Priority setting for adult malnutrition and nutritional screening in healthcare: a James Lind Alliance.

BACKGROUND: Malnutrition is one the greatest global health challenges of our generation, leading to the increased utilisation of healthcare resources, as well as morbidity and mortality. Research has primarily been driven by industry, academia and clinical working groups and has had little involvement from patients and carers. The project described in the present study aimed to establish a priority setting partnership allowing patients, carers and healthcare professionals an opportunity to influence the research agenda. METHODS: A national survey was conducted to gather malnutrition uncertainties and identify key issues (i.e. areas within scope where an evidence-base is lacking) from those with experience of malnutrition. Uncertainties were analysed according to themes. Similar questions were grouped and summary questions were developed. A second survey was conducted and respondents were asked to choose their 10 most important summary questions. A workshop was conducted to finalise the top 10 research priorities from the most frequently indicated uncertainties on the interim survey. RESULTS: Overall, 1128 uncertainty questions were submitted from 268 people. The interim survey had 71 responses and a list of the top 26 questions was generated for the workshop. There were 26 questions discussed, ranked and agreed by healthcare professionals, carers and patients at the workshop. The top 10 research priorities were then chosen. These included questions on oral nutritional supplements, vulnerable groups, screening, community care, use of body mass index and technology. CONCLUSIONS: The top 10 research priorities in malnutrition and nutritional screening have been identified from a robust process involving patients, carers and healthcare professionals.

Tumor therapy with targeted atomic nanogenerators.

A single, high linear energy transfer alpha particle can kill a target cell. We have developed methods to target molecular-sized generators of alpha-emitting isotope cascades to the inside of cancer cells using actinium-225 coupled to internalizing monoclonal antibodies. In vitro, these constructs specifically killed leukemia, lymphoma, breast, ovarian, neuroblastoma, and prostate cancer cells at becquerel (picocurie) levels. Injection of single doses of the constructs at kilobecquerel (nanocurie) levels into mice bearing solid prostate carcinoma or disseminated human lymphoma induced tumor regression and prolonged survival, without toxicity, in a substantial fraction of animals. Nanogenerators targeting a wide variety of cancers may be possible.

A Dual Mode Propulsion System for Small Satellite Applications‡.

This study focused on the development of a chemical micropropulsion system suitable for primary propulsion and/or attitude control for a nanosatellite. Due to the limitations and expense of current micropropulsion technologies, few nanosatellites with propulsion have been launched to date; however, the availability of such a propulsion system would allow for new nanosatellite mission concepts, such as deep space exploration, maneuvering in low gravity environments and formation flying. This work describes the design of "dual mode" monopropellant/bipropellant microthruster prototype that employs a novel homogeneous catalysis scheme. Results from prototype testing are reported that validate the concept. The micropropulsion system is designed to be fabricated using a combination of additively-manufactured and commercial off the shelf (COTS) parts along with non-toxic fuels, thus making it a low-cost and environmentally-friendly option for future nanosatellite missions.

Definition of essential sequences and functional equivalence of elements downstream of the adenovirus E2A and the early simian virus 40 polyadenylation sites.

In addition to the highly conserved AATAAA sequence, there is a requirement for specific sequences downstream of polyadenylic acid [poly(A)] cleavage sites to generate correct mRNA 3' termini. Previous experiments demonstrated that 35 nucleotides downstream of the E2A poly(A) site were sufficient but 20 nucleotides were not. The construction and assay of bidirectional deletion mutants in the adenovirus E2A poly(A) site indicates that there may be redundant multiple sequence elements that affect poly(A) site usage. Sequences between the poly(A) site and 31 nucleotides downstream were not essential for efficient cleavage. Further deletion downstream (3' to +31) abolished efficient cleavage in certain constructions but not all. Between +20 and +38 the sequence T(A/G)TTTTT was duplicated. Function was retained when one copy of the sequence was present, suggesting that this sequence represents an essential element. There may also be additional sequences distal to +43 that can function. To establish common features of poly(A) sites, we also analyzed the early simian virus 40 (SV40) poly(A) site for essential sequences. An SV40 poly(A) site deletion that retained 18 nucleotides downstream of the cleavage site was fully functional while one that retained 5 nucleotides downstream was not, thus defining sequences required for cleavage. Comparison of the SV40 sequences with those from E2A did not reveal significant homologies. Nevertheless, normal cleavage and polyadenylation could be restored at the early SV40 poly(A) site by the addition of downstream sequences from the adenovirus E2A poly(A) site to the SV40 +5 mutant. The same sequences that were required in the E2A site for efficient cleavage also restored activity to the SV40 poly(A) site.

