Complete Dose-response Curves Research Articles

θ-Defensins Prevent HIV-1 Env-mediated Fusion by Binding gp41 and Blocking 6-Helix Bundle Formation

Retrocyclin-1, a -defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 microm) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

Decreased repopulation as well as increased reoxygenation contribute to the improvement in local control after targeting of the EGFR by C225 during fractionated irradiation

Background and purpose Inhibition of repopulation and enhanced reoxygenation has been suggested to contribute to improvement of local tumour control after fractionated irradiation combined with inhibitors of the epidermal growth factor receptor (EGFR). The present study addresses this hypothesis in FaDu human squamous cell carcinoma. For this tumour model marked repopulation and incomplete reoxygenation during fractionated irradiation has previously been demonstrated. Furthermore, the anti-EGFR monoclonal antibody C225 has been shown to significantly improve the results of fractionated irradiation in this tumour. Materials and methods FaDu tumours in nude mice were irradiated with 18 fractions in 18 days (18f/18d) or 18 fractions in 36 days (18f/36d). Three Gy fractions were given either under ambient or under clamp hypoxic conditions. C225 or carrier was applied four times during the course of treatment. Fractionated irradiations were followed by graded top-up doses to obtain complete dose–response curves for local tumour control. Tumour control dose 50% (TCD 50) was determined at day 120 after end of treatment. Results Significant repopulation and reoxygenation occurred during fractionated irradiation of FaDu tumours ( P-values between 0.028 and <0.001). Application of C225 significantly decreased TCD 50 for 18f/36d under ambient conditions ( P=0.04). Bootstrap analysis revealed decreased repopulation and increased reoxygenation after application of C225 ( P=0.06 for the combined effect). This was further corroborated by a significant effect of C225 on the ‘repopulated’ dose under ambient conditions which is influenced by both, reoxygenation and repopulation ( P=0.012). Conclusions Our study provides evidence that both decreased repopulation as well as increased reoxygenation contribute to the improvement of local control after targeting of EGFR by C225 during fractionated irradiation of FaDu tumours.

Does heterogeneity of pimonidazole labelling correspond to the heterogeneity of radiation-response of FaDu human squamous cell carcinoma?

Background and purpose Pimonidazole is a marker for hypoxic cells which are radioresistant and thereby important for the outcome of radiotherapy. The present study evaluates heterogeneity in pimonidazole binding within and between tumours and relates the results to the heterogeneity of radiation response in the same tumour cell line. Materials and methods FaDu, a poorly differentiated human squamous cell carcinoma line, was transplanted subcutaneously into the right hind-leg of NMRI nude mice. Tumours were irradiated with graded single doses either under ambient or clamped blood flow conditions and local tumour control was evaluated after 120 days. Complete dose–response curves for local tumour control were generated and the slope, a measure of heterogeneity of radiation response, was determined. In parallel, 12 unirradiated tumours were examined histologically. Seven serial 10 μm cross-sections per tumour were evaluated using fluorescence microscopy and computerised image analysis to determine the pimonidazole hypoxic fraction (pHF). Heterogeneity in pHF was quantified by its coefficient of variation (CV). Poisson-based model calculations considering the intertumoural heterogeneity of pHF were performed and the slopes of the predicted and the observed dose–response curves were compared. Results The mean pHF was 11% [CV 50%] when one central section per tumour was evaluated. Measurements of multiple sections per tumour resulted in a mean pHF of 12% [CV 46%] ( P=0.7). Intertumoural heterogeneity in pHF was more pronounced than heterogeneity in individual tumours by a factor of 2. Model calculations based on the variability in pHF resulted in similar slopes of the dose–response curve for local tumour control in comparison with the observed slope when the heterogeneity in an unknown and arbitrarily chosen additional radiobiologically relevant parameter, in this example clonogen density, was taken into account. Conclusions While the average pimonidazole hypoxic fraction in FaDu tumours corresponds well to the radiobiological hypoxic fraction, the variability of pHF in FaDu tumours was not sufficient to explain the heterogeneity of radiation response in the same tumour line. Information on at least one additional parameter is expected to substantially enhance the predictive power of histological markers of tumour hypoxia.

