End-point Dilution Assay Research Articles

Tumor necrosis factor-alpha induces circular forms of human immunodeficiency virus type-1 DNA in the persistently infected low-level expressing cell line, ACH-2

The low human immunodeficiency virus type-1 (HIV-1) expressing T-cell line, ACH-2, was used to investigate accumulation of the circular, extrachromosomal form of HIV DNA (HD) after tumor necrosis factor-alpha (TNF-α) induction. We chose the 2 long terminal repeat (LTR) circular form to analyze unintegrated HD by polymerase chain reaction (PCR), using primer pairs which flank the 2 LTR HD. Approximately a 10-fold increase in 2 LTR HD was detected intracellularly in the TNF-α-induced ACH-2 cells using an end point-dilution assay. To examine the cellular compartment location of the 2 LTR HD accumulation, ACH-2 cells were fractionated into cytoplasmic and nuclear components and further subjected to PCR. A 4- to 5-fold increase in the 2 LTR HD signal was observed in the nuclear fraction. These results indicate that unintegrated HD increases in a chronically infected cell line after TNF-α induction. This phenomenon, which previously had been observed only with acute infections, may offer insight into basic pathogenic mechanisms.

Determination of cell dose-survival relationships from endpoint dilution assays.

Methods for fitting radiation survival curves to data obtained from endpoint-dilution assays are described. It is shown that for functional forms such as the linear-quadratic model the problem can be recast as a generalized linear model (GLM) and the data fitted using standard software. For functional forms which are not capable of being linearized, such as the multitarget model, the direct maximum likelihood (DML) techniques of Thames et al. (1986) can be used. Both these techniques produce exact maximum likelihood parameter estimates. Compared with the weighted least-squares (WLS) approach traditionally employed, these approaches avoid the need to approximate the binomial distribution of the number of negative wells by a normal distribution, and avoid the biases introduced by the need for arbitrary treatment of data points with 0 or 100% negative wells. The results of fittings using the novel GLM and DML approaches are compared with those obtained using the WLS method on a large series of datasets. For most datasets the WLS method performs well, compared with the exact method, but in a small number of cases the WLS predicted parameter estimates can be in error by as much as their estimated standard errors. A method for the use of a concurrent control to correct for interexperimental variation is outlined. The methods have been implemented in a Fortran computer program using the NAG subroutine library.

Biological comparison of wild-type and zidovudine-resistant isolates of human immunodeficiency virus type 1 from the same subjects: susceptibility and resistance to other drugs

We used a viral endpoint dilution assay to show changes in the proportion of zidovudine (azidothymidine; AZT)-resistant viruses within a heterogeneous mixture of human immunodeficiency virus type 1 (HIV-1) quasispecies isolated from patients on long-term AZT therapy. Several HIV-1 isolates, which could replicate in 10 microM AZT, were susceptible to both 2',3'-dideoxycytidine and a novel cytosine analog BCH-189, in which a sulfur atom replaces the 3' carbon of the pentose ring. In certain instances, cross-resistance was seen with 3'-didehydro-2',3'-dideoxythymidine. Although most strains of AZT-resistant HIV-1 displayed reduced susceptibility to 3'-azido-2',3'-dideoxyuridine, two strains were identified for which this was not the case.

A new perspective of microbial survival and dissemination in a prospectively contaminated air-fluidized bed model

A major concern of users of air-fluidized beds has been the possibility that such beds might be a source of microbial contamination. The purpose of this series of prospective, controlled experiments was to measure quantitatively the dissemination and survival of Bacillus subtilis, a species of bacterium that forms desiccation-resistant spores, as it was associated with circulating and clumped microbeads after challenge in an air-fluidized bed operating under decontamination conditions of heating at 48°C and microbead agitation by an air flow of 100% at 110 cu ft/min. Microbead samples collected after B. subtilis challenge from predesignated depths and locations within the air-fluidized bed at 0.25, 1, 2, 4, 24, and 48 hours were assayed for colony-forming units (CFU) of challenge bacteria by end point dilution and streak-plate assays. Results of three experiments with an average challenge of 1110 CFU of B. subtilis per gram of microbeads indicated that no more than 0.46 CFU of B. subtilis per gram of circulating microbeads and 0.78 CFU per gram of clumped microbeads were detected after 24 to 48 hours at decontamination conditions. Few microbes were detected during three control (sterile water) challenges of the air-fluidized bed operating at 33°C with 95% to 100% air flow. This study has demonstrated that an air-fluidized bed operating at the described decontamination conditions for 24 to 48 hours caused significant decreases, averaging a thousandfold per gram, in levels of microbead-associated challenge bacteria. These results suggest that mechanical abrasive forces between microbeads at high air flows, together with heat and desiccation, may be important in the air-fluidized bed in killing microorganisms from environmental, iatrogenic, and patient sources.

