Two quantitative trait loci are associated with recapping of Varroa destructor-infested brood cells in Apis mellifera mellifera.

Recapping of Varroa destructor-infested brood cells is a trait that has recently attracted interest in honey bee breeding to select mite-resistant Apis mellifera colonies. To investigate the genetic architecture of this trait, we evaluated a sample of A. mellifera mellifera colonies (N = 155) from Switzerland and France and performed a genome-wide association study, using a pool of 500 workers per colony for next-generation sequencing. The results revealed that two QTL were significantly (P < 0.05) associated with recapping of V. destructor-infested brood cells. The best-associated QTL is located on chromosome 5 in a region previously found to be associated with grooming behaviour, a resistance trait against V. destructor, in A. mellifera and Apis cerana. The second best-associated QTL is located on chromosome 4 in an intron of the Dscam gene, which is involved in neuronal wiring. Previous research demonstrated that genes involved in neuronal wiring are associated with recapping and varroa sensitive hygiene. Therefore, our study confirms the role of a gene region on chromosome 5 in social immunity and simultaneously provides novel insights into genetic interactions between common mite resistance traits in honey bees.

Identification of quantitative trait loci associated with calmness and gentleness in honey bees using whole-genome sequences.

The identification of quantitative trait loci (QTL) through genome-wide association studies (GWAS) is a powerful method for unravelling the genetic background of selected traits and improving early-stage predictions. In honey bees (Apis mellifera), past genetic analyses have particularly focused on individual queens and workers. In this study, we used pooled whole-genome sequences to ascertain the genetic variation of the entire colony. In total, we sampled 216 Apis mellifera mellifera and 28 Apis mellifera carnica colonies. Different experts subjectively assessed the gentleness and calmness of the colonies using a standardised protocol. Conducting a GWAS for calmness on 211 purebred A. m. mellifera colonies, we identified three QTL, on chromosomes 8, 6, and 12. The two first QTL correspond to LOC409692 gene, coding for a disintegrin and metalloproteinase domain-containing protein 10, and to Abscam gene, coding for a Dscam family member Abscam protein, respectively. The last gene has been reported to be involved in the domestication of A. mellifera. The third QTL is located 13 kb upstream of LOC102655631, coding for a trehalose transporter. For gentleness, two QTL were identified on chromosomes 4 and 3. They are located within gene LOC413669, coding for a lap4 protein, and gene LOC413416, coding for a bicaudal C homolog 1-B protein, respectively. The identified positional candidate genes of both traits mainly affect the olfaction and nervous system of honey bees. Further research is needed to confirm the results and to better understand the genetic and phenotypic basis of calmness and gentleness.

Survival curve of a human melanoma in nude mice.

Using cells from our tissue culture of human melanoma cell line Na 11, we transplanted 1 X 10)6) tumor cells sc into athymic nude mice. Tumors appeared after a latent period of 4-10 days; when they reached a mean volume of 100 mm3 we irradiated them with various doses of X-rays. Some tumors were irradiated while the mice were still alive; others were treated 10 minutes after the animals had been asphyxiated with nitrogen. All irradiation was done in the presence of oxygen. These tumors were excised, and cell suspensions were prepared; the cells formed colonies with a mean plating efficiency of 29%. In another series of experiments, we irradiated tumor cells in vitro 2 hours after excision, when most cells were fixed and presumably oxygenated. We then calculated survival curves for the tumor cells irradiated under these three conditions and found an average anoxic cell fraction of 85%, which was much higher than that reported in many other tumor systems. We explored several possible explanations for this phenomenon.

Kinetic parameters of aspartate transcarbamylase in human normal and tumoral cell lines.

The kinetic parameters of aspartate transcarbamylase activity were determined in dialyzed extracts coming from ten different human normal and tumoral cell lines (three fibroblasts, four melanomas, and three colorectal carcinomas). Specific activities do not correlate with the malignant character of the cells but rather with the fact that the cells divide actively or not. Growth curves show large variations in the cell sensitivity to N-(phosphonacetyl)-L-aspartate (PALA). However, no differences in substrate affinity or PALA sensitivity of the aspartate transcarbamylase activities present in the corresponding cell extracts could be detected. Thus, the different human cell susceptibilities to PALA do not result from an intrinsic property of aspartate transcarbamylase. Cell death under the influence of PALA does not correlate with the tumoral or normal character of the cells.

Variations in tumour oxygen tension (pO2) during accelerated radiotherapy of head and neck carcinoma.

