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Monte Carlo simulation of the biological effects of boron neutron capture irradiation with d(14)+Be neutrons in vitro.

It was shown that radiation effects in tumor cells treated with fast neutrons may be increased by the neutron capture reaction 10B(n, alpha)7Li. The classic approach for macroscopic dosimetry in fast-neutron therapy cannot be applied to the dose in boron neutron capture therapy (BNCT). The effectiveness of BNCT in killing tumor cells depends on the number of 10B atoms delivered to the tumor, the subcellular distribution of 10B and the thermal neutron fluence at the site of the tumor. Monte Carlo calculations of the energy depositions of short-range particles with high LET coming from 10B disintegrations were performed and compared to the observed biological effects. The simulation allows us to study the influence of the localization of intracellular 10B in the nucleus, cytoplasma, plasma membrane or extracellular space. The biological response function which describes the probability of the lethal effect produced by a single particle track through the cell nucleus was found by comparing the calculated microscopic dose distribution spectra for single events with the survival observed experimentally. Calculations for a human melanoma cell population treated as a monolayer in the presence or absence of boron with d(14)+Be neutrons will be demonstrated. Two different boron compounds enriched in 10B were investigated in this study: boric acid (H3 10BO3) and p-dihydroxyboryl phenylalanine (BPA). The study shows that a high fraction of BPA enters the cytoplasm while boric acid was found only in the extracellular space. The computer simulations indicate that BPA yields a higher potential effectiveness for inactivation of melanoma cells than boric acid.

Boron Neutron Capture Therapy↗

[Therapy by neutron capture of ocular melanoma: dosimetry and microdosimetry].

Neutron capture therapy (NCT) aims at destroying cancerous cells with the alpha and 7Li particles produced by the neutron capture reaction on 10B. This note reports on the study of the boron distribution in tissues on an animal model (nude mice) xenografted with a human ocular melanoma after an i.p.injection of 2g/kg of 10B-BPA and in cells cultured in the presence of 530 mumol/l of 10B-BPA. A concentration of 64 ppm of 10B in the active part of the tumour with a ratio of concentrations versus the skin of 3.7 are observed. Investigations on cells reveal the presence of boron in the cytoplasm. The biological, dosimetric and microdosimetric consequences of these findings are discussed.

Animals↗

Subcellular distribution of various boron compounds and implications for their efficacy in boron neutron capture therapy by Monte Carlo simulations.

Neutron capture therapy has a promising role in cancer treatment since it can achieve selectivity at the cellular level. The effect of this therapy depends on the subcellular localization of boron atoms in the target cell. Five boron compounds were investigated in this study: the monomeric and dimeric sulfhydryl boranes (BSH and BSSB), a boronated phenylalanine (BPA), and two porphyrin complexes (BOPP and VCDP). The study shows that when exponentially growing rat 9L gliosarcoma cells are exposed to an isoeffective concentration of each of the five compounds for 1 h, BOPP produces a much higher intracellular level of boron than the other four compounds; BSSB produces the second highest level, while exposure to BSH, VCDP, and BPA resulted in lower intracellular boron levels. Subcellular fractionation studies showed that most of the boron localized in the cytoplasm of the cells with all five compounds. A significantly higher boron concentration was found in the lysosomes of the cells, but the nuclei contained only minimal concentrations of boron. Computer simulations of neutron capture reactions with boron using a Monte Carlo simulation code indicated that BOPP would yield the highest potential effectiveness, followed by BSSB, BSH, VCDP, and BPA, in that order.

Animals↗

RBE in fast neutron therapy and in boron neutron capture therapy. A useful concept or a misuse?

In high-LET radiation therapy, radiation quality and radiation quality differences have to be taken into account and specified. In fast neutron therapy, an operational approach has been adopted which is based on the concept of "clinical RBE". The paper discusses the quantities of RBE (relative biological effectiveness), reference RBE and clinical RBE and their relationship and significance in radiation therapy. In particular, the difference between the well defined RBE in radiation biology and the clinical RBE which is based on the judgement of radiotherapists is elucidated and emphasized. The clinical RBE is based on the reference RBE and implicitly includes differences in physical selectivity of the irradiation beams and clinical experience. The particular aspects of radiation quality in boron neutron capture therapy (BNCT) are due to the poor penetration of the primary beam, the inhomogeneity of the spatial distribution of 10B nuclides in the irradiated tissue and the short range of the alpha-particles emitted after neutron capture in 10B. The related problems in accounting for radiation quality in BNCT and in the applicability of the clinical RBE concept are discussed.

