Single-hit Model Research Articles

Analytical investigation of the possibility of parameter invariant TCP-based radiation therapy plan ranking

Purpose. To analytically investigate the possibility of a parameter invariant ranking of radiotherapy (RT) plans based on comparing the tumor control probabilities (TCPs) produced by the competing plans for different values of the radiobiological model parameters determining the radiation response. Method. Individual TCP models based on the Single hit model of cell kill and on the linear-quadratic (LQ) model of cell damage, with and without repopulation, are considered. The tumor dose distributions in case of heterogeneous dose irradiation are described by a Gaussian distribution function on the basis of which a TCP expression is derived depending only on the mean dose to the tumor and its standard deviation and the TCP model parameters. Results. It is shown that in case of homogeneous dose to the tumor the plan ranking in terms of TCP is parameter invariant. In case of heterogeneous dose to the tumor there are cases when the plan ranking is parameter invariant and cases when the parameter invariance is violated. An interesting dependence of the extent of the parameter invariance violation on the model of cell kill as well as on the size and repopulation rate of the tumor is noted. Conclusion. We conclude that in many cases RT plan ranking in terms of TCP is parameter invariant. However, since there exist cases where the parameter invariance is lost an investigation of the specific plans to be ranked should be performed applying the proposed approach.

Study of Gafchromic® EBT film response over a large dose range

Presently Gafchromic EBT films are widely used for relative dose verification in standard radiation therapy using high-energy photons, inclusive IMRT. The use of films for dosimetry in medical ion beams is more complicated due to the strongly inhomogeneous dose deposition by ions on microscopic level. Track structure models, presently used to describe dosimeter response as a function of the ion field properties, are based on input information which can be obtained from the film response in photon beams. We therefore studied the performance of Gafchromic EBT films, ancestors of currently available EBT2 films, in 60Co photon beams. The dose–response curve was measured from 7.5 × 10−2 Gy to 3 × 104 Gy. The dynamic range, linearity and dose rate dependence of this calibration curve were studied. A high saturation dose of 3 × 103 Gy, and thus a large dynamic range, was observed. No signs of supralinearity and bleaching due to radiation were found. No dependence of the response on the dose rate at high dose rates and high doses was found. All those properties justify the use of simplified models of the film response to ions. Furthermore, fits of the calibration data by predictions of different models for signal creation mechanism of dosimetric materials were performed. The best description was found for the recently published gamma-distributed single-hit model which takes into account different sizes of the active centres.

Molecular mechanism of radiosensitizing effect of paclitaxel

Paclitaxel is a radiosensitizer which may stabilize microtubules, block the G2/M phase of the cell cycle and thus modulate the radioresponsiveness of tumor cells. However, its potential molecular mechanisms of radiosensitization have not been well understood yet. This study was to investigate the radiosensitizing effect of paclitaxel on human oral epithelium carcinoma (KB) cell line and to explore the molecular mechanism of radiosensitization. The survival of KB cells following the treatment with paclitaxel and/or radiation was determined by colony-forming assay. The radiosensitizing effect was evaluated by calculating the sensitizing enhancement ratio (SER) with multi-target single hit model. The cell cycle distribution was analyzed by flow cytometry. Differentially expressed genes related to paclitaxel radiosensitization were screened using human Oligo microarray. Expressions of protein regulating cytokinesis 1 (PRC1) and cyclin B2 genes were confirmed by real-time quantitative PCR. The proliferation of KB cells was significantly inhibited by paclitaxel combined with ionizing radiation. The SERD0 and SERDq were (2.40 +/- 1.87) and (12.23 +/- 2.81) respectively, when the concentration of paclitaxel was 20 nmol/l. After the treatment with paclitaxel in combination with irradiation, the percentage of G1 phase cells decreased from (48.32 +/- 2.40)% to (15.73 +/- 7.00)% (P<0.01), and the percentage of G2/M phase cells increased from (13.66 +/- 2.16)% to (52.51 +/- 5.02)% (P<0.01). In total 176 differentially expressed genes were identified to be related to paclitaxel radiosensitization. Ten genes were found to regulate cell division, two of which were up-regulated and eight were down-regulated after the treatment. Moreover, the expression of PRC1 and cyclin B2 was decreased. The radiosensitizing effect of paclitaxel on KB cells may be due to the down-regulated expression of PRC1 and cyclin B2, resulting in inhibition of mitotic spindle formation and cell necrosis.