Fluorescent in situ hybridization (FISH) in bone marrow and peripheral blood of leukemia patients: implications for occupational surveillance.

Although there has been a rapid rise in the application of fluorescent in situ hybridization (FISH) analysis of bone marrow tissue for the staging and prognosis determination of hematopoietic malignacies such as the chronic and acute leukemias, it's application as a surveillance tool for leukemogen exposed high risk occupational cohorts is understandably limited by the invasiveness of sample collection. While some small occupational studies have been performed using FISH in peripheral blood with promising results, some of the basic assumptions made in utilizing the FISH technique have not been fully explored. These include selection of the correct hematopoietic cell to assay (myeloid or lymphoid); selection of appropriate chromosomal markers and the sensitivity of peripheral blood FISH in detecting unbalanced genomic abnormalities. In this study, we performed a pilot 'validation' exercise utilizing the FISH technique and standard metaphase cytogenetics, comparing results in tandem pairs of peripheral blood with bone marrow cells, where clonal abnormalities arise. Samples were taken from patients with known chromosomal lesions associated with active leukemia. We carefully chose markers most frequently associated with leukemogen-inducing DNA damage and probes that have been utilized successfully in clinical practice. Ten de novo or therapy-related acute myeloid leukemia (t-AML) patients underwent bone marrow cell karyotyping and fluorescent in situ hybridization (FISH) analysis. Parallel peripheral blood samples were concommitently drawn and evaluated with FISH using the same probes. In six of eight paired samples treated with a 3-day phytohemagglutinin (PHA) stimulation, typically used to assay lymphocytes and their progenitors, we detected abnormal clones. In one of the two remaining cases, we identified an abnormal clone in both bone marrow and PHA-stimulated peripheral blood, although at a level in the peripheral blood sample that would typically be reported as "non-diagnostic" for clinical purposes. These results suggest that use of FISH in PHA stimulated peripheral blood samples with probes commonly employed in t-AML evaluations (chromosomes 5q, 7q, 8, 11q) to detect cytogenetic abnormalities in peripheral blood represents a potentially promising though as yet, under-utilized approach for the occupational surveillance of workers exposed to leukemogens, especially if it could be linked to automated high-throughput assays for increased sensitivity.

Response of LNCaP spheroids after treatment with an alpha-particle emitter (213Bi)-labeled anti-prostate-specific membrane antigen antibody (J591).

A theoretical drawback to alpha-particle therapy with 213Bi is the short range of the particle track coupled with the short half-life of the radionuclide, thereby potentially limiting effective cytotoxicity to rapidly accessible, disseminated individual tumor cells (e.g., as in leukemia). In this work, a prostate carcinoma spheroid model was used to evaluate the feasibility of targeting micrometastatic clusters of tumor cells using 213Bi-labeled anti-prostate-specific membrane antigen (PSMA) antibody, J591. In prostate cancer, vascular dissemination of tumor cells or tumor cell clusters to the marrow constitutes an important step in the progression of this disease to widespread skeletal involvement, an incurable state. Such prevascularized clusters are ideal targets for radiolabeled antibodies because the barriers to antibody penetration that are associated with the capillary basal lamina have not yet formed. Beta- and gamma-emitting radionuclides such as 131I, which are widely used in radioimmunotherapy, are not expected to be effective when targeting single cells or small cell clusters. This is because the range of the emissions is one to two orders of magnitude greater than the target size, and the energy deposited per traversal is insufficient to produce any significant radiobiological effect. Spheroids of the prostate cancer cell line, LNCaP-LN3, were used as a model of prevascularized micrometastases; their response to an anti-PSMA antibody, J591, radiolabeled with the alpha-particle emitter 213Bi (T(1/2), 45.6 min.) has been measured. The time course of spheroid volume reductions was found to be sensitive to the initial spheroid volume. J591 labeled with 0.9 MBq/ml 213Bi resulted in a 3-log reduction in spheroid volume on day 33, relative to control, for spheroids with an initial diameter of 130 microm; 1.8 MBq/ml were required to achieve a similar response for spheroids with an initial diameter of 180 microm. Equivalent spheroid responses were observed after 12 Gy of acute external beam photon irradiation. Monte Carlo-based microdosimetric analyses of the 213Bi decay distribution in individual spheroids of 130-microm diameter yielded an average alpha-particle dose of 3.7 Gy to the spheroids, resulting in a relative biological effectiveness factor of 3.2 over photon irradiation. The activity concentrations used in the experiments were clinically relevant, and this work supports the possibility of using 213Bi-labeled antibodies not only for disseminated single tumor cells, as found in patients with leukemia, but also for micrometastatic tumor deposits up to 180 microm in diameter (1200 cells).

