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Pre-clinical studies on boron neutron capture therapy.

The present report provides an overview of the multidisciplinary research effort on BNCT that currently is in progress at The Ohio State University. Areas under investigation include the preparation of boron containing monoclonal antibodies, the synthesis of boron containing derivatives of promazines and phathalocyanines, the development of a rat model for the treatment of glioblastoma by means of BNCT, the design of an accelerator-based neutron irradiation facility, and 10B concentration measurements using alpha track autoradiographic methods. Progress in each of these areas is described and the direction of future research is indicated.

Animals↗

Clinical considerations for neutron capture therapy of brain tumors.

The radiotherapeutic management of primary brain tumors and metastatic melanoma in brain has had disappointing clinical results for many years. Although neutron capture therapy was tried in the United States in the 1950s and 1960s, the results were not as hoped. However, with the newly developed capability to measure boron concentrations in blood and tissue both quickly and accurately, and with the advent of epithermal neutron beams obviating the need for scalp and skull reflection, it should now be possible to mount such a clinical trial of NCT again and avoid serious complications. As a prerequisite, it will be important to demonstrate the differential uptake of boron compound in brain tumor as compared with normal brain and its blood supply. If this can be done, then a trial of boron neutron capture therapy for brain tumors should be feasible. Because boronated phenylalanine has been demonstrated to be preferentially taken up by melanoma cells through the biosynthetic pathway for melanin, there is special interest in a trial of boron neutron capture therapy for metastatic melanoma in brain. Again, the use of an epithermal beam would make this a practical possibility. However, because any epithermal (or thermal) beam must contain a certain contaminating level of gamma rays, and because even a pure neutron beam causes gamma rays to be generated when it interacts with tissue, we think that it is essential to deliver treatments with an epithermal beam for boron neutron capture therapy in fractions in order to minimize the late-effects of low-LET gamma rays in the normal tissue. I look forward to the remainder of this Workshop, which will detail recent progress in the development of epithermal, as well as thermal, beams and new methods for tracking and measuring the uptake of boron in normal and tumor tissues.

Animals↗

Automatic control of anesthesia using two feedback variables.

A new controller of an ON/OFF type was implemented for halothane anesthesia. A proportional-plus-integral controller with time-delay compensation proved not to be robust enough for the known clinical situation, as shown both in computer simulations and in animal trials. The ON/OFF controller proved to be less sensitive to parameter mismatches, and repeated animal trials showed a short response time and acceptable steady-state tracking. A method for switching the controlled effect of the drug was also developed, since anesthetic agents have multiple effects. Mean arterial blood pressure and a measure of EEG frequency were chosen as controlled variables, both being depressed by halothane. A coordinator forces the system state as near the desired values of these variables as possible, given that only one drug is used.

Algorithms↗

A simple technique for on-line measurement of contractions of single smooth muscle fibers under current or voltage clamp.

This paper describes a simple technique for routine on-line measurement of length and unloaded, isotonic contractions of single smooth muscle fibers during electrophysiological experiments. The fiber is held by the recording electrode itself, stretched straight in a fast-flowing stream of solution. The video image of the fiber is measured on-line by a simple computer program. Unlike other optical methods of tracking fiber length, the technique does not require a rigid one-dimensional contraction of the fiber. The technique is reliable and easy to use, and readily compatible with current clamp, voltage clamp, and rapid reversible application of neurotransmitters and drugs.

Algorithms↗

[Bispectral index and desflurane concentration below 1 MAC].

