Detection of thymidine kinase activity using an assay based on the precipitation of nucleoside monophosphates with lanthanum chloride

A new assay method for the measurement of thymidine kinase (TK) is described. Cytosols were prepared from TK- and TK+ cells and evaluated for TK activity using an assay which is based on the phosphorylation of [125I]-iododeoxyuridine, [125I]-iododeoxycytidine, or [3H]thymidine and the precipitation of the monophosphates of these nucleosides by lanthanum chloride. The specificity, reproducibility, sensitivity, and convenience of this assay are demonstrated.     

Detection of immunologically cross-reacting capsid protein of alphaviruses on the surfaces of infected L929 cells.

Hyperimmune, but not normal immune, monospecific antiserum made to capsid protein of Sindbis virus (SIN) was found to cause cytolysis equally well of both SIN- and Semliki Forest virus-infected L929 cells in antibody-dependent, complement-mediated cytotoxicity assays. The cell surface reactivity of the hyperimmune antiserum was also demonstrated by solid-phase radioimmune assays with unfixed infected cells or infected cells fixed with low concentrations of glutaraldehyde (0.025%) before reactivity with antisera. Higher concentrations of glutaraldehyde lowered the sensitivity of detection. Purified SIN capsid protein specifically inhibited antibody-dependent, complement-mediated cytotoxicity by the monospecific anti-capsid protein serum on SIN- and Semliki Forest virus-infected target cells. That hyperimmune anti-SIN serum also cross-reacts with capsid protein on the surface of Semliki Forest virus-infected cells was suggested by the fact that capsid protein inhibited cross-cytolysis in the antibody-dependent, complement-mediated cytotoxicity assay. The latter antiserum was collected after repeated injections of purified virions over a 9-month period. The results suggest that hyperimmune monospecific antisera made to SIN capsid protein or hyperimmune antisera to SIN or Semliki Forest virions detect homologous and cross-reacting capsid protein determinants on the surface of infected cells.     

Characterization of Discrete Subpopulations of Progenitor Cells in Traumatic Human Extremity Wounds

Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics.     

Biotelemetry: a mechanistic approach to ecology

Remote measurement of the physiology, behaviour and energetic status of free-living animals is made possible by a variety of techniques that we refer to collectively as 'biotelemetry'. This set of tools ranges from transmitters that send their signals to receivers up to a few kilometers away to those that send data to orbiting satellites and, more frequently, to devices that log data. They enable researchers to document, for long uninterrupted periods, how undisturbed organisms interact with each other and their environment in real time. In spite of advances enabling the monitoring of many physiological and behavioural variables across a range of taxa of various sizes, these devices have yet to be embraced widely by the ecological community. Our review suggests that this technology has immense potential for research in basic and applied animal ecology. Efforts to incorporate biotelemetry into broader ecological research programs should yield novel information that has been challenging to collect historically from free-ranging animals in their natural environments. Examples of research that would benefit from biotelemetry include the assessment of animal responses to different anthropogenic perturbations and the development of life-time energy budgets.     

Abundance and characteristics of the recreational water quality indicator bacteria Escherichia coli and enterococci in gull faeces

AIMS: To evaluate the numbers and selected phenotypic and genotypic characteristics of the faecal indicator bacteria Escherichia coli and enterococci in gull faeces at representative Great Lakes swimming beaches in the United States. METHODS AND RESULTS: E. coli and enterococci were enumerated in gull faeces by membrane filtration. E. coli genotypes (rep-PCR genomic profiles) and E. coli (Vitek GNI+) and enterococci (API rapid ID 32 Strep and resistance to streptomycin, gentamicin, vancomycin, tetracycline and ampicillin) phenotypes were determined for isolates obtained from gull faeces both early and late in the swimming season. Identical E. coli genotypes were obtained only from single gull faecal samples but most faecal samples yielded more than one genotype (median of eight genotypes for samples with 10 isolates). E. coli isolates from the same site that clustered at >/=85% similarity were from the same sampling date and shared phenotypic characteristics, and at this similarity level there was population overlap between the two geographically isolated beach sites. Enterococcus API(R) profiles varied with sampling date. Gull enterococci displayed wide variation in antibiotic resistance patterns, and high-level resistance to some antibiotics. CONCLUSIONS: Gull faeces could be a major contributor of E. coli (10(5)-10(9) CFU g(-1)) and enterococci (10(4)-10(8) CFU g(-)1) to Great Lakes recreational waters. E. coli and enterococci in gull faeces are highly variable with respect to their genotypic and phenotypic characteristics and may exhibit temporal or geographic trends in these features. SIGNIFICANCE AND IMPACT OF THE STUDY: The high degree of variation in genotypic or phenotypic characteristics of E. coli or enterococci populations within gull hosts will require extensive sampling for adequate characterization, and will influence methods that use these characteristics to determine faecal contamination sources for recreational waters.     

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.     

Introduction to the Symposium: Ontogenetic Strategies of Invertebrates in Aquatic Environments

This symposium presents different ecological and physiologicalstrategies used by invertebrates to successfully adapt to aquaticenvironments. Adaptation has been studied mainly in adult animals,but the papers comprising the symposium emphasize ontogeneticstrategies, starting from the principle that natural selectionacts on all stages of development. Adaptive strategies may thusdiffer strikingly between developmental stages of the same organism.Invertebrates offer a wide array of ecophysiological modelsfor study, and these are exemplified by the contributions tothe symposium, which are briefly summarized. Future researchin the field will 1, expand the number of models for comparativepurposes; 2, examine the strategies, not only of larvae andjuveniles, but also of embryos, eggs and reproductive cells;and 3, investigate the genetic basis of ontogenetic strategies.     

Nonlinear dynamical law governs magnetic field induced changes in lymphoid phenotype.

The results of many different types of animal and human studies dealing with the biological effects of exposure to low frequency electromagnetic fields (EMFs) have consistently been both positive and negative. We addressed the question of why this pattern had occurred so commonly in biological studies involving exposure to EMFs and hypothesized that it stemmed from the prevalent use of a linear model to characterize what are inherently nonlinear input-output relationships. The hypothesis was tested by analyzing biological data using a novel statistical procedure that could be adjusted to detect either nonlinear or linear effects. The reliability of the procedure was established using positive and negative controls and by comparison with the results obtained from sampling a known nonlinear system. In four independent experiments, male and female mice were exposed continuously to 0.1 or 0.5 mT, 60 Hz, for 175 days, and the effect on 20 immune parameters was measured using flow cytometry and functional assays. In each experiment, EMF exposure resulted in statistically significant changes in lymphoid phenotype when and only when the response of the animals to the fields was analyzed as if it were governed by nonlinear laws. Our results suggest that the pattern of inconsistency in the EMF bioeffects studies is an artifact resulting from an incorrect choice of the conceptual model for the relation between the field and the biological effect it causally determines.