Identification and documentation of herbicide resistance

Proactive herbicide resistance management programs rely upon early detection of resistant populations and knowledge of which combinations of weed and herbicide are prone to the development of resistance. Annual weeds that are prolific seed producers, genetically diverse, and repeatedly exposed to a single herbicide mode of action, are prone to rapid development of resistance. When resistance is suspected, seed samples are collected and evaluated using a whole plant bioassay. Whole plant bioassays are conducted underfield, growth room, or Petri dish conditions. Complete dose response curves for the suspected resistant and a reference susceptible population are used to verify resistance. Bioassay, conducted in growth rooms, is the most reliable method for identification of new cases of herbicide resistance. Bioassays, based on the biochemical detection of a single mechanism of resistance, are not reliable for screening for new occurrences of resistance.

Microbial inhibition by pharmaceutical antibiotics in different soils—dose‐response relations determined with the iron(III) reduction test

Soil contamination from pharmaceuticals is an emerging problem, though quantitative data on their microbial effects are lacking. Thus, nine pharmaceutical antibiotics were tested for their effects on the microbial iron(III) reduction in six different topsoils. Complete dose-response curves were obtained and best-fit by sigmoidal Logit, Weibull, Box-Cox Logit, and Box-Cox Weibull equations (r2 0.73-1.00). The derived effective doses (ED [micromol/kg soil]) for the different antibiotics increased in the order (average ED50 in parentheses) chlortetracycline (53) < sulfadimethoxine (58) < oxytetracycline (170) < sulfadiazine (190) < sulfadimidine (270) = tetracycline (270) < sulfapyridine (430), though no effect was found for sulfanilamide and fenbendazole at doses up to 5,800 and 3,300 micromol/kg, respectively. Due to a strong soil adsorption, especially of the tetracyclines, the corresponding effective concentrations in the soil solution (EC50), derived from sorption experiments, were considerably smaller and ranged from 0.004 micromol/L (chlortetracycline) to 120 micromol/L (sulfapyridine). The effects of the antibiotics were governed by soil sorptive properties, especially the concentration of soil organic matter. The microbial inhibition was influenced indirectly by the soil pH, which affects the ionization status of the amphoteric antibiotics.

LGR8 Signal Activation by the Relaxin-like Factor

The relaxin-like factor (RLF) is thought to be responsible for the intra-abdominal migration of the testis during mammalian development. Our latest studies of RLF and LGR8 have revealed that the N-terminal region of the A chain is not required for receptor binding but is indispensable for cyclic AMP generation. RLF derivatives with six residues deleted from the N terminus of the A chain are active, whereas further truncation, up to the first A chain cysteine (A-10), yields tightly binding ligands devoid of signaling activity. These derivatives are specific competitive inhibitors (RLFi) of RLF. Although receptor binding is dependent upon B chain residues, the N-terminal region of the A chain is a generic trigger of the trans-membrane signaling activity.

Magnesium-induced vasodilation in the dorsal hand vein.

Magnesium affects blood pressure by modulating vascular tone and reactivity. In obstetric patients, magnesium is administered to prevent eclamptic seizures and as a tocolytic to treat preterm labour. Prior to studying vascular sensitivity in women with pre-eclampsia, we sought to determine the effect of magnesium on venous tone in healthy women of childbearing age. Dose-response study. Columbia-Presbyterian Medical Center, New York. Ten healthy non-pregnant women (age range 21-47 years). Vascular response to magnesium sulphate (MgSO4) was measured in a dorsal hand vein using the linear variable differential transformer (LVDT) technique. Complete dose-response curves to MgSO4 (0.0000275-0.088 mmol/min) were determined after 50% preconstriction of the vein with phenylephrine. The ED50 of MgSO4 (dose resulting in 50% of maximal effect, Emax) was determined. Blood samples from the contralateral upper extremity were obtained to assess total plasma magnesium levels at baseline and at the highest infused dose of magnesium. ED50 results are expressed as geometric mean (95% confidence interval, CI). Emax results and magnesium plasma concentrations are expressed as mean [SD]. The ED50 of MgSO4 was 0.000307 mmol/min (95% CI 0.138, 0.666) and Emax was 102% [20%]. Magnesium induces dose-dependent venodilation in healthy women in the absence of systemic haemodynamic effects. The dose resulting in vasodilation using the LVDT/hand vein model is two to three orders of magnitude less than the therapeutic doses of magnesium used for tocolysis or seizure prophylaxis. Studies of the effect of systemically administered therapeutic doses of magnesium on vascular reactivity in pre-eclampsia will be of interest.

