimide-activated microtiter wells. Interestingly, however, in the SEB electrochemiluminescence assay, double-stranded aptamers exceeded the performance of single-stranded aptamers. The detection limits of all aptamer assays were in the low nanogram to low picogram ranges.