Evidence of altered fatty acid metabolism in dogs with naturally occurring valvular heart disease and congestive heart failure.

INTRODUCTION: Myxomatous mitral valve disease (MMVD) is the most common cardiac condition in adult dogs. The disease progresses over several years and affected dogs may develop congestive heart failure (HF). Research has shown that myocardial metabolism is altered in cardiac disease, leading to a reduction in ß-oxidation of fatty acids and an increased dependence upon glycolysis. OBJECTIVES: This study aimed to evaluate whether a shift in substrate use occurs in canine patients with MMVD; a naturally occurring model of human disease. METHODS: Client-owned dogs were longitudinally evaluated at a research clinic in London, UK and paired serum samples were selected from visits when patients were in ACVIM stage B1: asymptomatic disease without cardiomegaly, and stage C: HF. Samples were processed using ultra-performance liquid chromatography mass spectrometry and lipid profiles were compared using mixed effects models with false discovery rate adjustment. The effect of disease stage was evaluated with patient breed entered as a confounder. Features that significantly differed were screened for selection for annotation efforts using reference databases. RESULTS: Dogs in HF had altered concentrations of lipid species belonging to several classes previously associated with cardiovascular disease. Concentrations of certain acylcarnitines, phospholipids and sphingomyelins were increased after individuals had developed HF, whilst some ceramides and lysophosphatidylcholines decreased. CONCLUSIONS: The canine metabolome appears to change as MMVD progresses. Findings from this study suggest that in HF myocardial metabolism may be characterised by reduced ß-oxidation. This proposed explanation warrants further research.

Metabolic Phenotypes of Carotid Atherosclerotic Plaques Relate to Stroke Risk: An Exploratory Study.

OBJECTIVE: Stroke is a major cause of death and disability. That three-quarters of stroke patients will never have previously manifested cerebrovascular symptoms demonstrates the unmet clinical need for new biomarkers able to stratify patient risk and elucidation of the biological dysregulations. In this study, the utility of comprehensive metabolic phenotyping is assessed to provide candidate biomarkers that relate to stroke risk in stenosing carotid plaque tissue samples. METHOD: Carotid plaque tissue samples were obtained from patients with cerebrovascular symptoms of carotid origin (n = 5), and from asymptomatic patients (n = 5). Two adjacent biological replicates were obtained from each tissue. Organic and aqueous metabolite extracts were obtained separately and analysed using two ultra performance liquid chromatography coupled to mass spectrometry metabolic profiling methods. Multivariate and univariate tools were used for statistical analysis. RESULTS: The two study groups demonstrated distinct plaque phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the two groups with a strong statistical significance (p = 10(-4)-10(-5)). Specifically, metabolites related to the eicosanoid pathway (arachidonic acid and arachidonic acid precursors), and three acylcarnitine species (butyrylcarnitine, hexanoylcarnitine, and palmitoylcarnitine), intermediates of the ß-oxidation, were detected in higher intensities in symptomatic patients. However, metabolites implicated in the process of cell death, a process known to be upregulated in the formation of the vulnerable plaque, were unaffected. CONCLUSIONS: Discrimination between symptomatic and asymptomatic carotid plaque tissue is demonstrated for the first time using metabolic profiling technologies. Two biological pathways (eicosanoid and ß-oxidation) were implicated in differentiating symptomatic from asymptomatic patients and will be further investigated. These results indicate that metabolic phenotyping should be further explored to investigate the chemistry of the unstable plaque, in the pursuit of candidate biomarkers for risk-stratification and targets for pharmacotherapeutic intervention.

Chemical mapping of the colorectal cancer microenvironment via MALDI imaging mass spectrometry (MALDI-MSI) reveals novel cancer-associated field effects.

Matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-MSI) is a rapidly advancing technique for intact tissue analysis that allows simultaneous localisation and quantification of biomolecules in different histological regions of interest. This approach can potentially offer novel insights into tumour microenvironmental (TME) biochemistry. In this study we employed MALDI-MSI to evaluate fresh frozen sections of colorectal cancer (CRC) tissue and adjacent healthy mucosa obtained from 12 consenting patients undergoing surgery for confirmed CRC. Specifically, we sought to address three objectives: (1) To identify biochemical differences between different morphological regions within the CRC TME; (2) To characterise the biochemical differences between cancerous and healthy colorectal tissue using MALDI-MSI; (3) To determine whether MALDI-MSI profiling of tumour-adjacent tissue can identify novel metabolic 'field effects' associated with cancer. Our results demonstrate that CRC tissue harbours characteristic phospholipid signatures compared with healthy tissue and additionally, different tissue regions within the CRC TME reveal distinct biochemical profiles. Furthermore we observed biochemical differences between tumour-adjacent and tumour-remote healthy mucosa. We have referred to this 'field effect', exhibited by the tumour locale, as cancer-adjacent metaboplasia (CAM) and this finding builds on the established concept of field cancerisation.