Effects of different methods of purification on aggregation of scrapie infectivity.

High levels of scrapie infectivity were found in detergent-insoluble residues of hamster brain purified by either repeated pelleting in 10% NaCl or by separation in Nycodenz gradients. Titres determined by the method of incubation interval assay were 100-fold higher than titres measured by endpoint dilution assay. The protein profiles and end-labelled RNA examined by one-dimensional polyacrylamide gel electrophoresis were not different from samples prepared from uninfected brain. Preparations produced by repeated pelleting were treated with RNase A and/or 7 M-urea with no loss of scrapie infectivity. However, the infectivity of samples prepared by gradient centrifugation in Nycodenz were reduced by 2 to 3 log10 LD50 by treatment with RNase A alone but not in combination with SDS. These results suggest that the scrapie agent may be aggregated by methods of purification employing pelleting in high concentrations of salt, or by adding polycations to disaggregated samples.

Direct analysis of quantal radiation response data.

A direct analysis is proposed for quantal (all-or-nothing) responses to fractionated radiation and endpoint-dilution assays of cell survival. As opposed to two-step methods such as the reciprocal-dose technique, in which ED50 values are first estimated for different fractionation schemes and then fit (as reciprocals) against dose per fraction, all raw data are included in a single maximum-likelihood treatment. The method accommodates variations such as short-interval fractionation regimens designed to determine tissue repair kinetics, tissue response to continuous exposures, and data obtained using endpoint-dilution assays of cell survival after fractionated doses. Monte-Carlo techniques were used to compare the direct and reciprocal-dose methods for analysis of small-scale and large-scale studies of response to fractionated doses. Both methods tended toward biased estimates in the analysis of the small-scale (3 fraction numbers) studies. The alpha/beta ratios showed less scatter when estimated by the direct method. Most important, the 95 per cent confidence intervals determined by the direct method were more appropriate than those determined by reciprocal-dose analysis, for which 18 per cent (small-scale study) or 8 per cent (large-scale study) of the confidence intervals did not include the 'true' value of alpha/beta.

End-point Dilution and Plaque Assay Methods for Titration of Cricket Paralysis Virus in Cultured Drosophila Cells

Cricket paralysis virus, an insect picorna-like virus, caused a distinct c.p.e. in cultured Drosophila melanogaster cells, allowing the development of titration methods based on end-point dilution or plaque assay methods. The end-point dilution (TCD50) method is more sensitive and economical than plaque assays and is easily scored. The data indicate a minimum infectivity/particle ratio of about 1/2000.

Determination of subgroup-specific feline oncornavirus-neutralizing antibody

A microneutralization assay was developed for antibody-to-subgroup-specific feline oncornaviruses. This study combines the economic advantage of a microtiter system and the quantitative focus reduction method which permits contruction of multiplicity curves for determination of virus-neutralizing titers. A twofold increase in Synder-Theilen feline sarcoma virus (ST-FeSV) on feline embryo cells decreased by approximately twofold the titer of reference goat serum prepared against Kawakami-Theilen feline leukemia virus. Similar dose effects with FeLV serotype virus preparations were not observed. An assay system utilizing FeLV serotypes on sarcoma-positive leukemia-negative cells demonstrated slightly greater sensitivity than one employing ST-FeSV on FE cells. Differential antibody responses to the three subgroup-specific feline oncornaviruses (A, B ,and C) were observed in reference goat sera. This test demonstrated good reproducibility as well as sensitivity and constitutes a significant improvement over end point dilution assay systems.