Boron Neutron Capture Therapy↗

Physical and tumor biological aspects and calculation model of dosage in boron neutron capture therapy (BNCT).

Fundamentally different aspects apply to dosage in boron neutron capture therapy (BNCT) compared to that in the case of normal radiotherapy with photons, electrons or heavy particles such as neutrons. The reason is that the latter only requires a knowledge of the stochastic distribution of the absorbed dose within cells, radiation quality and atomic composition of tissue in the regions of interest, whereas for the former the absolute concentration and microscopic distribution of 10B atoms in inter- and intracellular spaces of tumor and healthy cells is additionally of equal importance. The effects of radiation without 10B must always be superimposed on those of heavy particles resulting from neutron capture reactions on 10B atoms. Complex geometrical calculations are necessary with respect to ranges of the heavy particles smaller than a cell diameter. Apart from the direct effects of radiation without 10B, the dosage therefore depends on thermal neutron fluence, 10B concentration, its extreme inhomogeneous macroscopic distribution in the tumor tissue, the cellular localization of the 10B atoms in the large intercellular space, the cell membrane, within cytoplasm or the cell nucleus, the geometrical probability of hitting the cell nucleus, and that such a hit finally results in a cell killing, and a Poisson statistical enhancement factor, which describes the dose-effect relation for cell survival. The calculations necessary are demonstrated in the case of a normal and a tumor cell type, each with representative cell diameter and nucleus size. It is evident that the microscopic distribution of 10B atoms is one of the most critical parameters which is still insufficiently known.

Boron Neutron Capture Therapy↗

In vitro and in vivo evaluation of o-carboranylalanine as a potential boron delivery agent for neutron capture therapy.

o-Carboranylalanine (B10H10C2CH2CHNH2COOH) is a carborane-containing amino acid, which has been synthesized as a potential capture agent for boron neutron capture therapy (BNCT) of cancer. The purpose of the present study was to develop a rational approach for the in vitro and in vivo evaluation of boron containing compounds that possibly might be used for BNCT. The in vitro uptake of carboranylalanine (CBA) was evaluated using two cell lines, the human melanoma MRA 27, and the murine Harding-Passey melanoma. Uptake of CBA by MRA 27 cells ranged from 135-551 micrograms B/10(9) cells following 3 hrs incubation with medium containing 100-113 micrograms B/ml and was not reduced by exposing the tumor cells to either rotenone, an inhibitor of electron transport, or by culturing them at ambient temperature (approximately 22 degrees C). Cellular uptake and elution of CBA occurred rapidly under in vitro conditions. Uptake of CBA was slightly greater than that of boronophenylalanine (BPA). Following a 3 hr incubation with CBA at a concentration of 106 micrograms B/ml, cell boron content was 255 micrograms B/10(9) MRA 27 cells, compared to 192 micrograms B/10(9) cells when cells were incubated with BPA at a concentration of 95 micrograms B/ml. In vivo studies initially were carried out using the Harding-Passey melanoma, which had been implanted intramuscularly (i.m.) into the right flank of BALB/c mice. Tumors were allowed to grow for 14 days at which time mice were injected intraperitoneally (i.p.) with either CBA or BPA (1.25 mgB/mouse), and were killed 3, 6 and 8 hrs later. CBA attained a low tumor to blood ratio(1.0-1.4), and the tumor boron levels ranged from 15.7-26.2 micrograms B/g at 3 hrs and 3.3-19.9 micrograms B/g at 6 hrs. Higher blood and lower tumor boron levels were observed at all time points with CBA compared to BPA, suggesting that CBA was not taken up selectively by the melanoma. Similar studies, carried out in rats bearing intra-cerebral gliomas, failed to reveal detectable amounts of boron in the tumor. From the present study, it can be concluded that CBA does not appear to possess the requisite properties to be useful as a boron delivery agent for BNCT.