Comparison of cleaning efficacy between in-use disinfectant and electrolysed water in an English residential care home.

BACKGROUND: Infection control in hospitals and care homes remains a key issue. They are regularly inspected regarding standards of hygiene, but visual assessment does not necessarily correlate with microbial cleanliness. Pathogens can persist in the inanimate environment for extended periods of time. AIM: This prospective study compared the effectiveness of a novel sanitizer containing electrolysed water, in which the active ingredient is stabilized hypochlorous acid (Aqualution™), with the effectiveness of the quaternary ammonium disinfectant in current use for microbial removal from hand-touch surfaces in a care home. The study had a two-period crossover design. METHODS: Five surfaces were cleaned daily over a four-week period, with screening swabs taken before and after cleaning. Swabs were cultured in order to compare levels of surface microbial contamination [colony-forming units (cfu)/cm(2)] before and after cleaning with each product. FINDINGS: Cleaning with electrolysed water reduced the mean surface bacterial load from 2.6 [interquartile range (IQR) 0.30-30.40] cfu/cm(2) to 0.10 (IQR 0.10-1.40) cfu/cm(2) [mean log(10) reduction factor 1.042, 95% confidence interval (CI) 0.79-1.30]. Cleaning with the in-use quaternary ammonium disinfectant increased the bacterial load from 0.90 (IQR 0.10-8.50) cfu/cm(2) to 93.30 (IQR 9.85-363.65) cfu/cm(2) (mean log(10) reduction -1.499, 95% CI -1.87 to -1.12) (P < 0.0001). Using two proposed benchmark standards for surface microbial levels in hospitals, electrolysed water resulted in a higher 'pass rate' than the in-use quaternary ammonium disinfectant (80-86% vs 15-21%, P < 0.0001). CONCLUSION: Electrolysed water exerts a more effective bacterial kill than the in-use quaternary ammonium disinfectant, which suggests that it may be useful as a surface sanitizer in environments such as care homes.

In vitro receptor binding assays: general methods and considerations.

The development of receptor-targeting radiopharmaceuticals commonly involves the use of peptides, antibodies or small molecules. Resulting from the numerous modifications that can be made to these basic targeting agents, research in this field often generates a series of compounds as potential ligands for in vivo investigation. However, measuring each variant of a series in vivo can be both costly and time-consuming. Therefore, a number of in vitro assays, to study interactions between the targeted receptor and the ligands of interest, are frequently used to quickly and inexpensively narrow the field and identify a lead compound(s) for further investigation. For example, in saturation binding studies, the amount of radioligand required to saturate the receptors is measured and analyzed to determine the radioligand equilibrium dissociation constant (Kd), a useful gauge of the receptor binding affinity of a radioligand. In competitive binding experiments, a ligand of interest competes for available receptor sites with a standard radioligand of known high receptor affinity. Competition data are analyzed to yield another indicator of receptor affinity, called an IC50 value, which can be used to rank the relative receptor binding affinities for a series of ligands. In internalization and efflux studies, the rate and extent of receptor-mediated radioligand taken into and subsequently released from cells is measured, providing insight into cellular uptake and retention of the radioligand. Individually or taken together, these in vitro receptor binding assays are useful tools in radiopharmaceutical development.

Local photodynamic therapy for equine squamous cell carcinoma: evaluation of a novel treatment method in a murine model.

The objective of this study was to evaluate the effect of local photodynamic therapy (PDT) with verteporfin on tumor growth inhibition of squamous cell carcinoma (SCC) in a murine model. SCC was implanted in 85 nude mice by subcutaneous injection of A-431 SCC cells. Treatment groups (10 mice/group) received an intra-tumoral injection of verteporfin dissolved in dimethyl sulfoxide (DMSO) or 5% dextrose solution at a dose of 0.01 or 0.1mg/cm3. Controls received only solvent, or no injectate. All groups received identical light illumination (100J/cm2). Relative change in tumor volume (RCTV) at day 30 was compared between groups using the Wilcoxon rank sum test (P< 0.05). Local PDT with verteporfin at a dose of 0.1mg/cm3 resulted in significantly lower RCTV at day 30 compared to controls. Choice of solvent (DMSO versus D5W) did not affect the results. Local PDT may be an effective adjunctive therapy for the treatment of periocular equine SCC.

Xenotransplantation of cryopreserved equine squamous cell carcinoma to athymic nude and SCID mice.