The study was performed to assess the effect of accelerated radiotherapy on oxygenation of primary tumours and metastatic nodes in patients with advanced head and neck tumours. In 14 patients with head and neck tumour, oxygen tension (pO2) was evaluated in normal tissues and tumours (primary tumour or metastatic neck node) before (0 Gy) and after 2 weeks (32 Gy) of accelerated radiotherapy (70 Gy in 3.5 weeks, with three daily fractions). Radiotherapy was combined with carbogen breathing in 5 patients. pO2 was measured using a polarographic technique. For pooled normal tissues, median pO2 was 38 mmHg before treatment and 46 mmHg after 2 weeks. For tumours, very low values (< 2 mmHg) represented 20% of the recorded values before treatment and 10% after 2 weeks. The relative increase in tumour oxygenation was more pronounced for primary tumours (median pO2 12 mmHg before treatment versus 26 mmHg after 2 weeks, P < 0.05) than for metastatic nodes (respectively, 20 and 27 mmHg P = 0.1). For the 5 patients who breathed carbogen during accelerated radiotherapy, the median pO2 was 44 mmHg at 2 weeks, compared with 13.5 mmHg before treatment (P = 0.05). Very low pO2 values, corresponding to tumour hypoxia, were found in the tumours (primary and metastatic neck nodes) prior to accelerated treatment. During the first 2 weeks of accelerated treatment, an increase in median pO2 was found in nine of the 14 tumours, together with a decrease in the frequency of very low values.

Feasibility of measuring oxygen tension in uterine cervix carcinoma.

Cellular hypoxia is a cause of radioresistance. The oxygen tension (pO2) in normal tissues and in tumours can be measured by polarography. In this feasibility study we have measured the tissue pO2 of 10 patients suffering from uterine cervix carcinoma, using the Eppendorf histograph. The measurements were performed at the time of the brachytherapy after external radiotherapy. The machine was found to be reliable and no adverse effect was noted. The mean pO2 values in tumours were lower than those of normal tissues.

Chemical manipulations of tissue oxygenation for therapeutic benefit.

Many new approaches are currently being explored to improve cancer treatment through hypoxic cell radiosensitization. Oxygen carriers, calcium antagonists, some drugs which shift the oxygen hemoglobin curve to the right improve the oxygen availability in the tumors. An alternative way to improve radiotherapy or chemotherapy is to increase tumor hypoxia either by shifting the hemoglobin oxygen dissociation curve to the left or by using vasodilators. This paper summarizes main results obtained in that field. The relevance of data obtained from animal experiments to clinical practice will be discussed.

Radiosensitivity of Nall human melanoma transplanted into nude mice: repair, reoxygenation and dose fractionation.

Split and fractionated gamma-rays experimental have been performed on a human melanoma transplanted into nude mice using an in vitro colony assay. Repair of potentially lethal observed after a single dose of 20 Gy was found to no longer occur when 7 daily doses of 2.5 Gy were administered. In split-dose experiments, the increase in survival level probably can not be explained by repair of sublethal damage. When a single high dose of radiation is administered a certain reoxygenation is observed; however there is no reoxygenation when low radiation doses are delivered daily.

Radiosensitizing effects of misonidazole and SR 2508 on a human melanoma transplanted in nude mice: influence on repair of potentially lethal damage.

The radiosensitizing effects of misonidazole and SR 2508 (1 mg/g) were compared on a human melanoma (Na11) containing 85% hypoxic cells transplanted into nude mice. For both drugs, the enhancement ratios (ER) were 1.7 after immediate plating and 2.1 after delayed plating. This difference in ERs is related to a lack of PLD repair in tumors in the presence of the sensitizer. The effect of misonidazole was also investigated in another human melanoma (Be11) containing 40% hypoxic cells. After immediate plating, the ER was similar to that observed with the Na11 tumor (1.7), but PLD repair was not reduced. A comparative analysis of the influence of misonidazole on the response (survival curve - PLD repair) of Na11 melanoma to different ionizing radiations was attempted.

Does the direct measurement of oxygen tension in tumors have any adverse effects?

A new polarographic histograph was tested in tumors to assess its potential adverse effects in mice. The tumor cell lines used were: the lewis lung carcinoma (3LL) and two human xenografts (Na11+ and HRT18). The C57BL/6 mice survival and number of pulmonary metastases were not altered after pO2 measurements. Whatever the cell line, tumor doubling times were not changed after pO2 measurements. The new polarographic histograph was found to be reliable.

Radiosensitization by the combination of etanidazole (SR-2508) and pimonidazole (Ro 03-8799) in human tumor xenografts.