Tests of the Single-hit DNA Damage Model

The algebra of target theory for damage by radiation was laid out by Atwood and Norman in 1949. Their equations provide a widely embraced framework for distinguishing single-hit and multi-hit mechanisms of damage. The present work asks whether in vitro damage to DNA duplexes by different agents affects amplification by the polymerase chain reaction (PCR) in a single-hit manner. Real-time monitoring of fluorescent PCR product (qPCR) was used to measure the fraction of DNA ( S) surviving doses ( D) of three damaging agents: γ irradiation, DNase I, and UV radiation. The log fraction surviving was compared to the best-fit straight line predicted for a random single-hit model (ln S = kD). Human DNA targets for analysis were segments of multiple (nested) DNA lengths from the nuclear and the mitochondrial genomes within 10% of 150, 250, 350, 450, 650, 1000 and 2000 bases. For γ irradiation, the results were consistent with a single-hit model for all segment sizes. In the case of DNase I, the shortest segment (150 bp), for both genomic and mitochondrial DNA, experienced more damage at low concentrations of DNase than the random single-hit model predicted. Conversely, in the case of UV, all segments of the nuclear target gene were less damaged at low doses and more damaged at high doses than predicted by the one hit model. These deviations from the predictions of a random single-hit model were interpreted as evidence for concerted activity in the case of DNase and of a multi-hit, sequence-dependent mechanism in the case of UV, perhaps due to the accumulation of lesions that slowed but did not entirely block Taq polymerase elongation.

Limiting-dilution analysis of T cell reactivity to mycobacterial antigens in peripheral blood and synovium from rheumatoid arthritis patients.

Limiting-dilution analysis (LDA) was used to quantify the frequency of Mycobacterium bovis BCG- and 65-kD-reactive T cells in paired samples of peripheral blood and synovial tissue from patients with rheumatoid arthritis. The frequency of BCG-reactive T cells detected in the peripheral blood of patients ranged from 1/585 to 1/7639 versus a control frequency range of 1/480 to 1/6773. The frequency of such cells in the synovium was found to be much lower than it was in peripheral blood; in fact, in 80% of patients synovial BCG-reactive T cells were not detected. The frequency of 65-kD-reactive cells in the peripheral blood of each individual was lower than the frequency of BCG-reactive cells (range 1/3738 to 1/55,324), as would be expected. However, no synovial 65-kD-reactive cells were detected from any of the patients studied. The LDA assay for the 65-kD antigen was consistent with the single hit model, that for BCG was not. The relatively high proportion of mycobacterial-reactive precursors seen in the peripheral blood of non-vaccinated individuals may reflect a population of cells induced either by natural environmental exposure to mycobacteria or, given the highly conserved nature of heat shock proteins across phylogeny, by some other infection. The results also suggest that the frequent finding of reactivity to proteins such as the 65-kD heat shock protein contained within BCG may not be a generalized phenomenon in rheumatoid synovium.

Quantitative risk assessment for Escherichia coli O157:H7 in frozen ground beef patties consumed by young children in French households

A quantitative risk assessment for Escherichia coli O157:H7 in frozen ground beef patties consumed by children under 10 years of age in French households was conducted by a national study group describing an outbreak which occurred in France in 2005. Our exposure assessment model incorporates results from French surveys on consumption frequency of ground beef patties, serving size and consumption preference, microbial destruction experiments and microbial counts on patties sampled from the industrial batch which were responsible for the outbreak. Two different exposure models were proposed, respectively for children under the age of 5 and for children between 5 and 10 years. For each of these two age groups, a single-hit dose-response model was proposed to describe the probability of hemolytic and uremic syndrome (HUS) as a function of the ingested dose. For each group, the single parameter of this model was estimated by Bayesian inference, using the results of the exposure assessment and the epidemiological data collected during the outbreak. Results show that children under 5 years of age are roughly 5 times more susceptible to the pathogen than children over 5 years. Exposure and dose-response models were used in a scenario analysis in order to validate the use of the model and to propose appropriate guidelines in order to prevent new outbreaks. The impact of the cooking preference was evaluated, showing that only a well-done cooking notably reduces the HUS risk, without annulling it. For each age group, a relation between the mean individual HUS risk per serving and the contamination level in a ground beef batch was proposed, as a tool to help French risk managers.