An alpha-particle emitting antibody ([213Bi]J591) for radioimmunotherapy of prostate cancer.

A novel alpha-particle emitting monoclonal antibody construct targeting the external domain of prostate-specific membrane antigen (PSMA) was prepared and evaluated in vitro and in vivo. The chelating agent, N-[2-amino-3-(p-isothiocyanatophen-yl)propyl]-trans-cyclohexane-1, 2-diamine-N,N',N',N'',N''-pentaacetic acid, was appended to J591 monoclonal antibody to stably bind the 213Bi radiometal ion. Bismuth-213 is a short-lived (t 1/2 = 46 min) radionuclide that emits high energy alpha-particles with an effective range of 0.07-0.10 mm that are ideally suited to treating single-celled neoplasms and micrometastatic carcinomas. The LNCaP prostate cancer cell line had an estimated 180,000 molecules of PSMA per cell; J591 bound to PSMA with a 3-nM affinity. After binding, the radiolabeled construct-antigen complex was rapidly internalized into the cell, carrying the radiometal inside. [213Bi]J591 was specifically cytotoxic to LNCaP. The LD50 value of [213Bi]J591 was 220 nCi/ml at a specific activity of 6.4 Ci/g. The potency and specificity of [213Bi]J591 directed against LNCaP spheroids, an in vitro model for micrometastatic cancer, also was investigated. [213Bi]J591 effectively stopped growth of LNCaP spheroids relative to an equivalent dose of the irrelevant control [213Bi]HuM195 or unlabeled J591. Cytotoxicity experiments in vivo were carried out in an athymic nude mouse model with an i.m. xenograft of LNCaP cells. [213Bi]J591 was able to significantly improve (P < 0.0031) median tumor-free survival (54 days) in these experiments relative to treatment with irrelevant control [213Bi]HuM195 (33 days), or no treatment (31 days). Prostate-specific antigen (PSA) was also specifically reduced in treated animals. At day 51, mean PSA values were 104 ng/ml +/- 54 ng/ml (n = 4, untreated animals), 66 ng/ml +/- 16 ng/ml (n = 6, animals treated with [213Bi]HuM195), and 28 ng/ml +/- 22 ng/ml (n = 6, animals treated with [213Bi]J591). The reduction of PSA levels in mice treated with [213Bi]J591 relative to mice treated with [213Bi]HuM195 and untreated control animals was significant with P < 0.007 and P < 0.0136, respectively. In conclusion, a novel [213Bi]-radiolabeled J591 has been constructed that selectively delivers alpha-particles to prostate cancer cells for potent and specific killing in vitro and in vivo.

Radioimmunotherapy for model B cell malignancies using 90Y-labeled anti-CD19 and anti-CD20 monoclonal antibodies.