Metastatic lung disease to the central nervous system: in vitro response to chemotherapeutic agents.

Metastatic lung disease to the central nervous system (CNS) comprises a significant percentage of cranial metastases. For those cases where chemotherapy may be of palliative or therapeutic benefit, in vitro chemoresponse testing may identify the agent(s) most likely to be effective clinically. Tumor-derived cell cultures were established from 14 surgically excised lung lesions metastatic to the CNS. In vitro chemoresponse testing was performed with a variety of anticancer agents on the tumor cells that grew out of the cultured tissue specimens. Drug concentrations and exposure times were adjusted to bracket approximate average peak plasma levels that are observed typically in vivo. For each tumor-derived cell culture, a complete dose-response curve was established for each chemotherapeutic agent tested. Approximately 80% of the 14 tumor cell cultures had a definitive response to one or more chemotherapeutic agents in vitro, with approximately one-third of these cultures displaying a response to at least three of the drugs tested. There was considerable heterogeneity in the response of individual tumor cell cultures to the chemotherapeutic drugs. The agents that showed the highest cytotoxic response rate against the individual tumor cell cultures included lomustine, carboplatin, cisplatin and etoposide. The tumor cells isolated from individuals with lung lesions metastatic to the brain demonstrated differential chemoresponses to the agents tested. No single agent was effective against every tumor cell culture. These data suggest that in vitro chemoresponse testing of cultured tumor cells may be useful to identify biologically effective chemotherapeutic agents for individual patients, thereby addressing at least one factor in this complex therapeutic challenge.

Measuring the mitotic index in chemically-treated human lymphocyte cultures by flow cytometry

In the human lymphocyte chromosome aberration assay, the mitotic index (MI) is the standard cytotoxic parameter for determining which test concentrations will be evaluated for chromosome aberrations. Assessment of the MI is performed microscopically by determining the frequency of mitotic cells in a population of 1000 cells. With the commercial availability of antibodies to the mitosis-specific marker, phosphorylated-histone H3 at serine 10, automating the assessment of the MI using flow cytometry is now possible [Cytometry 32 (1998) 71]. Our laboratory has utilized and validated this technology to measure the mitotic index of chemically-treated human lymphocyte cultures. Comparisons between the microscopic and flow MI frequencies from 24 h treatments with mitomycin-C, aphidicolin, eugenol, etoposide, hydroxyrurea, potassium cyanide, staurosporine, ethyl alcohol, noscapine and colcemid ® are presented. Our results show that the mitosis specific H3-P marker is excellent for measuring the MI frequency in human lymphocyte cultures treated up to toxic concentrations. In addition, this study demonstrates that automation of analysis by flow cytometry is an excellent alternative to the microscopic method of analysis producing less variability than the microscopic scoring and a more complete dose response curve.

Discriminant analysis in allergic rhinitis and asthma: methacholine dose–response slope allows a good differentiation between mild asthma and rhinitis