INTRODUCTION: We investigated the relationship between bispectral index (BIS, A-2000, Aspect Medical Systems, USA) and end-tidal desflurane concentrations below 1 MAC which is especially the range of interest if desflurane is combined with remifentanil for fast-track anaesthesia. METHODS: With institutional review board approval and written informed consent we investigated 50 adult ASA I-III patients scheduled for minor orthopaedic surgical procedures. The BIS electrode (BIS-Sensor, Aspect Medical Systems, USA) was positioned on the patient's forehead as recommended by the manufacturer. All patients were premedicated with 0.15 mg x kg(-1) diazepam orally in the evening and on the morning before surgery. Induction of anaesthesia was started with a remifentanil infusion at 0.4 micro g x kg(-1) x min(-1) and 5 min later 2 mg x kg(-1) propofol was given for hypnosis. Immediately after intubation, remifentanil was reduced to 0.2 micro g x kg(-1) x min(-1), and the depth of anaesthesia was adjusted according to clinical needs by regulating the desflurane concentration. Starting 20 min after induction, a total of 1,483 data pairs, i.e. end-tidal desflurane concentrations with corresponding BIS values, was recorded. During surgery data pairs were analysed by linear regression analysis for each patient separately. During emergence from anaesthesia, desflurane effect compartment concentrations were simulated and non-linear regression analysis was applied. RESULTS: We found a relationship between BIS ranges and mean end-tidal desflurane concentrations: A BIS range of 100-85 was related to 1.1+/-0.7 vol%, BIS 84-65 to 1.5+/-1.2 vol%, BIS 64-50 to 2.9+/-1.3 vol% and BIS 49-40 to 3.1+/-0.9 vol%. In particular, end-tidal desflurane concentrations related to these BIS ranges were significantly different from each other (ANOVA; P<0.05). Intraoperatively, we obtained a correlation coefficient of R=0.42+/-0.24 (mean+/-SD, range 0.05-0.96), during emergence from anaesthesia the correlation coefficient was R=0.84+/-0.12 (range 0.61-0.99). CONCLUSION: We could demonstrate a relationship between bispectral index and end-tidal desflurane concentrations below 1 MAC with a BIS range of 49-40 being associated with approximately 0.5 MAC of desflurane during desflurane-remifentanil-anaesthesia.

Adult↗

CpG-Oligodeoxynucleotides activate tyrosinase-related protein 2-specific T lymphocytes but do not lead to a protective tumor-specific memory response.

PURPOSE: Peritumoral CpG-oligodeoxynucleotide (ODN) treatment has been successful in tumor mouse models expressing strong antigens to induce activation of tumor-specific CD8+ T lymphocytes which contribute to the control of tumor growth. To get near to clinical reality, the tumor-specific CD8+ response was investigated in mice bearing the weakly immunogenic B16 melanoma tumor and using the melanocyte differentiation tyrosinase-related protein 2 (TRP-2) as a tracking antigen. METHODS: The expansion and activation of TRP-2-specific T lymphocytes by CpG-ODNs was analyzed by tetramer staining and IFN-gamma production assays, while the activity of these cells in both memory and primary response was evaluated in vivo. RESULTS: After CpG-ODN treatment, the number of TRP-2 tetramer-stained CD8+ T lymphocytes was not significantly modified, but these cells produced higher levels of interferon gamma (IFN-gamma) in response to the antigen than those from untreated mice. Mice possessing these activated T lymphocytes, when evaluated for their antitumor memory response, showed marginal protection against intravenous (i.v.) and subcutaneous (s.c.) tumor rechallenge. These cells were not crucial for the control of primary tumor growth since strong reduction of subcutaneous tumor was observed after CpG-ODN treatment in both CD8+ T cell depleted or nondepleted mice. On the contrary, NK cell depletion markedly reduced CpG-ODN-induced tumor growth inhibition. CONCLUSIONS: Altogether, these data indicate the CpG treatment activates tumor-reactive effector CD8+ T lymphocytes, but, paralleling recent clinical observations, our model indicates that the mere activation of antitumor T cells is insufficient to result in a clinical response.

Animals↗

Influence of in vitro low-level gamma-radiation on the UV-induced DNA repair capacity of human lymphocytes--analysed by unscheduled DNA synthesis (UDS) and comet assay.

Unscheduled DNA synthesis (UDS) induced by ultraviolet radiation (UV) was studied in human lymphocytes after exposing blood samples in vitro to doses ranging between 1 and 10 mGy gamma-radiation, by way of measuring tritiated thymidine (3H-TdR) uptake in the DNA of these lymphocytes. The results indicate that samples pre-exposed to gamma-ray doses ranging between 2.5 and 4 mGy show higher UDS levels compared with those pre-exposed to doses of less than 2.5 or more than 4 mGy. These results were verified by studying the rate of removal of UV-induced photoproducts using the comet assay. The reason for the increase in DNA repair capacity in this dose range is discussed in comparison with earlier reports on this phenomenon. The DNA repair capacity with respect to inter-individual variability and age is also analysed. The study implies that the comet assay is a simple and sensitive visual method to track nucleotide excision repair and hence can be used to estimate UV-induced DNA repair in the place of the more reliable yet cumbersome and time-consuming, grain-counting autoradiographic technique.