In recent years, radioimmunotherapy (RIT) with beta(-) particle emitting radionuclides targeting the CD20 antigen on B cells in the treatment of non-Hodgkin's lymphoma has provided the most compelling human clinical data for the success of RIT. CD19, like CD20, is an antigen expressed on the surface of cells of the B lineage, and CD19 may provide an alternative target for radioimmunotherapy of B cell neoplasms. CD19 has been largely overlooked as a target for conventional 131I RIT, because the antigen rapidly internalizes upon binding of antibody, resulting in catabolism and significant release of 131I. Such modulation may be an advantage to RIT with radiometals such as 90Y, 177Lu, 213Bi and 225Ac. Herein, we have compared beta(-) particle RIT with antibodies targeting either CD19 or CD20. The anti-CD19 and anti-CD20 antibodies, B4 or C2B8, respectively, were appended with the SCN-CHX-A''-DTPA bifunctional chelating agent and labeled with 90Y. In the tumor model used, there were three times as many CD20 target sites on lymphoma cells as compared to CD19 sites (62000 vs 20000 binding sites, respectively). We compared the efficacy of the 90Y-labeled antibodies to reduce lymphoma in a nude mouse xenograft solid tumor model, after measurable lymphoma appeared. Reduction in tumor size began at day 3 in all three 90Y-treated groups, but tumor began to recur in many animals 9 days after the treatments. There was one cure in each specific treatment group. In contrast, the tumor in the two control groups showed no regression. There was a significant prolongation of median survival time from xenograft (P < 0.0001) in all the 90Y-labeled antibody construct-treated groups (32 days for 0.15 mCi 90Y-B4; 26 days for 0.20 mCi 90Y-C2B8, and 23 days for 0.15 mCi 90Y-C2B8) in comparison to the two control groups (11 days for 0.02 mg of C2B8 and 9 days for untreated growth controls). Specificity of the radioimmunotherapy was also shown. In conclusion, 90Y-labeled anti-CD19 antibody has efficacy comparable to 90Y-labeled anti-CD20 antibody in the treatment of mice bearing human lymphoma xenografts. These data suggest that CD19-targeted RIT merits further study.

Zyn-Linker delivery of antirheumatic agents.

Despite our increasing ability to manage rheumatoid arthritis through systemic medication, refractory joints require local administration of more aggressive therapy in a substantial number of patients. These studies tested whether a new class of molecules designated Zyn-Linkers could deliver and retain therapeutics in a joint. Zyn-Linkers are synthetic lipid-like molecules designed to insert into cell membranes and enhance drug delivery to cells. After intra-articular injection into the knee of NZW rabbits, Zyn-Linkers bound rapidly and homogenously to synovial lining cells. Chelating Zyn-Linkers which contained Re-186 or Y-90 were synthesized to evaluate localization and retention after intra-articular injection. Initial studies using Re-186 Zyn-Linker gave excellent localization as evaluated by whole-body imaging: counts in the knee region represented > 90% of counts present in the whole body for at least 4-6 days postinjection. Similar results were obtained using a Y-90 Zyn-Linker and this agent was used for biodistribution studies due to its greater stability and ease of preparation. Efficacy and safety of Y-90 Zyn-Linker as a potential radiation synovectomy agent were estimated by extrapolation of biodistribution data to humans. A therapeutically effective dose of 8,000 cGy to synovium was calculated to require intra-articular injection of 3.4 mCi Y-90 Zyn-Linker, a value less than or equal to doses of particulate Y-90 agents used clinically in Europe. The predicted safety profile for Y-90 Zyn-Linker was excellent, with estimated doses to nontarget organs and tissues falling well within FDA-recommended safety levels for research-only radiopharmaceuticals. In addition to exhibiting desirable localization and retention properties, Zyn-Linkers may also be synthesized to release antirheumatic drugs such as methotrexate at controlled rates. This suggests substantial potential for these drug delivery molecules as chemical synovectomy agents which may be used concurrently with systemic chemotherapy to improve management of refractory joints.

Evaluation of biological response modifiers in the enhancement of tumor uptake of technetium-99m labeled macromolecules. A preliminary report.