Asthma and rhinitis frequently coexist in allergic patients, but nasal symptoms may predominate, leading to asthma underdiagnosis and undertreatment. Discriminant analysis obtains the best differentiation between groups using one or one set of variables. Our aim was to identify the laboratory test [allergen exposure, total and specific serum IgE, lung function, blood eosinophils and, bronchial response and sensitivity to methacholine (Mth) and allergen] or combination of them that allowed the best differentiation between mild asthma and allergic rhinitis. A cross-sectional analysis was performed in 86 Dermatophagoides pteronyssinus allergic rhinitis patients, who were classified according to clinical data as rhinitis plus mild asthma ( n =62) or “pure” rhinitis ( n =24). Bronchial symptoms had been exhaustively evaluated during a 2-years pre-inclusion period. Patients underwent skin tests and bronchial challenge with Mth and allergen. The exposure to D. pteronyssinus allergen (Der p1) was quantified in dust samples. Dose–response curves with Mth [until the FEV 1 fell by 40% or the maximal dose (200mg/ml) was inhaled] were attained. We developed multiple models of discriminant analysis in order to evaluate the capacity of the above variables to differentiate groups. Asthma patients had higher total and specific IgE levels and a greater sensitivity (PD 20 values) and response [dose–response slope (DRS)] to both Mth and allergen. The model entering these variables was the one that correctly classified more patients (79.2%). The discriminative power of the model that only included Mth-DRS values was similar to the above (78.8%). Bronchial response to Mth is quantitatively different in allergic rhinitis patients who display mild asthma symptoms when compared to those that only report rhinitis, suggesting a distinct bronchial intrinsic behavior. The utilization of complete dose–response curves with Mth allows a good separation between mild asthma and “pure” rhinitis patients and might be useful in the diagnosis of mild asthma. Whether the early detection and treatment of these patients prevents the development of symptomatic asthma needs further evaluation.

An approach for assessing estrogen receptor-mediated interactions in mixtures of three chemicals: a pilot study.

Most studies investigating interactions among endocrine-active chemicals have been limited to binary mixtures. This study reports on the preliminary evaluation an in vitro MCF-7 cell ER-alpha reporter gene system, coupled with a statistical methodology adapted for assessing interactions within ternary (3-chemical) mixtures. Two mixtures were initially chosen for assessment of the in vitro system's ability to detect additivity (mixture A) as well as greater-than-additive (mixture B) responses. Mixture A was composed of 17beta-estradiol (E2), ethinyl estradiol, and diethylstilbestrol and served as a control for additivity, whereas mixture B (E2, epidermal growth factor, insulin-like growth factor-I) was selected to model greater-than-additive interactions based on previous in vitro studies. After generating complete dose-response curves for each chemical, ternary mixtures were then tested in a full factorial design (4 concentrations per chemical, 64 treatment groups). A response surface was estimated using a nonlinear mixed model, and the observed responses were statistically analyzed for departures from the responses expected under the assumption of additivity. Mixture A exhibited additivity in vitro when the chemicals were present at concentrations in the linear range of their individual dose-response curves. For mixture B, in vitro analysis resulted in the additivity hypothesis being rejected (p < 0.001) because of a greater-than-additive interaction, as expected. A limited in vivo evaluation of mixture A was performed in the immature mouse uterotrophic assay (27 treatment groups), which agreed with the in vitro assessment of no significant departure from additivity ( p = 0.903). These findings demonstrate the ability of this in vitro methodology to detect additive, greater-than-additive, and less-than-additive interactions within ternary mixtures, which now allows for the assessment of environmentally relevant mixtures.

Effect of muscarinic antagonists on micturition pressure measured by cystometry in normal, conscious rats

Objectives. To establish an in vivo model to screen new muscarinic antagonists for the treatment of overactive urinary bladder and to calculate the respective ID 50 values. Methods. The conscious rat cystometry model was modified to determine a complete dose-response curve in each animal. Spontaneous micturition was induced by infusion of room-temperature saline into rat bladders at a constant rate of 12 mL/hr. Cumulative doses of muscarinic antagonists administered in the femoral vein caused dose-dependent inhibition of the urinary bladder contraction measured as the micturition pressure. In addition, the in vitro pK B values for atropine, PNU-200577 (DD01), tolterodine, oxybutynin, and terodiline were determined in carbachol-contracted rat bladder strips. Results. The rank order of the in vivo ID 50 values were atropine (14 ± 4 nmol/kg), PNU-200577 (22 ± 12 nmol/kg), tolterodine (94 ± 20 nmol/kg), oxybutynin (175 ± 89 nmol/kg), darifenacin (236 ± 144 nmol/kg), desethyloxybutynin (313 ± 209 nmol/kg), propiverine (4561 ± 2079 nmol/kg), and terodiline (18,339 ± 5348 nmol/kg). Tolterodine and PNU-200577 caused a parallel shift of the in vitro concentration-response curve to the right and did not alter the maximal contraction. The ID 50 values correlated significantly with the in vitro rat pK B and human bladder pA 2 values. Conclusions. The present results suggest that the rat cystometry model can be used in in vivo screening for new muscarinic antagonists.