The effect of p21 antisense oligodeoxynucleotides on the radiosensitivity of nasopharyngeal carcinoma cells with normal p53 function

p21(WAF1/CIP1) is transcriptionally activated by p53 and is required for G1 to S phase progression. p21 plays a critical role in DNA repair after DNA damage. Thus, cells with defective p21 may result in an enhancement of radiation induced apoptosis and improved radiosensitivity. We tested the hypothesis that p21 antisense oligodeoxynucleotides (p21 AS ODNs) can be used to reduce p21 expression level and increase radiosensitivity in CNE-1-wtp53 nasopharyngeal carcinoma cell line with normal p53 function. The p21 antisense oligodeoxynucleotides (p21 AS ODNs) and the random control oligodeoxynucleotides (p21 RD ODNs) were synthesized. p21 AS ODNs sequence: 5'-TGTCATGCTGGTCTGCCGCC-3'; p21 RD ODNs sequence: 5'-CCGGTGAACGAGCGAGCACA-3'. p21 AS ODNs and p21 RD ODNs were transfected into CNE-1-wtp53 nasopharyngeal carcinoma cell line. The protein expression levels of P21 were evaluated using Western blotting analysis. Cell cycle progression and apoptotic cells were assessed by flow cytometric analysis. The clonogenic survival assay was performed to determine the survival fraction. The parameters D0, Dq, and N for the single-hit multitarget model and the parameters alpha, beta, alpha/beta, and SF2 for the linear-quadratic model were calculated. BALB/c nude mice were used to investigate the effect of p21 AS ODNs on the radiosensitivity of nasopharyngeal xenografts in vivo. p21 AS ODNs were detected mainly in plasma with fluorescence microscopy investigation. P21 protein level dramatically decreased and the amount of apoptotic cells increased in p21 AS ODNs transfected cells than in p21 RD ODNs transfected cells after irradiation. The percentage of G1 arrest decreased in p21 AS ODNs transfected cells 24 h after radiation, then G2 arrest decreased 48 h after radiation. The values of D0, Dq, SF2 decreased and alpha value increased in p21 AS ODNs transfected cells than in control cells. The inhibition rate in tumor xenografts exposed to X ray of 10 Gy alone was 39.1%, while it was 51.4% in xenografts injected with p21 AS ODNs before exposure to radiation. Unfortunately, there was no significant difference between these two groups (P < 0.05). p21 Antisense oligodeoxynucleotides led to inhibition of P21 protein expression, loss of G1 arrest, increase of apoptosis in CNE-1-wtp53 nasopharyngeal carcinoma cell line in vitro and inhibited tumor growth in vivo. Antisense oligodeoxynucleotides may become a promising strategy to enhance radiosensitivity in nasopharyngeal carcinoma cells with normal p53 function.

Radiation damage, repopulation and cell recovery analysis of in vitro tumour cell megacolony culture data using a non-Poissonian cell repopulation TCP model

The effects of radiation damage, tumour repopulation and cell sublethal damage repair and the possibility of extracting information about the model parameters describing them are investigated in this work. Previously published data on two different cultured cell lines were analysed with the help of a tumour control probability (TCP) model that describes tumour cell dynamics properly. Different versions of a TCP model representing the cases of full or partial cell recovery between fractions of radiation, accompanied by repopulation or no repopulation were used to fit the data and were ranked according to statistical criteria. The data analysis shows the importance of the linear–quadratic mechanism of cell damage for the description of the in vitro cell dynamics. In a previous work where in vivo data were analysed, the employment of the single hit model of cell kill and cell repopulation produced the best fit, while ignoring the quadratic term of cell damage in the current analysis leads to poor fits. It is also concluded that more experiments using different fractionation regimes producing diverse data are needed to help model analysis and better ranking of the models.