Imaging tumors with radioactive monoclonal antibodies remains attractive but continues to be challenging. With the hypothesis that the use of biological response modifiers (BRMs) may augment the tumor uptake, technetium-99m(99mTc)-labeled tumor necrosis factor (TNF) and nuclear histone specific TNT-1-F(ab')2 were evaluated in tumor bearing mice given a single dose of interferon (IFN). Ukrain or pokeweed mitogen as BRMs. As early as 1.5 h post injection (p.i.) of the radioactive macromolecules, the absolute tumor uptake (% administered dose/g) of each agent was enhanced (e.g., TNF, control = 1.8 +/- 0.4, Ukrain = 3.2 +/- 0.5, P = 0.006) and tumor to muscle ratios were elevated (e.g., TNF, control a 4.1 +/- 2.2, interferon 8.3 +/- 2.7, P = 0.01). The absolute tumor uptake remained practically unchanged at 4 h p.i. Generally with BRMs, the blood clearance was rapid and tumor/blood ratios and tumor/muscle ratios were higher than in the control group, increasing to greater than 200% for IFN as a BRM. The early enhancement in tumor uptake of macromolecules, leading to excellent delineation of tumors by scintigraphy is highly encouraging and warrants further studies to explore the full potential of BRMs.

Rhenium-186-labeled monoclonal antibodies for radioimmunotherapy: preparation and evaluation.

Rhenium-186 has been determined to be a leading radionuclide for radioimmunotherapy. However, the use of 186Re has been limited due to the lack of a convenient and efficient method by which the radionuclide can be bound to monoclonal antibodies. We have developed a simple technique to label IgM, IgG, fragmented antibodies and tumor necrosis factor-alpha with 186Re. This technique uses ascorbic acid (AA) for controlled reduction of antibody disulfide groups to sulfhydryls and SnCl2 in citric acid for the reduction of 186ReO4-. The labeling yields as determined by instant thin-layer chromatography, molecular filtration and gel filtration were greater than 95% and the colloid formation was less than 5%. The labeled antibodies were stable when challenged with 100 and 250 molar excess of DTPA and HSA for 24 hr at 37 degrees C. SDS-PAGE analysis and autoradiography of labeled IgM, IgG and F(ab')2 monoclonal antibodies indicated uniform labeling and that no fragmentation of the monoclonal antibodies had taken place during the labeling procedure. Immunospecificity of 186Re-labeled human neutrophil specific IgM, as determined by in vitro antigen excess assay, was comparable to that of indium-111-labeled c-DTPA-IgM and technetium-99m-labeled-IgM. A nuclear histone specific 186Re-TNT-1-F(ab')2 was evaluated in mice bearing experimental tumors. The tumor/muscle ratios at 4 and 24 hr were 5.9 +/- 0.21 and 13.8 +/- 6.7, respectively compared to that of 2.4 +/- 0.3 at 4 hr p.i. with a nonspecific protein. The labeling technique is simple, reliable and has already been adapted to a single-vial kit preparation.

Preparation of alpha-emitting 213Bi-labeled antibody constructs for clinical use.

UNLABELLED: Preclinical evaluation of alpha particle-emitting 213Bi-labeled antibody constructs have demonstrated the specificity and potency of these agents in a variety of cancer systems. The transition of a 213Bi-radiolabeled antibody from a preclinical construct to a clinical drug represented a difficult task that involved development of reliable and validated methods to provide multiple MBq quantities of a pure, immunoreactive agent that met pharmaceutical standards to treat patients. METHODS: The methods used for the preparation of (213Bi)CHX-A-diethylenetriamine pentaacetic acid (DTPA)-HuM195, an alpha particle-emitting anti-CD33 antibody construct for therapy of myeloid leukemias, is used as a specific example. This article describes methods for reagent purification, drug labeling, radioprotection and chromatographic purification. Quality of the drug is evaluated using radiochemical incorporation and purity assays with instant thin-layer chromatography (ITLC) and high-performance liquid chromatography (HPLC), determination of cell-based antibody total immunereactivity, small animal safety, pyrogen level, sterility and radionuclidic purity. RESULTS: Sixty-seven doses were prepared. Individual doses ranged from 148 to 814 MBq. Specific activities ranged from 329 to 766 MBq/mg. The radiolabeling efficiency (median +/- SD) of CHX-A-DTPA-HuM195 with 213Bi was 81% +/- 9% (n = 67) after 9 min. The construct was purified by size-exclusion chromatography and was found to be 99% +/- 2% pure (n = 67) by either ITLC or HPLC methods. The immunoreactivity of (213Bi)CHX-A-DTPA-HuM195 was 89% +/- 9% (n = 44) and was independent of the specific activity. The formulated pharmaceutical was found to contain < or =4 +/- 1 EU/mL pyrogens (n = 66); all samples examined were sterile. An 225Ac radionuclidic impurity was present at a level of 0.04 +/- 0.03 x 10(-6)/mL (n = 10) in a product volume of 7.4 +/- 0.5 mL (n = 67). Each of the 67 doses was injected intravenously into patients without complication as part of a phase I clinical trial. CONCLUSION: These data show that 213Bi-labeled antibody constructs can be prepared and administered safely to humans at a wide range of therapeutic levels.