Dose-volume effects in the rat cervical spinal cord after proton irradiation

Purpose : To estimate dose-volume effects in the rat cervical spinal cord with protons. Methods and Materials : Wistar rats were irradiated on the cervical spinal cord with a single fraction of unmodulated protons (150–190 MeV) using the shoot through method, which employs the plateau of the depth-dose profile rather than the Bragg peak. Four different lengths of the spinal cord (2, 4, 8, and 20 mm) were irradiated with variable doses. The endpoint for estimating dose-volume effects was paralysis of fore or hind limbs. Results : The results obtained with a high-precision proton beam showed a marginal increase of ED 50 when decreasing the irradiated cord length from 20 mm (ED 50 = 20.4 Gy) to 8 mm (ED 50 = 24.9 Gy), but a steep increase in ED 50 when further decreasing the length to 4 mm (ED 50 = 53.7 Gy) and 2 mm (ED 50 = 87.8 Gy). These results generally confirm data obtained previously in a limited series with 4–6-MV photons, and for the first time it was possible to construct complete dose-response curves down to lengths of 2 mm. At higher ED 50 values and shorter lengths irradiated, the latent period to paralysis decreased from 125 to 60 days. Conclusions : Irradiation of variable lengths of rat cervical spinal cord with protons showed steeply increasing ED 50 values for lengths of less than 8 mm. These results suggest the presence of a critical migration distance of 2–3 mm for cells involved in regeneration processes.

A robust method for evaluation of NANC transmission in human sigmoid colon muscle in vitro

Introduction: Human tissues are notoriously difficult to work with, giving results that are quantitatively variable within and between studies. Hence, previous investigations of nonadrenergic, noncholinergic (NANC) relaxation in human colon muscle report both partial and complete inhibitions of the NANC response by specific competitive inhibitors of nitric oxide (NO) production. We have established a robust and reproducible model to examine the contribution of NO during NANC relaxation assay in human sigmoid colon muscle strips. Methods: Complete control curves to long-train, stepwise, frequency-dependent, continuous electrical field stimulation (EFS) relaxation using vertical platinum electrodes connected to a biphasic pulse train stimulator generated NANC responses in fresh human sigmoid colon circular muscle strips set up in Bennett baths. A second complete curve was generated on the same strip in the presence of specific drugs to determine the contribution of NO to NANC relaxation. Responses to NO were also generated in muscle strips. Results were fitted to the Hill equation. Results: The first and second frequency–response curves without test drugs could be fitted to the Hill equation, resulting in similar midpoint locations ([ f] 50), maximal asymptotes ( α), and midpoint slope ( n) parameters. l- N G-nitro-arginine ( l-NOARG), TTX, and haemoglobin produced a tonic contraction in the muscle strips. NANC relaxations to EFS were inhibited by l-NOARG (30–37%), TTX (56–62%), and haemoglobin (48–90%). NO relaxations were concentration dependently inhibited by haemoglobin. Haemoglobin was equipotent in mediating tonic contraction and inhibiting NO relaxation. Discussion: We established reproducible assays for human colon muscle strips by the generation of two complete dose–response curves to long-train EFS, thus enabling a “within-preparations” study. The results suggest that NO contributes but is not the sole mediator of relaxations to long-train EFS in human sigmoid colon muscle. Moreover, a basal production of NO may serve to regulate tone of human colonic muscle.

Amphetamine-induced locomotor activation in 5-HT(1B) knockout mice: effects of injection route on acute and sensitized responses.