Animals↗

Evaluation of in vitro cytotoxicity of carboranyl amino acids, their chemical precursors and nido carboranyl amino acids for boron neutron capture therapy.

The purpose of the present study was to define the in vitro cellular toxicity of three carborane-containing amino acids: p-(o-carboran-yl)-phenylalanine (CBPA), O-(o-carboran-1-ylmethyl)-tyrosine (CBT), and o-carboranylalanine (CBA), which are analogues of phenylalanine, tyrosine, and alanine respectively. In addition, two of their chemical precursors: CBACN (B10H11C2-CH2CHNH2CN) and CBTCN (B10H11C2-CH2OC6H4CH2CHNH2CN) and nido CBA were evaluated for their toxicity on human MRA 27 melanoma cells. Hydroxypropyl-beta-cyclodextrin (beta-CD) initially was used to solubilize all the compounds except nido CBA in the toxicity assays Cells were incubated with the test compounds at varying concentrations for 24 hrs, following which the proliferative activity of surviving cells was determined by pulsing with tritiated thymidine ([3H]-TdR) for an additional 18 hrs. CBT at a concentration of 280 micrograms/ml was non-toxic when solubilized with beta-CD. CBA at a concentration of 350 micrograms/ml was non-toxic when solubilized with beta-CD, but when solubilized with DMSO produced a 50% reduction in uptake of [3H]-TdR at a concentration of 75 micrograms/ml. CBPA, solubilized with beta-CD, was nontoxic at a concentration of 400 micrograms/ml, while CBTCN and CBACN at concentrations of 50 micrograms/ml and 40 micrograms/ml, respectively, were both toxic, even when solubilized with beta-CD. Nido CBA at a concentration of 400 micrograms/ml in medium was non-toxic. Although the toxicity of these boron compounds precludes their use as capture agents for Neutron Capture Therapy, they may have some potential for cytoreductive chemotherapy of cancer, and further evaluation may be warranted.

Amino Acids↗

Boron neutron capture irradiation of the rat spinal cord: histopathological evidence of a vascular-mediated pathogenesis.

A histopathological study was carried out on the spinal cord of rats after boron neutron capture (BNC) irradiation. Rats were irradiated with thermal neutrons alone or in combination with borocaptate sodium (BSH) or p-boronophenylalanine (BPA). Spinal cords were examined 1 year after irradiation, or at earlier times in rats developing myelopathy. Considered overall, the pathology of the spinal cord after BNC irradiation was comparable with that reported previously after X irradiation of the spinal cord in the identical strain of rat. When BSH was used as the neutron capture agent, the biologically effective dose of radiation delivered to the CNS parenchyma was a factor of -2.7 lower than that delivered to the vascular endothelium. In effect, the blood vessels were selectively irradiated using this BNC modality. The resultant pathology was similar to that observed after irradiation with thermal neutrons alone or in the presence of BPA, situations in which the CNS vasculature was not selectively irradiated. This points to the vascular endothelium as being the critical target cell population, damage to which results in the development of the lesions seen in the spinal cord after BNC irradiation and, by inference, after irradiation with more conventional modalities.

Alpha Particles↗

Intratumoral delivery of boronated epidermal growth factor for neutron capture therapy of brain tumors.