Cryopreserved equine ocular squamous cell carcinoma (SCC) was inoculated subcutaneously into 15 athymic nude and 15 SCID mice. Xenotransplantation resulted in tumor growth in two athymic nude mice and 1 SCID mouse. Histological appearance and immunohistochemical characterization using cytokeratin 5/6 markers and p53 markers of the tumor grown in mice was in full accord with the original equine tumors. No evidence of metastasis was noted in any mouse. This model may serve as a relevant in vivo model for studying the biology of equine ocular SCC and for the testing of new therapeutic modalities.

Development of a two-antibody model for the evaluation of copper-64 radioimmunotherapy.

Copper-64 emits beta(+) and beta(-) particles suitable for positron emission tomography and radioimmunotherapy (RIT) of cancer. Copper-64-labelled antibodies have caused complete responses in laboratory animal RIT studies at far lower radiation doses than traditionally prescribed. The intracellular localization of copper radioisotopes may lead to cytotoxic effects by mechanisms beyond ionizing radiation damage. The purpose of this research was to develop a model using both internalizing and non-internalizing antibodies for direct comparison in future RIT studies using the same animal model of cancer. The monoclonal antibodies, cBR96 and cT84.66, were conjugated with N-hydroxysulfosuccinimidyl DOTA. All conjugates retained high immunoreactivity and labelled efficiently with (64)Cu with high specific activity and radiochemical purity. Twenty-four hour biodistributions determined in LS174T tumour-bearing nude mice demonstrated low organ and high tumour uptakes for both monoclonal antibodies. This model constitutes a promising system for elucidating whether internalization of (64)Cu is responsible for an enhanced tumour cytotoxicity in vivo.

Fibrinogen and preclinical echocardiographic target organ damage: the strong heart study.

Relations of fibrinogen to preclinical target organ damage, such as left ventricular hypertrophy, systolic dysfunction, and increased arterial stiffness while accounting for traditional risk factors, are unknown in a population-based sample free of clinically overt coronary heart disease. Therefore, we studied clinical and echocardiographic characteristics of 2709 American Indians participating in the Strong Heart Study without symptomatic atherosclerosis. The study sample was divided into tertiles of fibrinogen (cut-points, 3.24 and 3.83 g/L). Mean age, body mass index, proportion of women, and prevalences of hypertension and diabetes increased from the first to third tertile of fibrinogen. After adjustment for covariates, systolic and pulse pressures did not significantly differ among tertiles of fibrinogen, whereas diastolic pressure was slightly lower in the third than in lower tertiles of fibrinogen. HDL cholesterol was lower and plasma creatinine and urinary albumin/creatinine ratio was higher in the third tertile of fibrinogen. Left ventricular mass index, pulse pressure/stroke index, an estimate of arterial stiffness, and cardiac index were higher and left ventricular systolic function and total peripheral resistance were lower in the third than in two lower tertiles of fibrinogen independent of major covariates. In multiple regression analyses, left ventricular mass and pulse pressure/stroke index were positively associated with, and stress-corrected midwall shortening negatively associated with fibrinogen, independent of major covariates. Participants with fibrinogen >3.83 g/L were more likely to have at least 1 preclinical cardiovascular abnormality such as left ventricular hypertrophy, elevated arterial stiffness, or systolic myocardial dysfunction independent of covariates including renal dysfunction (adjusted odds ratio, 1.38; P<0.001). Thus, in a population sample of adults without clinically overt coronary heart disease, elevated fibrinogen is an independent correlate of prognostically relevant cardiovascular target organ damage.

Remotely sensed biological production in the equatorial Pacific.

A combination of ship, buoy, and satellite observations in the tropical Pacific during the period from 1992 to 2000 provides a basin-scale perspective on the net effects of El Niño and La Niña on biogeochemical cycles. New biological production during the 1997-99 El Niño/La Niña period varied by more than a factor of 2. The resulting interannual changes in global carbon sequestration associated with the El Niño/La Niña cycle contributed to the largest known natural perturbation of the global carbon cycle over these time scales.

Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.

The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.

Ultraviolet-radiation-induced keratinocyte apoptosis in C1q-deficient mice.