SU-FF-T-249: Small Electron Field Cutout Output Factors Measured Using a 2D Ion Chamber Array Compared to Radiographic Film

Purpose: To compare the electron cutout output factor (COF) of small fields measured by two methods: radiographic film (Kodak X-Omat V) and the Seven29 (PTW) 2D small volume ion chamber array. Method and Materials: The COFs for four small electron fields were measured on a Varian-2100C accelerator with a 10 cm × 10 cm cone at 6 MeV. Radiographic film and Seven29 array ion chamber were set perpendicular to the central axis of the beam. The film and effective point of measurement for the 2D array were both set at dmax. Solid water was used for build up in both cases, and 100 cm SSD was set at the top surface of the solid water. The data were measured using 10 to 400 monitor units (MU) for Seven29 and 30 to 200 MU for the film. The open 10 cm × 10 cm insert data from film measurement was used to compute the film parameters of maximum optical density (OD) and sensitivity based on a single hit model. These parameters were used later to convert the measured cutout data from OD to dose for the COF calculation. The OD was read from a Digital Densitometer II. Results: The Seven29 ion chamber array behaved linearly as a function of MU as expected, which provided an identical COF regardless of the number of MU's used (less than 1% difference). The COF results from the Seven29 and from film measurements showed a maximum difference of 1.6%. Conclusion: The 2D ion chamber array can be used to measure the COF for a small electron fields. Using the Seven29 to measure small field COF will save measurement time compared to using film dosimetry, and in addition, this is also beneficial to filmless departments.

SU-FF-T-373: Fitting the Zaider-Minerbo TCP Model to Cell Megacolony Culture Dose Response in Vitro Data

Purpose: To investigate the effects of radiation damage, tumor repopulation and cellular sublethal damage repair. Method and Materials: An expression of the Zaider-Minerbo model obtained by Stavreva et al. is used to fit published dose-response in vitro data from two cellular megacolony cultures. Results: The data analysis shows the importance of the linear-quadratic mechanism of cell damage for the description of in vitro cell dynamics. In a previous work, where in vivo data were analyzed, the employment of the single hit model and cell repopulation produced the best fit, while ignoring the quadratic term in the current analysis leads to poor fits. Also, the best-fit value of the probability of sublethal damage repair, τ, for both cell cultures tends to infinity, indicating that full recovery of the cells occurs between any two consecutive fractions. Conclusion: We conclude that the Zaider-Minerbo model assuming full recovery of the cells between fractions accompanied by cell repopulation best fits the data from both cellular cultures investigated in the current work. However, a model assuming no repopulation returned a fit statistically indistinguishable from the fit produced by the Zaider-Minerbo model, though at the expense of unusual best fit values of the cell radiosensitivity characteristics and a large value of τ. Therefore, we recommend the design of experiments using different fractionation regimes producing diverse data to help better analyze the TCP models and rank the models in accordance with statistical criteria.

Silatecan DB-67 is a novel DNA topoisomerase I–targeted radiation sensitizer

The silatecan 7-tert-butyldimethylsilyl-10-hydroxy-camptothecin (DB-67) represents a new generation of camptothecin derivatives that exhibits a potent in vitro DNA topoisomerase I (TOP1)-mediated DNA-damaging activity, improved blood stability, and holds significant promise for the treatment of human cancers. In this study, we characterize the role of TOP1 in mediating the radiosensitization activity of DB-67. As examined by clonogenic survival assay, DB-67 exhibited potent radiosensitization activity at a concentration 10-fold lower than camptothecin in the human glioma D54-MG and T-98G cells, which harbor wild-type and mutant p53, respectively. Analyzed by the single-hit multitarget model, DB-67 induced radiosensitization by obliterating the "shoulder" of the radiation survival curve in the D54-MG cells. The in vivo targeting of TOP1 by DB-67 was investigated by immunoblot analysis. In a dose-dependent manner, DB-67 specifically stimulates covalent linking of TOP1 to chromosomal DNA at concentrations 10-fold lower than camptothecin in the D54-MG cells. The potency of in vivo targeting of TOP1 by DB-67 correlates well with its cytotoxicity and radiosensitization activity. Furthermore, DB-67 exhibited substantially less cytotoxicity and radiosensitization activity in the TOP1 mutant Chinese hamster lung fibroblast DC3F/C-10 cells than in their parental DC3F cells. Together, our data show that DB-67 exhibits potent cytotoxicity and radiosensitization activity by targeting TOP1 in mammalian cells and has great potential for being developed to treat human cancers.

Characteristics of sensitometric curves of radiographic films.