The two current second generation radiosensitizers SR-2508 (etanidazole) and Ro 03-8799 (pimonidazole) have different dose-limiting toxicities in humans. SR-2508 produces peripheral neuropathy, whereas Ro 03-8799 causes acute CNS toxicity. Overall toxicity can be minimized while increasing maximal radiosensitization by combining the two sensitizers. The additivity of their radiosensitizing properties was tested using clinically relevant SR-2508 and Ro 03-8799 concentrations in two human tumor xenografts: a rectal adenocarcinoma (HRT18) and a pigmented melanoma (Na11+). Drug concentrations were determined by a High Performance Liquid Chromatography method. Tumor response was assayed by clonogenic cell survival. The maximal radiosensitizing effect was determined using a single dose of radiation and high doses of drugs. For HRT18, the maximal radiosensitization was achieved when SR-2508 and Ro 03-8799 were given 45 and 30 min, respectively, before irradiation. For Na11+, the maximal radiosensitizing effect was lower than for HRT18 and not significant, but a trend was observed at about 30 min before irradiation for both drugs. Using clinically relevant doses, mean Sensitizing Enhancement Ratio (SER) values for HRT18 were: 1.3 (Ro 03-8799: 0.1 mg/gram body weight (gbw) or SR-2508: 0.2 mg/gbw), and 1.5 (Ro 03-8799: 0.1 mg/gbw + SR-2508: 0.2 mg/gbw). Mean SER values for Ro 03-8799 0.2 mg/gbw, SR-2508 0.4 mg/gbw, and Ro 03-8799 0.2 mg/gbw + SR-2508 0.4 mg/gbw were 1.5, 1.5, and 1.8, respectively. No significant radiosensitizing effect was observed on Na11+ with either drug administered singly or in combination and at the same concentrations. Our results suggest that the effectiveness of the two sensitizers administered alone or in combination may be tumor-dependent and that melanomas may not be good candidates.

New high O2 carrying perfluorochemical emulsions and/or carbogen: reactions of a human tumor xenograft to irradiation.

The effects of two new perfluorochemical emulsions based on F-66E and PFOB which carry, in combination with carbogen, more oxygen than Fluosol-DA 20% were investigated on a human tumor xenograft (HRT18). Tumor bearing mice were pretreated with 15 ml/kg of F-66E emulsion (50 w/v%) or PFOB (100 w/v%) emulsion in the presence of carbogen for 1 hr prior to and during irradiation. F-66E emulsion was non toxic in vitro. In vivo, the dose modifying factor was 1.8 for mice pretreated with carbogen and F-66E emulsion whereas it was 1.6 for carbogen-breathing mice. Results with PFOB emulsion were similar to those obtained with F-66E emulsion. The effect of F-66E emulsion was studied using a fractionated radiation treatment: 12 Gy were delivered in 6 fractions of 2 Gy with an interval of 8 hrs between fractions. A single dose of F-66E emulsion was injected before the first irradiation. Surviving fractions were 0.35 for air-breathing mice, 0.091 for carbogen-breathing mice and 0.099 for carbogen-breathing mice pretreated with F-66E emulsion. These results showed that carbogen alone or carbogen plus F-66E or PFOB emulsion were very efficient even with low doses of radiation.

Ro 03-8799: preferential relative uptake in human tumor xenografts compared to a murine tumor: comparison with SR-2508.

The pharmacokinetics of Ro 03-8799 and SR-2508 were studied in 6 tumor cell lines (5 human, 1 murine) transplanted into athymic nude mice. The human tumors were rectocolic adenocarcinomas (HRT18, HT29) and melanomas (Be11, Na11+, Na11-), the rodent tumor was a mammary tumor (EMT6). The concentrations of drugs in tumor, blood, plasma, and red cells were measured by HPLC 15, 30, and 45 minutes after the simultaneous i.v. injection of 0.1 mg/g of each compound. Little or no difference was found between the concentrations in the plasma, blood, and red cells for either drug; SR-2508 concentration was higher than that of Ro 03-8799. Both compounds were concentrated in all the tumors, the concentration increases or decreases as a function of the time, depending on the cell line; there was more Ro 03-8799 in the pigmented melanomas than in the other tumors. The relative uptake of Ro 03-8799 and SR-2508 in the tumor was evaluated by the ratio of drug concentrations in the tumor and in the blood. The results suggest that Ro 03-8799 and SR-2508 accumulated in all the tumors, the relative uptake of Ro 03-8799 was higher than that of SR-2508. For human tumors, the ratios increased between 15 and 45 minutes. They ranged from 1.2 to 3.6 for SR-2508 and from 3.1 to 14.6 for Ro 03-8799. For Ro 03-8799, the highest ratios were found for the melanomas; the uptake was higher in the pigmented Na11+ tumors than in the non-pigmented Na11-. The ratios for EMT6 were about 2.3 for SR-2508 and 4.7 for Ro 03-8799; these ratios varied slightly between 15 and 45 minutes.