Pharmacokinetics and dosimetry of an alpha-particle emitter labeled antibody: 213Bi-HuM195 (anti-CD33) in patients with leukemia.

UNLABELLED: Data from nine patients with leukemia participating in a phase I activity-escalation study of HuM195, labeled with the alpha-particle emitter 213Bi (half-life = 45.6 min), were used to estimate pharmacokinetics and dosimetry. This is the first trial using an alpha-particle emitter in humans. The linear energy transfer of alpha particles is several hundredfold greater than that of beta emissions. The range in tissue is approximately 60-90 microm. METHODS: The activity administered to patients ranged from 0.6 to 1.6 GBq. Patient imaging was initiated at the start of each injection. Thirty 1-min images followed by ten 3-min images were collected in dynamic mode; a 20% photopeak window centered at 440 keV was used. Blood samples were collected until 3 h postinjection and counted in a gamma counter. Contours around the liver and spleen were drawn on the anterior and posterior views and around a portion of the spine on the posterior views. No other organs were visualized. RESULTS: The percentage injected dose in the liver and spleen volumes increased rapidly over the first 10-15 min to a constant value for the remaining hour of imaging, yielding a very rapid uptake followed by a plateau in the antibody uptake curves. The kinetic curves were integrated to yield cumulated activity. The mean energy emitted per nuclear transition for 213Bi and its daughters, adjusted by a relative biologic effectiveness of 5 for alpha emissions, was multiplied by the cumulated activity to yield the absorbed dose equivalent. Photon dose to the total body was determined by calculating a photon-absorbed fraction. The absorbed dose equivalent to liver and spleen volumes ranged from 2.4 to 11.2 and 2.9 to 21.9 Sv, respectively. Marrow (or leukemia) mean dose ranged from 6.6 to 12.2 Sv. The total-body dose (photons only) ranged from 2.2 x 10(-4) to 5.8 x 10(-4) Gy. CONCLUSION: This study shows that patient imaging of 213Bi, an alpha-particle emitter, labeled to HuM195 is possible and may be used to derive pharmacokinetics and dosimetry. The absorbed dose ratio between marrow, liver and spleen volumes and the whole body for 213Bi-HuM195 is 1000-fold greater than that commonly observed with beta-emitting radionuclides used for radioimmunotherapy.

Alpha-emitting bismuth cyclohexylbenzyl DTPA constructs of recombinant humanized anti-CD33 antibodies: pharmacokinetics, bioactivity, toxicity and chemistry.