Knockout mice lacking serotonin(1B) (5-hydroxytryptamine(1B); 5-HT(1B)) receptors (1BKO) exhibit increased sensitivity to the stimulant and reinforcing effects of indirect dopamine (DA) agonists and are more reactive to mild stressors such as handling and the injection procedures that commonly occur when using the intraperitoneal (i.p.) route of drug administration. Since the intravenous (i.v.) route of administration allows minimal handling and injection-induced stress, the present study was designed to evaluate the effect of the administration route on amphetamine-induced locomotor activation and behavioural sensitization in 1BKO mice. For this purpose, 1BKO and wild-type (WT) control mice were administered i.p. or i.v. amphetamine in a within-session design, which allows evaluation of a complete dose-response curve within a single session. The locomotor stimulant effects of i.p. (0.5-4.0 mg/kg) and i.v. (0.6-1.2 mg/kg) amphetamine were investigated both acutely and following repeated treatments (four treatments at 48 h intervals). The results showed that acute i.p. amphetamine injection induced a significant higher horizontal activity peak effect in 1BKO mice, while this difference was less profound after acute i.v. injection. However, repeated i.p. or i.v. administration of amphetamine induced significantly higher locomotion in 1BKO mice. We conclude that the stimulant effects of amphetamine can be influenced by the route of administration in a genotype-dependent manner, and that route of drug administration (and associated variables) should be considered an important factor in studies of psychostimulant action in knockout mice.

Drug evaluations using a novel microphysiometer based on cell-based biosensors

This paper presents a novel microphysiometer based on cell-based biosensors for simultaneous measurements of several extracellular ion concentrations in living cells when treated with drugs. It can measure the extracellular acidification rate (ECAR) under the effects of the drug. Thus the relation between the cells’ metabolism and the activity of receptors can be explored, and the function of the drug can be analyzed. We used our system to measure the effects of some drugs on the nephridium and cardiac muscle cells of the suckling rat. As the microphysiometer works under regular cell culture conditions, cells can be repeatedly simulated with drugs to complete dose–response curve within a few hours. With the detection of a general parameter (extruded protons and ions), the system can be used to monitor the real-time process of the cells’ metabolism, observe the functional responses of different kinds of membrane-bound receptors and evaluate the drugs.

A novel microphysiometer based on MLAPS for drugs screening

This paper presents a novel microphysiometer for simultaneous measurements of several extracellular ions concentrations in living cells based on MLAPS (multi-light addressable potentiometric sensor). In the microphysiometer, different sensitive membranes are illuminated in parallel with n light sources at different frequencies, the response amplitudes of each frequency component can be measured on-line by parallel processing algorithm. By the experiments, we can analyze the relations of the extracellular environmental H +, Na +, K +, Ca 2+ under the effects of western medicines (dilantin, phenobarbital sodium, penicillin sodium) and Chinese drugs (scutellaria, medlar, hemlock parsley), and estimate the effects of several drugs. As the novel microphysiometer works under regular cell culture conditions, cells can be repeatedly simulated with drugs to complete dose–response curve within a few hours. With the detection of a general parameter (extruded protons and ions), the system can be used to monitor the real-time process of the cells’ metabolism, observe the functional responses of different kinds of membrane-bound receptors, evaluate the drugs.

Dose and duration-dependence of ganciclovir treatment against murine cytomegalovirus infection in severe combined immunodeficient mice

The present study investigates the full dose–response curve and treatment duration dependence of ganciclovir (GCV) against murine cytomegalovirus (MCMV) infection in severe combined immunodeficiency (SCID) mice. Animals inoculated intraperitoneally with 6.3×10 3 pfu of MCMV per mouse developed typical wasting syndrome rapidly and died around day 12 post-inoculation. Once-daily treatment with subcutaneous GCV for 5 days dose dependently delayed MCMV-induced wasting syndrome and mortality at a dose range of 1–80 mg/kg per day, whereas a dose of 160 mg/kg per day induced reversible side-effects. The effect of GCV treatment on mean death day (MDD) was significantly correlated to reductions of viral titers in the lung ( r=0.969, P<0.05). Treatment duration dependence was examined at the dose of GCV at 80 mg/kg per day for 1, 5, 8 and 12 days. The protective duration, over vehicle-treated mice, was constantly 3–4 days plus the duration of GCV treatment, as evidenced by the delay of viral replication, wasting syndrome and death. At a sub-optimally effective dose of 10 mg/kg per day of GCV, maximum protection was achieved with a 8-day treatment regimen. Prolongation of this treatment to 12 days failed to further delay mean death day and wasting syndrome that started on day 10, indicative of insufficient suppression of viral replication. Treatment with a single dose of GCV failed to show a complete dose–response curve since only minimal protective effects were observed at the dose of 80 mg/kg while side-effects were associated with the dose of 160 mg/kg. The treatment duration dependence and requirement for sufficient dosage of GCV against CMV infection observed in the current model are consistent with clinical observations. It also suggests that 5–8 days treatment duration may be a good balance considering the opportunity for identifying active compounds and speeding up the turnaround time in drug evaluations.