The gene for epidermal growth factor receptor (EGFR) is amplified or overexpressed in high-grade gliomas but is low or undetectable in normal brain. Recently, there has been increasing interest in using epidermal growth factor (EGF)-based bioconjugates as targeting agents for brain tumors. In the present study, we have investigated the potential use of boronated EGF as a delivery agent for boron neutron capture therapy, which is based on the capture reaction that occurs when 10B, a stable isotope, is irradiated with low-energy thermal neutrons. A fourth generation starburst dendrimer was boronated and linked to EGF using heterobifunctional reagents. Either wild-type or EGFR gene transduced C6 glioma cells (C6EGFR), which expressed 10(5)-10(6) receptor sites/cell, were stereotactically implanted into the right cerebral hemisphere of Fischer rats. Four weeks later, the rats received either i.v. or intratumoral (i.t.) injection of 131I-labeled boronated starburst dendrimer (BSD) or BSD-EGF. The biodistribution of 131I-BSD-EGF and 131I-BSD was studied by means of whole-body scintigraphy, autoradiography, and gamma scintillation counting. Following i.t. injection of 131I-BSD-EGF, 21.8% of the injected dose per gram tissue (% ID/g) was localized in C6EGFR tumors at 24 h and 16.3% at 48 h compared to 5 and 1.3% ID/g in C6 wild-type tumors, respectively, and 0.01 and 0.006% ID/g, respectively, for i.v. injected animals at the corresponding times. In contrast, following i.t. injection of BSD-EGF, only 0.01-0.1% ID/g was localized in the liver and spleen at 24 and 48 h compared to 5-12% ID/g following i.v. injection. Our data indicate that direct i.t. injection can selectively deliver BSD-EGF to EGFR-positive gliomas and suggest that intracerebral administration may be the most effective way for delivering EGF-based bioconjugates to EGFR-positive brain tumors.

Animals↗

Positron emission tomography-based boron neutron capture therapy using boronophenylalanine for high-grade gliomas: part I.

Determination of tumor boron-10 (10B) levels is required for accurate neutron dosimetry during boron neutron capture therapy. We assessed a new method for quantitative measurement of boronated drug uptake in high-grade gliomas. This method uses positron emission tomography (PET) with fluorine-18-labeled L-fluoroborono-phenylalanine (L-18F-10B-FBPA), which was synthesized as an analogue of L-boronophenylalanine. We studied the accumulation of L-18F-10B-FBPA by PET in patients with high-grade gliomas. Dynamic PET studies of brain tumors revealed that L-18F-10B-FBPA accumulated gradually after bolus injection, and the value of PET activity divided by the integrated plasma activity reached a constant level 42 min after injection, which was defined as the incorporation constant (Ic*). This constant reflected the appropriate L-18F-10B-FBPA accumulation in tumor tissue. Based on the Ic* constant, the methods for estimating tumor 10B concentration were devised. With this method, the estimated values of 10B concentration in gliomas were very close to the 10B levels in surgical specimens. This method was based solely on PET and can potentially provide data that would assist in the selection of patients for future treatment with boron neutron capture therapy after surgical resection of their brain tumors.

Boron↗

Monitoring of the boron neutron-capture reaction by a simple animal model.

Sodium borocaptate (BSH, Na2Bl2HllSH), a slow neutron-capture compound, was injected into the left forebrain ventricle of 1-week-old rats (150 microg BSH/3 microl phosphate buffered saline). After 90 min, the animals were irradiated by epithermal neutrons (LVR-15 nuclear reactor in Rez near Prague, flux density 8.8 x 10(7) neutrons cm-2 s-1, 8 MW reactor power, 8.2 cGy/min) for 5, 10 or 20 min. The brains were examined histologically 8 h after irradiation. In animals irradiated for 5 to 10 min (41 and 82 cGy-Eq, respectively) lethal damage of cells was found in the external granular layer of the cerebellum and the subependymal layer of the forebrain. Irradiation for 20 min (164 cGy-Eq) caused more extensive destruction of cell populations in these regions and, in addition, dead cells appeared also in the more differentiated postmitotic compartments, namely the deeper layers of the cerebellum, layers II/III of the cerebral cortex and corpus callosum. In the forebrain periventricular layer, the extent of cell damage was declining towards the olfactory bulbs. In intact animals, as well as in those injected only with the 150 microl phosphate buffered saline, the radiation damage was low and limited only to the most sensitive dividing populations of the cerebellum and the forebrain. The study demonstrates a differentiation-dependent damage of the rat brain cells by alpha particles and presents a simple model for evaluation of the biological effectiveness of slow neutron beams constructed for neutron-capture therapy of tumors.

Animals↗

Neutron capture therapy with a new boron-porphyrin compound in the rat 9L glioma model.