Distribution of radiation sensitivities for human tumor cells of specific histological types: comparison of in vitro to in vivo data.

The radiosensitivities of human tumor cell lines, grouped into 6 histological categories, have been studied using data from the published literature. The parameters alpha, beta, n, D0, D, and the surviving fraction to 2 Gy (S2) and 8 Gy (S8) were calculated. Only the two parameters mainly derived from the initial part of the survival curve, alpha and D, together with S2, provided data which were correlated with the clinical radioresponsiveness of each histological group. Thus, there are intracellular factors which influence clinical radioresponsiveness whose relative importance varies from one histological cell type to another. The value of D gave the most precise characterization of the average group radiosensitivity. It was possible to compare the in vivo radiosensitivities of non-severely hypoxic cells with those of tumor cells irradiated in vitro for 7 tumor lines grown as xenografts in mice. The average radiosensitivity was 1.9 times less in vivo than in vitro. This difference indicates that, in addition to the intrinsic factors of radioresistance demonstrated in vitro, and independently of severe hypoxia, there are other factors which specifically reduce radiosensitivity in vivo.

Partial hypoxia as a cause of radioresistance in a human tumor xenograft: its influence illustrated by the sensitizing effect of misonidazole and hyperbaric oxygen.

While previous studies with three human tumor xenografts suggest that contact-resistance plays a major role in the response of these tumors to radiation, it remains possible that partial hypoxia may provide an alternate explanation. The present study was carried out to check this possibility by investigating the influence of misonidazole (MISO) and hyperbaric oxygen (HBO) on both the initial and distal components of the survival curves of HRT18 tumor cells. The effect of a challenge dose of radiation on the initial radioresistance of this tumor was also studied. To assess the effects of MISO and HBO, tumor cell survival was determined by excision assay in two groups of tumor-bearing mice, one given MISO (1 mg/g body weight, i.p.) 45 min before irradiation and the other exposed to HBO (3.5 bars). MISO treatment caused greater sensitization than HBO. The enhancement ratios at the 5.10(-1) level were 1.7 (MISO) and 1.7 (HBO); at the 10(-1) level, they were 1.6 (MISO) and 1.4 (HBO); while at 10(-2), they were 1.6 (MISO) and 1.4 (HBO). These two sensitizing effects favor the hypothesis that solid tumors contain a compartment of partially hypoxic cells. To study the effect of a challenge radiation dose on initial radioresistance, tumors were given a challenge dose of 8 Gy, followed 24-48 hr later by doses ranging from 2-12 Gy. The challenge dose did not modify the shape of the survival curve.

Radiobiological evaluation of a newly synthesized cysteamine derivative.

A new cysteamine-based compound, I109 (N-glycylglycyl-S acetylcysteamine trifluoroacetate) was tested on both normal tissues and tumors to evaluate its radioprotective potential. I109, which is three times less toxic than WR-2721, was injected at a dose approximately equal to half the LD50(30 days). The Protection Factor (PF = gamma ray dose ratio) after whole body irradiation was 1.3-1.4 for intestinal death and 1.4-1.5 for hemopoietic death when intervals of 40 or 20 min elapsed between injection and the end of irradiation. A crypt cell assay was done for both I109 and WR-2721; PFs were 1.1 and 1.4, respectively. I109 was then tested on five solid tumors. For each cell line (4 human, 1 murine) one dose of radiation was delivered. Surviving fraction ratios with and without drug ranged between 2.4 and 14.1 when an interval of 20 min elapsed between injection and the end of irradiation. The degree of radioprotection proved time dependent for EMT6 and HRT18; radioprotection afforded by WR-2721 on these tumors is either similar or greater than radioprotection afforded by I109 depending on the time interval between injection and irradiation.

Radioprotection of EMT6 tumor by a new class of radioprotectors based on a pseudo-peptide cysteamine combination.