Adult↗

A simple approach for the analysis of intracellular movement of oxidant-producing intracellular compartments in living human neutrophils.

In human neutrophils, superoxide is generated primarily within specialized oxidant-producing intracellular compartments. The present study employs a simple methodological approach to evaluate the intracellular movement of these structures in living human neutrophils. Using a CCD camera system, we monitored fluorescence in cells loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate, which is nonfluorescent until oxidized by reactive oxygen species. Fluorescence-positive intracellular compartments became detectable after neutrophils were stimulated with phorbol myristate acetate for 1 min. Further stimulation increased the intracellular compartments in both number and size in a time-dependent manner. Upon stimulation with phorbol myristate acetate, no fluorescence was seen in intracellular compartments of neutrophils isolated from patients with X-linked chronic granulomatous disease lacking gp91-phox, a membrane component of NADPH oxidase. The method enables tracking of the movement of a single oxidant-producing intracellular compartment following cell stimulation and visualization of the intracellular structures formed by fusion of oxidant-producing intracellular compartments with endocytotic vesicles and phagosomes. Therefore, it is considered to be an informative tool for evaluation of the intracellular dynamics of oxidant-producing intracellular compartments in living human neutrophils and may have a diagnostic value.

Biological Transport↗

On the control of visual spatial attention: evidence from human electrophysiology.

We used electrophysiological methods to track the deployment of visual spatial attention while observers were engaged in concurrent central attentional processing, using a variant of the attentional blink paradigm. Two visual targets (T1, T2) were presented at a stimulus onset asynchrony of either 200 ms or 800 ms. T1 was a white digit among white letters presented on a dark background using rapid serial visual presentation at fixation. T2 was another digit that was presented to the left or right of fixation simultaneously with a distractor digit in the opposite visual field, each followed by a pattern mask. In each T2 display, one digit was red and one was green. Half of the subjects reported the red digit and ignored the green one, whereas the other half reported the green digit and ignored the red one. T1 and T2 were reported in one block of trials, and only T2 in another block (order counterbalanced across subjects). Accuracy of report of T2 was lower at short SOA than at long SOA when both T1 and T2 were reported, but was similar across SOA when only T2 was reported. The electrophysiological results focused on the N2pc component, which was used as an index of the locus of spatial attention. N2pc was reduced in amplitude when subjects reported T1, and particularly so at the short SOA. The results suggest that attention to T1 interfered with the deployment of visual spatial attention to T2.

Attention↗

Genetic diversity of Cryptosporidium spp. in cattle in Michigan: implications for understanding the transmission dynamics.

Epidemiological and molecular data on 248 bovine, 17 human, and 16 water samples of Cryptosporidium spp. collected from the lower peninsula of Michigan between 1997 and 2000 were analysed. Cryptosporidium parvum bovine genotype and Cryptosporidium andersoni were found in 56 and four cattle samples, respectively. A total of six C. parvum subgenotypes were found in 34 bovine samples, and five of the eight farms had two or three subgenotypes in cattle. Six water samples from these farms had C. andersoni, five had the C. parvum bovine genotype, and one had Cryptosporidium muris. In contrast, four PCR-positive human samples produced the C. parvum bovine genotype and two had the C. parvum human genotype. Among the C. parvum bovine genotype samples, two human samples and one water sample had subgenotypes identical to those found on cattle farms. The results of this study demonstrate the potential use of molecular methods in tracking the transmission of Cryptosporidium.

Agriculture↗

Strongyloides ratti: thermokinesis of glycolytic enzyme- and lectin-treated third-stage infective larvae in vitro.