UNLABELLED: The alpha-particle-emitting radionuclides have several physical characteristics that make them attractive candidates for radioimmunotherapy: (a) high linear energy transfer; (b) short path lengths (50-80 microm); and (c) limited ability of cells to repair damage to DNA. This article describes the pharmacokinetic, bioactivity, toxicity and chemical characteristics of alpha-particle-emitting, 213Bi and 212Bi radiometal conjugated HuM195 (anti-CD33) constructs. Conjugation of HuM195 to SCN-CHX-A-DTPA resulted in the attachment of up to 10 chelating ligand molecules per antibody. RESULTS: Radiolabeling efficiency of the CHX-A-DTPA-HuM195 construct with 213Bi was 78%+/-10% (n = 46) after 10 min at specific activities of up to 1110 MBq/mg. The immunoreactivity of the 213Bi-labeled CHX-A-DTPA-HuM195 construct was 84%+/-10% (n = 28) and was independent of the specific activity. The bismuth-labeled CHX-A-DTPA-HuM195 construct was rapidly internalized into the cell in a time-dependent manner ranging from 50% at 1 h to 65% at 24 h. 205Bi/206Bi-labeled constructs were stable for at least 2 d in vitro in the presence of human serum at 37 degrees C. After injection into mice, there was no uptake or loss of bismuth to mouse tissues, which do not express CD33, or to the kidney, which has avidity for free bismuth. Mice injected intraperitoneally with doses of (213Bi)CHX-A-DTPA-HuM1 95 ranging from 18.5 to 740 MBq/kg showed no toxicity, but at 2590 MBq/kg, two of the three mice died within 2 wk and a third mouse showed significant reductions in white blood cell counts. Mice injected intravenously with doses of (213Bi)CHX-A-DTPA-HuM195 up to 370 MBq/kg exhibited little toxicity, but 666 MBq/kg was above the MTD for mice. Leukemia cell killing in vitro with bismuth-labeled HuM1 95 showed dose- and specific activity-dependent killing of CD33+ HL60 cells; approximately 50% killing was observed when two bismuth atoms (50 fM radiolabeled antibody) were initially bound onto the target cell surface. CONCLUSION: Alpha-emitting antibodies are among the most potent cytotoxic agents known, yet are specific and appear safe in vivo. The physical and biochemical characteristics of the 213Bi isotope and its generation, as well as the biochemistry of the 213Bi-labeled CHX-A-DTPA-HuM195 construct, make it possible to use the constructs safely and feasibly in humans at therapeutic levels.

Interleukin-1 genetic association with periodontitis in clinical practice.

BACKGROUND: Periodontitis is a bacterial disease modified by multiple risk factors. The pro-inflammatory cytokine interleukin- (IL-1) is a key regulator of the host responses to microbial infection and a major modulator of extracellular matrix catabolism and bone resorption. It has been reported that variations in the IL-1 gene cluster on chromosome 2 are associated with increased susceptibility to severe adult periodontitis. METHODS: The present study evaluated the association between a composite IL-1 genotype, including allele 2 at each of two loci (IL-1A +4845 plus IL- B +3954), and a broad spectrum of periodontally healthy to diseased patients in a population that is typically encountered in a dental practice setting. Ninety patients, non-smokers or former smokers with less than 10 pack-year (pk/yr) history, were recruited from a private dental practice. The major outcome variable was bone loss determined by computerized linear measurements of radiographs. Genotypes were analyzed from finger-stick blood samples using previously reported methods. RESULTS: Multivariate logistic regression models demonstrated that patient age, former smoking history, and the IL-1 genotype were significantly associated with severity of adult periodontitis. For non-smokers or former light smokers (<5 pk/yr), IL-1 genotype positives were at increased odds ratio of having moderate to severe periodontal disease of 3.75 (95% CI: 1.04-13.50) to 5.27 (95% CI: 1.23-22.70), depending on ethnicity, compared to IL-1 genotype negatives. Former moderate smokers (>5 pk/yr and <10 pk/yr) who were IL-1 genotype negative were at increased odds ratio of having moderate to severe periodontal disease of 7.43 (95% CI: 1.20-46.20) compared to non-smokers or former light smokers who were IL-1 genotype negative. In addition, past smoking history was also a significant effect modifier as demonstrated by the statistically significant interaction between past smoking history status and IL-1 genotype status. CONCLUSIONS: This study demonstrates that the composite IL-1 genotype is significantly associated with the severity of adult periodontitis. It also confirmed that both IL-1 genotyping and smoking history provide objective risk factors for periodontal disease in a private practice environment.

Visual stimulus compounding with pigeons.

Eight pigeons responded to successively presented color and line stimuli correlated with different likelihoods of reinforcement (100, 50 and 10%). An intertriai interval was included during training for half of the birds. In Experiment 1, tests with superimposed compounds resulted in variable responding, but overall, rates of responding were intermediate to the rates controlled by the individual component stimuli. In Experiments 2a and 2b, tests with compounds of spatially separated stimuli yielded rates of responding that were intermediate to, and in some cases lower than, responding controlled by the individual component stimuli. These results suggest limitations to the generality of additive models of stimulus compounding when visual stimulus combinations are employed.