A new type of radiographic film, EDR (extended dose range) film, has been recently become available for film dosimetry. It is particularly attractive for composite isodose verification of intensity modulated radiation therapy because of its low sensitivity relative to the more common Kodak XV film. For XV film, the relationship between optical density and dose, commonly known as the sensitometric curve, depends linearly on the dose at low densities. Unlike XV film, the sensitometric curve of EDR film irradiated by megavoltage x rays is not linearly dependent on the dose at low densities. In this work, to understand the mechanisms governing the shape of the sensitometric curves, EDR film was studied with kilovoltage x rays, 60Co gamma rays, megavoltage x rays, and electron beams. As a comparison, XV film was also studied with the same beams mentioned above. The model originally developed by Silberstein [J. Opt. Soc. Am. 35, 93-107, 1945)] is used to fit experimental data. It is found that the single hit model can be used to predict the sensitometric curve for XV films irradiated by all beams used in this work and for EDR films exposed to kilovoltage x rays. For EDR film irradiated by 60Co gamma rays, megavoltage x rays, and electron beams, the double hit model is used to fit the sensitometric curves. For doses less than 100 cGy, a systematic difference between measured densities and that predicted by the double hit model is observed. Possible causes of the observed differences are discussed. The results of this work provide a theoretical explanation of the sensitometric behavior of EDR film.

Mg2+ ions do not induce expansion of the melted DNA region in the open complex formed by Escherichia coli RNA polymerase at a cognate synthetic Pa promoter. A quantitative KMnO4 footprinting study.

Footprinting studies of prokaryotic open transcription complexes (RPO), based on oxidation of pyrimidine residues by KMnO4 and/or OsO4 at a single oxidant dose, have suggested that the extent of DNA melting in the transcription bubble region increases in the presence of Mg . In this work, quantitative KMnO4 footprinting in function of the oxidant dose of RPO, using Escherichia coli RNA polymerase (E(sigma)70) at a fully functional synthetic promoter Pa having -35 and -10 consensus hexamers, has been used to determine individual rate constants of oxidation of T residues in this region at 37degrees C in the absence of Mg2+ and in the presence of 10 mM MgCl2, and to evaluate therefrom the effect of Mg2+ on the extent of DNA melting. Population distributions of end-labeled DNA fragments corresponding to oxidized Ts were quantified and analyzed according to the single-hit kinetic model. Pseudo-first order reactivity rate constants, ki, thus obtained demonstrated that Mg2+ ions bound to RPO merely enhanced the reactivity of all 11 oxidizable thymines between the +3 and -11 promoter sites by a position-dependent factor: 3-4 for those located close to the transcription start point +1 in either DNA strand, and about 1.6 for those located more distantly therefrom. On the basis of these observations, we conclude that Mg2+ ions bound to RPO at Pa do not influence the length of the melted DNA region and propose that the higher reactivity of thymines results mainly from lower local repulsive electrostatic barriers to MnO4 diffusion around carboxylate binding sites in the catalytic center of RPO and promoter DNA phosphates.

The Beta Poisson dose-response model is not a single-hit model.

The choice of a dose-response model is decisive for the outcome of quantitative risk assessment. Single-hit models have played a prominent role in dose-response assessment for pathogenic microorganisms, since their introduction. Hit theory models are based on a few simple concepts that are attractive for their clarity and plausibility. These models, in particular the Beta Poisson model, are used for extrapolation of experimental dose-response data to low doses, as are often present in drinking water or food products. Unfortunately, the Beta Poisson model, as it is used throughout the microbial risk literature, is an approximation whose validity is not widely known. The exact functional relation is numerically complex, especially for use in optimization or uncertainty analysis. Here it is shown that although the discrepancy between the Beta Poisson formula and the exact function is not very large for many data sets, the differences are greatest at low doses--the region of interest for many risk applications. Errors may become very large, however, in the results of uncertainty analysis, or when the data contain little low-dose information. One striking property of the exact single-hit model is that it has a maximum risk curve, limiting the upper confidence level of the dose-response relation. This is due to the fact that the risk cannot exceed the probability of exposure, a property that is not retained in the Beta Poisson approximation. This maximum possible response curve is important for uncertainty analysis, and for risk assessment of pathogens with unknown properties.