Neutron capture therapy with a new boron-porphyrin compound was tested in a rat brain tumor model. Although the concentration of boron in the tumor was too low to elicit a therapeutic effect, prominent histopathologic changes, such as necrosis, congestion and bleeding were observed in the tumors of the rats which were administered the boron neutron capture therapy.

Animals↗

Capture techniques and morphological measurements of the mona monkey (Cercopithecus mona) on the island of Grenada, West Indies.

Morphological measurements were collected from 12 wild and 12 captive mona monkeys (Cercopithecus mona) on the Caribbean island of Grenada. Mona monkeys were introduced to Grenada from Africa approximately 200 to 300 years ago during the slave trade era. Wild monkeys were captured using either 1) a baited treadle-door trap and anesthetic-filled darts fired from a blowpipe, or 2) rifle-fired anesthetic-filled darts. All wild monkeys were released back into the forest after capture and were seen with their original groups within 24 hours of release. Captive monkeys were anesthetized using blowpipe-fired darts. A Ketaset/Rompun mixture was the most effective anesthetic for wild monkeys while Ketaset alone was suitable for captive monkeys. Responses to and recovery times from both drugs varied among individuals. Data on eight linear body measurements, canine length, testicle size, and weight were collected from all monkeys. Adult monkeys were significantly sexually dimorphic across all measurements. Mean adult male weight (mean = 4.7, SD = 0.9, n = 13) was almost twice that of adult females (mean = 2.8, SD = 0.8, n = 7). No significant differences in weight or measurements were found between adult wild and captive males. Preliminary comparisons with morphometrics for African C. mona from the literature showed the upper limit of Grenada mona body length and weight to be smaller than that of African monas for both sexes. These differences may be due to genetic divergence, ecological adaptation, inter-African geographic variation, and/or small sample sizes.

Africa↗

Human papillomavirus detection by hybrid capture in paired cervicovaginal lavage and cervical biopsy specimens.

Infection of the uterine cervix with human papillomavirus (HPV) is associated with dysplastic lesions that may progress to malignancy. Certain HPV types are associated with higher risk of cervical cancer than other genital HPVs. The goal of this study was to determine if cells obtained by cervicovaginal lavage contain similar HPV types as paired cervical biopsy in women referred because of abnormal cervical cytology. Thirty-four paired lavage and biopsy samples were analyzed for HPV DNA by hybrid capture, using "low risk" (HPV types 6. 11, and related types and "high risk" group (HPV types 16, 18, and related types) HPV. HPV was detected in 24 lavage samples and 18 biopsies. High risk types were predominant. In 14 of 18 HPV-positive biopsies, the paired lavage was also positive for the same HPV group. Four biopsies were HPV-positive at low levels, and the paired lavage was HPV-negative. The mean viral copy numbers of the biopsies from patients with positive and negative lavage samples were 2.7 and 0.1, respectively (P = .02). Ten low level HPV infections were detected by lavage that were not detected by biopsy. HPV detection by hybrid capture in cells obtained by cervicovaginal lavage reflects the results of HPV testing in cervical biopsies.

Cell Biology↗

Concanavalin A-immobilized polystyrene nanospheres capture HIV-1 virions and gp120: potential approach towards prevention of viral transmission.

To establish an effective tool for the prevention of HIV-1 transmission, lectin-immobilized polystyrene nanospheres were synthesized and examined for their HIV-1 capture activity. When concanavalin A (Con A) was immobilized on the surface of polystyrene nanospheres (400 nm in diameter) with poly(methacrylic acid) branches and incubated with HIV-1 suspension at room temperature for 60 min, the nanospheres (Con A-NSs) achieved a >3.3 log and a 2.2 log reduction of viral infectivity in HIV-1 (IIIB strain) suspension at a concentration of 2 and 0.5 mg/ml, respectively. Meanwhile, Con A-free nanospheres, which were not immobilized with Con A, achieved only a 0.29 log reduction at 0.5 mg/ ml. Con A-NSs (2 mg/ml) could also reduce the gp120 level of III(B) and HE strains to <7.1% and 5.5% of each control, respectively. The combination of Con A-NS treatment followed by filtration with a microporous membrane efficiently removed virion-free gp120 as well as infectious viral particles from HIV-1 suspension. Electron microscopic examination demonstrated that HIV-1 virions were trapped on the surface of Con A-NSs. Thus, Con A-NSs can capture HIV-1 virions and gp120 with high affinity, and may have potential as an effective tool for the prevention of HIV-1 transmission.