Tolerance of rat spinal cord to continuous interstitial irradiation

Purpose : To study the kinetics of repair in rat spinal cord during continuous interstitial irradiation at different dose rates and to investigate the impact of a rapid dose fall off over the spinal cord thickness. Materials and Methods : Two parallel catheters were inserted on each side of the vertebral bodies from the level of T 10 to L 4 . These catheters were afterloaded with two 192Ir-wires of 4 cm length each (activity 1–19 mCi/cm) or connected to the HDR- microSelectron. Experiments have been carried out to obtain complete dose response curves at 7 different dose rates: 0.53, 0.90, 1.64, 2.56, 4.4, 9.9 and 120 Gy/h. Paralysis of the hindlegs after 5–6 months and histopathological examination of the spinal cord of each animal were used as experimental endpoints. Results : The distribution of the histological damage was a good reflection of the rapid dose fall — off over the spinal cord, with white matter necrosis or demyelination predominantly seen in the dorsal tracts of the spinal cord or dorsal roots. With each reduction of the dose rate, spinal cord tolerance was significantly increased, with a maximum dose rate factor of 4.3 if the dose rate was reduced from 120 Gy/h (ED 50 of 17.3 Gy and 75.0 Gy, respectively). Estimates of the repaire parameters using different types of analysis are presented. For the direct analysis the best fit of the data was obtained if a biexponential function for repair was used. For the 100% dose prescribed at the ventral side of the spinal cord the α β ratio is 1.8 – 2.8) and two components of repair are observed: a slow component of repair of 2.44 h (1.18 - ∞) and fast component of 0.15 h (0.02 - ∞). The proportion of the damage repaired with the slow component is 0.59 (0.18 – 1). For the maximum of 150% of the prescribed dose at the dorsal side of the spinal cord the α β ratio is 2.7 Gy (1.5 – 4.4); the two components for the kinetics of repaire remain the same. Conclusions : Spinal cord radiation tolerance is significantly increased by a reduction in dose rate. Depending on the dose prescription, the α β ratio is 1.8 or 2.7 Gy for the 100% and 150% of the reference dose (rate), respectively; for the kinetics of repair a biphasic pattern is observed, with a slow component of 2.44 hours and a fast component of 0.15 hours, which is independent of the dose prescription.

Environmental endotoxin measurement: interference and sources of variation in the Limulus assay of house dust.

Studies suggest that endotoxin levels in offices and homes may be associated with sick building syndrome or increased asthma severity. However, reported endotoxin levels in these studies were highly inconsistent, suggesting incompatible measurements from various laboratories. Therefore, an investigation of Limulus assay for endotoxin in house dust was undertaken. Interference with the assay was common and could produce endotoxin estimates varying by a factor of > 100, depending on the dilution used. Analysis of dose-response curves allowed detection of two types of interference: dilution-dependent and dilution-independent. Dilution-dependent interference persisted when samples were reassayed, but valid estimates could be obtained by appropriate dilution and data analysis. Valid estimates could not be obtained from assay showing dilution-independent interference. However, dilution-independent interference was frequently overcome by repeating the assay. Estimates based on a single sample dilution produced reasonable results on average when a dilution factor of 900 was used. However, the results were less precise from a single dilution (median coefficient of variation [CV] 40%) than from the complete dose-response curve (median CV 23%) and ranged from 11 to 240% of the estimates based on complete dose-response data. A comparison of extraction methods found that sonication in a phosphate-triethylamine buffer produced greater endotoxin activity than extraction in buffer with addition of saponin or sodium dodecyl sulfate. Endotoxin activity was stable for 8 to 10 weeks in dust samples stored at 4 and -20 degrees C but was not stable in extracts. The lot of Limulus amebocyte lysate reagent and method of detecting a response in the kinetic Limulus assay had significant effects on endotoxin estimates.