Antibody and Virus: Binding and Neutralization

During infection with an enveloped virus, antibodiesare elicited to envelope proteins. Some of these antibod-ies bind to envelope spikes on the virion, some bind tononvirion forms of the envelope (“viral debris”), and somebind to both. The relative amounts of different antibodytypes elicited varies from virus to virus. There is generalagreement that only anti-envelope antibodies that bind tothe envelope spike on the virion will be neutralizing orshow antiviral activity. However, there is less agreementabout whether all antibodies that bind to the envelopespike will neutralize virus, i.e., are there antibodies thatbind well to envelope spikes but do not neutralize virus?The question is more than academic. If all antibodiesthat bind neutralize, then the envelope spike has therequisite antigenic properties of an ideal vaccine candi-date. On the other hand, if the envelope spike can inducenonneutralizing antibodies, then it may not be an optimalantigen. In particular, the induction of nonneutralizingantibodies that can bind to envelope spikes and inhibitthe binding of neutralizing antibodies would be undesir-able.Our experience, most particularly in studies on humanmonoclonal antibodies to HIV-1, RSV, and Ebola virus,has been that there is an excellent correlation betweenbinding to envelope spikes and neutralization, which inthese studies was measured as binding to infected cells(i.e., cell-associated virus). We have not encounteredmonoclonal antibodies that bind well to envelope spikeson infected cells but do not neutralize virus. Furthermore,we have generally seen a close correlation betweenhalf-maximal antibody binding and antibody concentra-tion required to give 50% neutralization suggesting thatneutralization is directly related to occupancy of sites onthe virion. For HIV-1, neutralization is incremental withincreasing antibody occupancy, irrespective of theepitope recognized, leading to increased inhibition ofinfectivity. This is consistent with a multihit neutralizationmodel rather than with a single-hit model or modelswhich predict a neutralization threshold. We have viewedantibody neutralization as a process in which virionsbecome coated with antibody and are thereby stericallyinhibited from attachment to the target cell or fusion withthe membrane of the target cell. The antibody moleculeis typically similar in size to an envelope spike, e.g., forHIV-1 the extracellular trimer has a molecular weight ofabout 450 kDa, similar to that of three IgG molecules,making steric obstruction a likely scenario. In this view, itis relatively difficult to imagine a virion coated with non-neutralizing antibodies.However, in contrast to these observations and thisdiscussion, there is a strong tradition in virology of “bind-ing (i.e., to the virion) but nonneutralizing” antibodies. Wehave wondered why we have not found evidence forsuch antibodies. Of course this could simply reflect theirabsence in the systems that we have studied. We ques-tion the evidence for nonneutralizing antibodies that bindwell to the virus.Early studies described a “nonneutralizable” fraction ofvirus which persisted even at high antibody concentra-tions. Addition of anti-antibody could reduce infectivity ofthis fraction. It seems that virus aggregation may havebeen responsible for this phenomenon. We also note thatnonneutralizable fractions have been described for hep-atitis A and hepatitis C as a result of virus associationwith lipids or lipoproteins.Undoubtedly, some descriptions of binding, nonneu-tralizing antibodies arose because of a failure to appre-ciate that antibodies that bind to isolated envelope mol-ecules do not necessarily, and very often do not, bind toenvelope spikes. A classic example is HIV-1 where manyantibodies have been described which bind with highaffinity to monomeric gp120 or unprocessed gp160, veryfew of these however showing substantial affinity forenvelope spikes.Antibody-mediated enhancement of infection is a phe-nomenon that at first glance illustrates the existence ofnonneutralizing antibodies since the antibodies involvedmust bind to virions. However, a key observation here isthat enhancement, when described, appears to occur forneutralizing antibodies at subneutralizing concentra-

Design of limiting dilution analysis experiments for helper T lymphocyte precursor frequency determination in the context of allogeneic bone marrow transplantation

Helper, interleukin 2 (IL-2) producing, T lymphocyte precursor (HTLp) frequency determination by limiting dilution analysis (LDA) is of value for quantifying alloreactivity in allogeneic bone marrow transplantation (BMT). LDA assays are labour-intensive and time-consuming to perform and the numbers of donor and recipient cells available are limited. It is therefore important that the design of the experiment yields reliable frequencies with a minimum of effort and a realistic cell requirement. We have critically evaluated the methods proposed for LDA design by Strijbosch et al. [Strijbosch, L.W., Buurman, W.A., Does, R.J., Zinken, P.H., Groenewegen, G., 1987. Limiting dilution assays. Experimental design and statistical analysis. J. Immunol. Methods 97, 133] and by Blackett and Gordon [Blackett, N.M., Gordon, M.Y., 1996. Optimizing limiting dilution assays: frequency and 'ability' measurements of haemopoietic progenitor cells. Br. J. Haematol. 92, 507 (see comments)] and found them inadequate for this application. The estimation of the HTLp frequency is traditionally based on the single-hit Poisson model and the adequacy of this model was compared with that of a double-hit model. The results were in favour of the single-hit model. Ten different LDA experimental designs were explored by Monte Carlo simulations. The optimal design exploits the maximal numbers of cells that can be obtained for analysis to estimate HTLp frequencies in the range 1:1,000,000–1:20,000 with a coefficient of variation of 10–20% and with a minimum of manual labour.