Effects of signaled versus unsignaled delay of reinforcement on choice.

Pigeons chose between 5-s and 15-s delay-of-reinforcement alternatives. The first key peck to satisfy the choice schedule began a delay timer, and food was delivered at the end of the interval. Key pecks during the delay interval were measured, but had no scheduled effect. In Experiment 1, signal conditions and choice schedules were varied across conditions. During unsignaled conditions, no stimulus change signaled the beginning of a delay interval. During differential and nondifferential signal conditions, offset of the choice stimuli and onset of a delay stimulus signaled the beginning of a delay interval. During differential signal conditions, different stimuli were correlated with the 5-s and 15-s delays, whereas the same stimulus appeared during both delay durations during nondifferential signal conditions. Pigeons showed similar, extreme levels of preference for the 5-s delay alternative during unsignaled and differentially signaled conditions. Preference levels were reliably lower with nondifferential signals. Experiment 2 assessed preference with two pairs of unsignaled delays in which the ratio of delays was held constant but the absolute duration was increased fourfold. No effect of absolute duration was found. The results highlight the importance of delayed primary reinforcement effects and challenge models of choice that focus solely on conditioned reinforcement.

Rapid preparation of short-lived alpha particle emitting radioimmunopharmaceuticals.

Alpha emitting radionuclides are of considerable interest for targeted radioimmunotherapy. Generator supplied 213Bi emitting 8.5 MeV alpha particles with a 45.6 min half-life has been conjugated to a monoclonal antibody (HuM195-CHX-A-DTPA) for targeted therapy of leukemia in a clinical trial. The clinical dose preparation of pharmaceutical formulation by a pair of skilled radiochemists took 25 min, which corresponds, to an overall decay loss of 30% of the initial 213Bi activity eluted from the generator. In order to allow more widespread and practical clinical use of targeted 213Bi alpha particle therapy, we developed a new procedure that is simpler, more rapid and adaptable to a hospital pharmacy. The new 10 min process includes a tandem elution and labeling, and an anion exchange column purification method that can be reproducibly used.

Breakthrough of 225Ac and its radionuclide daughters from an 225Ac/213Bi generator: development of new methods, quantitative characterization, and implications for clinical use.

Bisumth-213, a short-lived alpha particle emitting radionuclide, is generated from the decay of 225Ac, which has a half-life of 10 days. The development of a clinical 225Ac/213Bi generator and the preparation of a 213Bi radiolabeled antibody for radioimmunotherapy of leukemia have been reported. The 225Ac decay scheme is complex; therefore a thorough understanding of the impact of both the parent 225Ac and its daughters on radiolabeling, purification, and quantification is necessary for optimal use of the generator system. This paper reports: (i) unique new methods to measure 221Fr, 213Bi, and 209Pb, the prominent daughters of 225Ac; and (ii) a quantitative evaluation of 225Ac/213Bi generator breakthrough and the radionuclidic purity of 213Bi labeled radiopharmaceutical dose formulations. A quantitative multi-dimensional proportional scanning method was employed to distinguish and measure specific daughter radionuclides. This method combines thin layer chromatography in two perpendicular directions with attenuated collimation as a function of time for data collection and analysis. Francium-221 and 213Bi eluted differentially from the generator, and 221Fr contributed minimally to unchelated 213Bi in the reaction and final products. Lead-209 was present in the reaction solution, but not strongly bound by the chelating moiety either (i) under the 213Bi labeling reaction conditions or (ii) following chelated 213Bi decay. As a consequence of incorporating several new procedures to the operation of the generator, 225Ac breakthrough in the final product was further reduced and represented a trivial contaminant in the final drug formulations.

An 225Ac/213Bi generator system for therapeutic clinical applications: construction and operation.

A method for construction and operation of an 225Ac/213Bi generator capable of producing 25-100 mCi of 213Bi suitable for clinical antibody labeling is described. The generator has been designed to have an effective lifetime of several weeks, producing up to six therapeutic doses of radionuclide per day. To date, 57 clinical doses have been prepared and injected into patients using the described 213Bi generator. Factors such as radiation damage, radioprotection, iodide eluate chemistry, radiolabeling chemistry and radionuclide purity are addressed.