Concanavalin A↗

Molecular identification of metastatic cancer to the skin using laser capture microdissection: a case report.

BACKGROUND: In the current study the authors report a 57-year-old woman with a scalp tumor and cervical lymphadenopathy who had a previously resected duodenal carcinoid. Histologic and immunophenotypic characteristics of the duodenal carcinoid differed from those of the scalp and cervical lymph node tumors, prompting the use of molecular methodologies to make the diagnosis. METHODS: Paraffin embedded tissues from the duodenal carcinoid, scalp, and lymph node tumors were dissected using microscopic visualization and laser capture microdissection. DNA was extracted and polymerase chain reaction (PCR) was performed to evaluate loss of heterozygosity and microsatellite alterations using primers flanking 22 polymorphic microsatellite markers from 9 chromosomal regions, including genes associated with MEN-1 (11q), CDKN2 (9p), p53 (17p), and bronchial carcinoid (3p). Microdissected lymphocytes from the three tissues were used as source of constitutional DNA (controls). RESULTS: Fourteen of the 22 markers were informative (heterozygous in control lymphocytes). A marker on 3p12 showed loss of the same parental allele in the three tumors. A different marker on 3p14.2 showed an identical shifted band in the three tumors indicative of a common microsatellite alteration. CONCLUSIONS: The shared molecular abnormalities among the three tumors indicated a common clonal origin, leading to a diagnosis of primary duodenal carcinoid with clear cell metastases to the scalp and cervical lymph nodes. These findings led to radiation therapy and immunotherapy rather than chemotherapy. This case illustrates the novel application of laser capture microdissection combined with PCR-based analyses of genomic markers for the identification of the origin of metastatic disease.

Alleles↗

Detection of human papillomavirus DNA in bronchopulmonary carcinomas by hybrid capture II: a study of 185 tumors.

BACKGROUND: Some human papillomaviruses (HPVs) are oncogenic in the cervix and are also associated with benign and malignant proliferations in other organs. Currently, the association of HPV with tumors of the lower respiratory tract is not so clearly defined because the studies are difficult to compare; series of cases reported from different geographic regions have used frozen or formalin fixed samples and a variety of techniques of HPV detection. METHODS: The authors studied the prevalence of HPV in a large series of 185 frozen bronchopulmonary tumor samples with a new solution hybridization technique, Hybrid Capture II assay. This test is largely applied in cervical pathology. Its sensitivity is very close to the sensitivity of PCR. It allows the detection of 18 mucosal HPV types, divided into 1 oncogenic and 1 nononcogenic group. RESULTS: Oncogenic HPV DNA was detected by the Hybrid Capture II assay in 5 cases (2.7%) of 185 (3 males and 2 females). In the rare positive cases detected, the authors could not find any consistent morphologic changes classically associated with HPV infection in anogenital lesions, such as koilocytosis. CONCLUSIONS: Oncogenic HPV DNA is detected in a small proportion of cases of bronchopulmonary carcinoma, and thus HPV infection appears to play a limited role in the tumorigenesis of most lung carcinomas.

Adenocarcinoma↗

Electron capture dissociation of substance P using a commercially available Fourier transform ion cyclotron resonance mass spectrometer.

Electron capture dissociation of the peptide Substance P is reported for the first time, with an unmodified, commercially available Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The fragmentation pattern is compared with that obtained with collisionally induced dissociation of the ions in the electrospray ion source, and note that electron capture dissociation gives a more easily interpreted spectrum, showing mainly C-fragments. With the exception of the proline residues, which require cleavage of two chemical bonds, we observe all C-fragmental we find the bias voltage of the electron gun not to be very critical.

Amino Acid Sequence↗