Replication of a nuclear polyhedrosis virus in a continuous cell culture of Spodoptera frugiperda: purification, assay of infectivity, and growth characteristics of the virus.

Nonoccluded virus, polyhedra, and occluded virus were purified from a continuous cell culture of Spodopera frugiperda infected with nuclear polyhedrosis virus. The optimal temperature for the replication and lateral transmission of infectivity for the nuclear polyhedrosis viruses (NPV) in cell culture was 27 C. End-point dilution and plaque assay procedures for the measurement of infectivity are described and compared. Dose-response data demonstrated that a single particle could initiate an infection, and the validity of the relationship of 0.7 PFU per mean tissue culture infective dose (TCID(5 0)) further substantiated the accuracy of these infectivity assays. Particle-infectious unit calculations gave a ratio of 62 to 310 nonoccluded virus particles TCID(5 0). Growth cycle and lateral transmission experiments indicated that infectious material was released from cells 12 h postinfection (p.i.) and approached a maximal titer 4 days p.i. The number of polyhedra, nonoccluded virions, and TCID(5 0) produced per cell was also presented. Typical yields of NPV produced per liter flask suggested that insect cell culture systems represent a feasible means by which the replication of these viruses could be investigated.

Studies of the cellular radiosensitivity of transplants of murine ependymomas irradiated in vivo.

SummaryCell-survival curves for in vivo tumour irradiation have been obtained for two subcutaneously transplantable murine ependymomas, using end-point dilution assays in isologous hosts.No significant transplantation immunity could be detected in either tumour system, but both exhibited a ‘Revesz effect’.The TD50 for No. 15 tumour was about 300 unirradiated cells in the initial assays, but in a later series this was reduced to 20. The TD50 of tumour No. 48 was about 100 cells. The survival curves gave D0 = 260 rads for tumour No. 15, and D0 = 288 rads for tumour No. 48, suggesting that a large fraction of the viable tumour cells were hypoxic in vivo.For unirradiated tumours, doubling-times of 21 hr for No. 48 were estimated both from the latency data and from caliper measurements of small tumours. After high radiation doses (3500 rads), these values were increased to as long as 5 days, although such long doubling-times were not always maintained as the tumours increased in size.

A lung-colony assay to determine the radiosensitivity of cells of a solid tumour.

SummaryAn assay system is described in which a number of single tumour cells are injected intravenously into recipient mice and grow in the lungs of these mice to give macroscopic tumour nodules in 16 to 20 days. The relationship between the number of tumour nodules observed and the number of cells injected has been found to be linear up to a mean of 50 colonies/lung for both non-irradiated and irradiated cells, and the inter-experiment consistency has been shown to be good. The number of tumour nodules observed has been found to be affected by the simultaneous injection of a large number of heavily-irradiated cells, provided they are injected intravenously. An aerobic radiation survival-curve has been established and agrees very well with one obtained for the same tumour using the end-point dilution assay method. The assay has the advantage that the time required for completion is short, compared with that required for end-point dilution assays.

Tumour reoxygenation during fractionated radiotherapy; studies with a transplantable mouse osteosarcoma

The oxygenation status of transplantable mouse osteosarcoma C 22LR was studied from the radiation dose survival curves. Cell suspensions were prepared from this tumour and the radiosensitivity of both the suspensions in vitro and the tumour in situ were determined by the endpoint dilution assay of Hewitt. From these survival curves, the mean anoxic cell fraction in the tumour in situ could be determined at 14%. After four daily doses of 250 rad to the tumour, the anoxic cell fraction rose to 32% and two days after a single dose of 1000 rad 88% anoxic cells were found. A calculation of the fractions of surviving cells shows that little or no reoxygenation of previously anoxic cells had occurred in this tumour after the preferential elimination of oxygenated cells by the radiation. This contrasts markedly with earlier observations for another tumour and it seems likely that different tumours may differ greatly in their capacity to reoxygenate anoxic cells during fractionated therapy. Since this capacity was calculated to be most important in determining the radiation dose needed to cure a tumour, a difference in reoxygenation may well be responsible for a large part of the variation in curability by radiotherapy between different tumours.