Although WR-2721 preferentially protects normal tissues against irradiation, it seemed desirable to find other drugs presenting a lower toxicity and the same radioprotective properties. A new compound, I 102, was selected; it was characterized on one hand by a coupling between cysteamine and an amino-acid, and on the other hand by an acetyl-group, which protects the thiol function. The effects of WR-2721 and of I 102 were studied on EMT6 tumors grafted on BALB/c mice. Whatever the size of the tumor, the cell survival increased as a function of the time elapsed between the injection of I 102 and the end of the irradiation (TI). In contrast, the radioprotection afforded by WR-2721 was found to be independent of TI. The survival curves suggest that, like WR-2721, I 102 protects essentially oxygenated cells.

Predictive assays of radiation response in patients with head and neck squamous cell carcinoma: a review of the Institute Gustave Roussy experience.

PURPOSE: The aim of the study was to present the updated Institut Gustave Roussy experience of the predictive value of three biological parameters in patients with squamous cell carcinoma of the Head and Neck (HNSCC) treated with radiation therapy. METHODS AND MATERIALS: Three parameters have been investigated independently: tumor cell kinetics (TS, Tpot and LI), oxygen tension measurements (PO2) and intrinsic radiosensitivity (SF2Gy). RESULTS: No relationship has been found between local-regional control and Tpot or LI in a series of 74 patients. Our data also support that the surviving fraction at 2 Gy, (SF2) was unlikely to predict the clinical outcome in a series of 92 patients. Differences in PO2 measurements have been observed between tumors, and tumor oxygenation was lower than that of normal tissue for the majority of patients. However PO2 measurements did not predict clinical outcome, but further investigations are needed to draw definitive conclusions, given the limited number of patients entered in our study (35 patients). In addition, we were able to measure the three parameters in 10 patients showing no correlation between PO2, SF2 and Tpot. CONCLUSIONS: The method used to evaluate Tpot and SF2 did not provide clinically relevant predictive parameters for this type of cancer. Further investigations are needed to assess the predictive value of PO2 measurements and of new biological parameters in a multiparametric approach, taking into account other possible clinical and biological confounding factors.

New high O2 carrying perfluorochemical emulsions: toxicity, radiosensitivity of GM-CFC and development of metastases in mice.

The effects of two new concentrated perfluorochemical emulsions based on F-66E and PFOB, which carry significantly more oxygen than Fluosol-DA 20%, were tested on normal tissues (toxicity and radiation response) and on the development of metastases from Lewis Lung Carcinoma (3LL) in female C57 BL/6 mice. Twenty one days after injection of F-66E or PFOB emulsions (15 ml/kg body weight), the spleen and liver weights were significantly increased but had returned to normal after 2-3 months. Splenomegaly already observed in 3LL bearing mice was significantly increased by F-66E emulsion injection. The radiosensitivity of GM-CFC was not altered when unanesthetized GM-CFC was not altered when unanesthetized mice were pretreated with F-66E emulsions and/or carbogen 1 hr prior to and during irradiation. The rate of tumor take and the period before detection of tumors were not modified when an emulsion of F-66E was injected simultaneously or 10 days after 3LL cells. Mean survival of mice, and the number of metastases on lung surfaces were similar in F-66E injected mice and control mice.

Biological dosimetry after total body irradiation (TBI) for hematologic malignancy patients.

PURPOSE: Biological dosimetry based on scoring chromosomal aberrations in peripheral lymphocytes was compared to physical dosimetry done for total body irradiation (TBI) before bone marrow transplantation (BMT) in patients with hematologic malignancies. PATIENTS AND METHODS: Fifteen patients undergoing TBI were included in the study. A total dose of 12 Gy in 2.5 days was fractionated into 2 or 3 daily doses of 1.8 Gy delivered by a 18 MV linear accelerator (dose rate: 15.8 cGy x min(-1)). Blood samples were obtained from patients before irradiation and after the first fraction of 1.8 Gy. A standard dose-effect curve was established by in vitro irradiation of healthy volunteer lymphocytes. Chromosomal aberrations were scored by the conventional cytogenetics (CCG) method for unstable anomalies and by fluorescent in situ hybridization (FISH) for stable anomalies. RESULTS: Healthy donor lymphocytes before irradiation yielded 0.1% dicentrics and 0.3% translocations of chromosome 4 (Chr. 4), that is 2.5% for the whole genome. Patients before irradiation had 2% of dicentrics and 1.1% of chromosome 4 translocations. The biologically estimated dose of the 15 patients after exposure to 1.8 Gy was 1.93 Gy (95% CI: 1.85-2.05) according to CCG, and 2.06 Gy (95% CI: 1.75-2.15) by FISH. CONCLUSION: The dose estimated by biological dosimetry, in this case of homogeneously distributed radiation of TBI agrees well with the absorbed radiation dose calculated by physical dosimetry.