The infective third-stage larvae (L3s) of a parasitic nematode of rodents, Strongyloides ratti, showed three types of thermokinesis on a temperature gradient using an in vitro agarose tracking assay method. These depended both on the pattern of gradient temperature and the prior culture temperature. Most L3s (> or = 80%) isolated from rat feces cultured at 25 degrees C and placed on a gradient at temperatures between 30 degrees C and 37 degrees C showed no directional response, at 22-29 degrees C more than 50% of the L3s showed positive thermokinesis, at 21 degrees C L3s showed positive, negative and no directional responses in the same ratio, while at 18-20 degrees C, L3s showed negative thermokinesis (approx. 40%) or no directional response (approx. 60%) as in our previous study. The present study describes the effects of glycolytic enzyme- and lectin-treated positive thermokinesis of L3s. alpha-Glucosidase or concanavalin A significantly exhibited inhibitory effects on thermokinesis.

Animals↗

Scatterscore: a reconnaissance method to evaluate changes in water quality.

Water quality data collected in periodic monitoring programs are often difficult to evaluate, especially if the number of parameters is large, the sampling schedule varies, and values are of different orders of magnitude. The Scatterscore Water Quality Evaluation was developed to yield a quantitative score, based on all measured variables in periodic water quality reports, indicating positive, negative or random change. This new methodology calculates a reconnaissance score based on the differences between up-gradient (control) versus down-gradient (treatment) water quality data sets. All parameters measured over a period of time at two or more sampling points are compared. The relationship between the ranges of measured values and the ratio of the medians for each parameter produces a data point that falls into one of four sections on a scattergram. The number and average values of positive, negative and random change points is used to calculate a Scatterscore that indicates the magnitude and direction of overall change in water quality. The Scatterscore Water Quality Evaluation, a reconnaissance method to track general changes, has been applied to 20 sites at which coal utilization by-products (CUB) were used to control acid mine drainage (AMD).

Coal↗

Characterization of Escherichia coli isolates from different fecal sources by means of classification tree analysis of fatty acid methyl ester (FAME) profiles.

Microbial source tracking (MST) methods need to be rapid, inexpensive and accurate. Unfortunately, many MST methods provide a wealth of information that is difficult to interpret by the regulators who use this information to make decisions. This paper describes the use of classification tree analysis to interpret the results of a MST method based on fatty acid methyl ester (FAME) profiles of Escherichia coli isolates, and to present results in a format readily interpretable by water quality managers. Raw sewage E. coli isolates and animal E. coli isolates from cow, dog, gull, and horse were isolated and their FAME profiles collected. Correct classification rates determined with leaveone-out cross-validation resulted in an overall low correct classification rate of 61%. A higher overall correct classification rate of 85% was obtained when the animal isolates were pooled together and compared to the raw sewage isolates. Bootstrap aggregation or adaptive resampling and combining of the FAME profile data increased correct classification rates substantially. Other MST methods may be better suited to differentiate between different fecal sources but classification tree analysis has enabled us to distinguish raw sewage from animal E. coli isolates, which previously had not been possible with other multivariate methods such as principal component analysis and cluster analysis.

Animals↗

A prospective biomonitoring campaign with honey bees in a district of upper-bavaria (Germany).

In order to establish a monitoring method to track long term changes of the amount of anthropogenic contamination in a district of Bavaria (Germany), a biomonitoring campaign with honey bees was performed in spring 2002. Expected anomalies from the industry or from residential areas in the sampled district could not be detected. An anomaly over a considerable part of the sampling area correlating with other phenomena lead to the hypothesis of a prehistoric cosmic impact. Moreover a principal component analysis of the data showed evidence for a biogenic, an anthropogenic and an unknown component hypothetically related to a possible cosmic impact.

Animals↗

Determining sources of fecal bacteria in waterways.