Relationships between colony forming efficiency and parameters of intrinsic radiosensitivity

Purpose: In an attempt to determine whether radiosensitivity is correlated with colony forming efficiency (CFE), a large amount of data have been analysed from the literature. Materials and methods: The survival curves of 446 human cell lines irradiated in exponentially growing phase in vitro are included in this study. Technical factors such as culture type and the use of feeder cells were considered cofactors in addition to the genetic and histological origin of the cells. Intrinsic radiosensitivity is expressed in terms of the parameters of the linear quadratic model and the single-hit multitarget model. Results: It is shown that low CFE is characteristic of cells plated in agar and cells from primary biopsies. Cells plated in the presence of feeder cells have, in general, higher CFE than cells plated without feeder cells. A positive correlation is observed between intrinsic radiosensitivity and CFE: the higher the CFE, the more resistant the cell line. This relationship is particularly obvious when radiosensitivity is expressed in terms of alpha, S 2 or D, parameters which essentially characterize the initial part of the survival curve. The correlation is also found within histological or genetic groups of cell lines. However, for a given cell line, there is no relationship between CFE and radiosensitivity among different experiments. Cells irradiated in the presence of feeder cells are less subject to this behaviour. Conclusions: CFE as well as radiosensitivity are intrinsic properties of a cell line. Experimental conditions determine the quality of the correlation between radiosensitivity and CFE.

A Single Hit Model of Polymicrobial Sepsis: Cecal Ligation and Puncture

A Single Hit Model of Polymicrobial Sepsis: Cecal Ligation and Puncture

Biologic synergism and parallelism.

In epidemiologic studies of two binary exposure factors, much attention has been given to the concept of synergism of the factors. The leading dictionary of epidemiology offers two definitions of synergism, one of which this author labels statistical and the other biologic. The epidemiologic literature has been largely concerned with statistical synergism, which is typically measured using additive or multiplicative interaction. This paper focuses on biologic synergism, on the related concept of biologic parallelism, and on the question of how much information can be gleaned about population amounts of biologic synergism and parallelism--information which is of vital interest to epidemiologists. A fundamental identity equates the difference between the amounts of biologic synergism and parallelism to the additive interaction. Two biologic models, the multistage model and the no-hit or immunity model, enhance the interpretation of multiplicative interaction as a measure of statistical synergism, but it is pointed out here that, unfortunately, both models incorporate the strong assumption that there is no parallelism. A third model, the single-hit or vulnerability model, makes the even stronger assumption that there is no biologic synergism and consequently that the additive interaction is equal to minus the amount of parallelism. A consequence of this fact is that a link which has been perceived in the literature to exist between the single-hit model and the additive interaction is false.

Cellular radiosensitivity of small-cell lung cancer cell lines

The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based on the construction of continuous exponential growth curves. Fifteen SCLC cell lines were studied, applying a slightly modified clonogenic assay and a growth extrapolation method. A dose-survival curve was obtained for each experiment and used for calculating several survival parameters. The multitarget single hit model was applied to calculate the cellular radiosensitivity (D0), the capacity for sublethal damage repair (Dq), and the extrapolation number (n). Values for alpha and beta were determined from best-fit curves according to the linear-quadratic model and these values were applied to calculate the surviving fraction after 2-Gy irradiation (SF2). In our investigation, the extrapolation method proved to be inappropriate for the study of in vitro cellular radiosensitivity due to lack of reproducibility. The results obtained by the clonogenic assay showed that the cell lines studied were radiobiologically heterogeneous with no discrete features of the examined parameters including the repair capacity. The results indicate that SCLC tumors per se are not generally candidates for hyperfractionated radiotherapy.