The microbiological contamination of waterways by pathogenic microbes has been, and is still, a persistent public safety concern in the United States and in most countries of the world. As most enteric pathogens are transmitted through the fecal-oral route, fecal pollution is generally regarded as the major contributor of pathogens to waterways. Fecal pollution of waterways can originate from wastewater treatment facilities, septic tanks, domestic- and wild-animal feces, and pets. Because enteric pathogens are derived from human or animal sources, techniques capable of identifying and apportioning fecal sources have been intensively investigated for use in remediation efforts and to satisfy regulatory concerns. Pollution of human origin is of the most concern, since human feces is more likely to contain human-specific enteric pathogens. Fecal indicator bacteria have been used successfully as the primary tool for microbiologically based risk assessment. However measurement of fecal indicator bacteria does not define what pathogens are present, or define the sources of these bacteria. Microbial source tracking (MST) methods that have the ability to differentiate among sources of fecal pollution are currently under development. These methods will ultimately be useful for risk assessment purposes and to aid regulatory agencies in developing strategies to remediate microbiologically impaired waterways.

Enterobacteriaceae↗

Three-dimensional movement analysis of lateral pterygoid electromyographic activity during mastication in the rat.

Simultaneous recordings of mandibular movement in three dimensions (frontal, horizontal and sagittal plane), and lateral pterygoid electromyographic (EMG) data during mastication of pellets and slurry, were collected from 5 rats by the opto-electronic mandibular tracking (OMT) method and computerized digital-processing of the EMG wave-forms. The microcomputer analyses allowed determination of frontal-, horizontal- and sagittal-plane movement displacements and velocities for whole chew cycles and, during lateral pterygoid activity, opening burst (OB) and closing burst (CB) latencies. There were significant (p less than or equal to 0.01) differences between duration, displacement and velocity data from slurry and pellet chew cycles. OB latencies were significantly (p less than or equal to 0.01) longer than CB, and OB displacements started significantly (p less than or equal to 0.01) farther from minimal gape than CB ones. Working-side OB activity occurred as the mandible moved inferiorly, laterally and posteriorly; working-side CB activity occurred as the mandible moved superiorly, medially and anteriorly. A secondary peak of OB activity, coinciding with anterior sagittal translation during the last third of mandibular depression, was detected; this may represent independent activity of the rat superior lateral pterygoid effecting anterior translation of the mandibular-joint disc and capsule. Rat lateral pterygoid activity has the same basic, bimodal pattern (active during both mandibular depression and elevation) as is found in humans.

Animals↗

Computer-assisted analysis of the vascular endothelial cell motile response to injury.

We have developed an automated, user-friendly method to track vascular endothelial cell migration in vitro using an IBM PC/XT with MS DOS. Analog phase-contrast images of the bovine aortic endothelial cells are converted into digital images (8 bit, 250 x 240 pixel resolution) using a Tecmar Video VanGogh A/D board. Digitized images are stored at selected time points following mechanical injury in vitro. FORTRAN and assembly language subroutines have been implemented to automatically detect the wound edge and the edge of each cell nucleus in the phase-contrast, light-microscope field. Detection of the wound edge is accomplished by intensity thresholding following noise reduction in the image and subsequent sampling of the wound. After the range of wound intensities is determined, the entire image is sampled and a histogram of intensities is formed. The histogram peak corresponding to the wound intensities is subtracted, leaving a histogram peak that gives the range of intensities corresponding to the cell nuclei. Rates of cell migration, as well as cellular trajectories and cell surface areas, can be automatically quantitated and analyzed. This inexpensive, automated cell-tracking system should be widely applicable in a variety of cell biologic applications.

Animals↗

The use of chemical reagents in the detection of DNA mutations.

As the analysis of the human genome proceeds at an ever-increasing pace, many genes have been identified which are the site for mutations responsible for inherited diseases. The identification of the mutations within these genes has become a major application of molecular biology technologies, and to this end a number of mutation detection systems have been developed for use in diagnostic and research laboratories. The uses of these mutation detection systems are in the diagnosis of inherited disease (both prenatal and neonatal) and in an understanding of the function of the affected protein by cataloguing the range of mutations. Two of these mutation detection systems are reviewed here. Both rely on chemical modification of mismatched nucleotides, by either carbodiimide or hydroxylamine and osmium tetroxide. The methods are termed the carbodiimide (CDI) and the Chemical Cleavage of Mismatch (CCM) methods. The history and evolution of the methods is tracked, illustrating the way in which they developed, both as suitable technology became available (for example, the polymerase chain reaction) and as a result of a specific need. The current methodologies are briefly discussed, followed by a discussion of their applications, especially in the realm of disease mutation detection.

Animals↗