Exposure to ultraviolet B radiation is an important trigger of both systemic and cutaneous disease flares in individuals with systemic lupus erythematosus. More than 90% of individuals with homozygous C1q deficiency develop a systemic-lupus-erythematosus-like illness, which is typically associated with a severe photosensitive rash. Apoptotic, human keratinocytes have been shown in vitro to bind C1q, in the absence of antibody. These observations, together with the hypothesis that a major source of the autoantigens driving the immune response in systemic lupus erythematosus comes from apoptotic cells, led us to investigate the effects of murine C1q deficiency on ultraviolet-radiation-induced keratinocyte apoptosis in vivo. In this work, we demonstrated C1q binding to apoptotic murine keratinocytes in vitro and showed for the first time that C1q is also present on sunburn cells in vivo. In addition to C1q, we detected C3 deposition on sunburn cells in both wild-type and C1q-deficient mice, suggesting activation of the alternative pathway. Following acute ultraviolet exposure in vivo, no difference in the rate of clearance of sunburn cells was found in C1q-deficient mice from three different genetic backgrounds, compared with strain-matched wild-type controls. Furthermore, chronic ultraviolet exposure did not result in the production of autoantibodies or the development of glomerulonephritis. Our findings suggest that C1q does not play a critical role in the physiologic clearance of apoptotic murine keratinocytes in vivo.

An improved method for conjugating monoclonal antibodies with N-hydroxysulfosuccinimidyl DOTA.

A simple, water-soluble procedure for conjugation of monoclonal antibodies to 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) has been improved by optimizing pH, buffer, and temperature conditions for the preparation of N-hydroxysulfosuccinimidyl DOTA and its conjugation to the human/murine chimeric anti-carcinoembryonic antigen antibody cT84.66. This improved method results in a 6-fold increase in conjugation efficiency, a 3-7-fold decrease in antibody cross-linking, a more homogeneous population of conjugate species, and a 5-fold decrease in the quantities of reagents needed for conjugation. The cT84.66-DOTA conjugate was labeled to high specific activity with 111In, 90Y, 88Y, 64Cu, and 67Cu, affording near-quantitative incorporation of the majority of these radiometals. This improved conjugation procedure facilitates large-scale production and radiometal labeling of cT84.66-DOTA for clinical radioimmunotherapy trials.

Conjugation of monoclonal antibodies with TETA using activated esters: biological comparison of 64Cu-TETA-1A3 with 64Cu-BAT-2IT-1A3.

A simple method for conjugation of monoclonal antibodies (mAbs) with the chelating agent 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), has been developed using commercially available reagents. This method involved activation of a single carboxyl group of TETA with N-hydroxysulfosuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The resulting activated ester of TETA was reacted with the anti-colorectal carcinoma mAb 1A3 at molar ratios ranging from 10:1 to 100:1 to give immunoconjugates modified with an average of 0.4 to 2.0 functional chelators per antibody molecule. The TETA-1A3 conjugate was labeled with 64Cu at specific activities as high as 15.4 microCi/microgram, and the radiolabeled mAb exhibited high in vitro serum stability and minimal loss of immunoreactivity. The biodistribution of 64Cu-labeled TETA-1A3 in hamsters bearing GW39 human colon carcinoma xenografts was compared to that of 64Cu-BAT-2IT-1A3 (BAT = 6-(p-bromoacetamidobenzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8,11- tetraacetic acid; 2IT = 2-iminothiolane). Both conjugates showed high tumor uptake (6.60-9.05% injected dose/gram) from 24 to 48 h post-injection and generally similar blood clearance and non-target organ uptakes. Human absorbed dose estimates derived from the hamster biodistribution data showed the critical organs for both conjugates to be the large intestine and the red marrow. Our results suggest that the in vitro and in vivo performance characteristics of 64Cu-TETA-1A3 compare favorably with those of 64Cu-BAT-2IT-1A3 and that further evaluation of the diagnostic and therapeutic efficacy of 64Cu-TETA-1A3 is warranted.

Longitudinal stability of coagulation, fibrinolysis, and inflammation factors in stored plasma samples.

We sought to assess the longitudinal stability of risk factors for atherosclerosis and thrombosis. including several coagulation. fibrinolysis, and inflammation factors, in frozen plasma samples stored at -70 degrees C for months or years. We reviewed data collected on 29 different control pools over periods ranging from 7 to 59 months for two functional assays (factor VII and fibrinogen) and seven antigen measurements (C-reactive protein. D-dimer, plasmin-alpha2-antiplasmin complex, plasminogen activator inhibitor-1, protein C, protein S, and tissue plasminogen activator), totaling more than 15,000 data points. Screening of the data using least squares regression revealed only sporadic associations between monthly means and time, with no consistent trends. Analysis by repeated measures and summary measure methods revealed no evidence of sample degradation over time for the factors studied. Our finding of longitudinal stability in the biochemical properties of frozen plasma strengthens the presumption of sample stability on which molecular epidemiologic studies are based.

Clustering of procoagulation, inflammation, and fibrinolysis variables with metabolic factors in insulin resistance syndrome.

The known metabolic cardiovascular disease risk factors associated with insulin resistance syndrome (IRS) do not adequately explain the excess cardiovascular disease risk attributed to this syndrome, and abnormalities in hemostatic variables may contribute to this excess risk. Using data from 322 nondiabetic elderly men and women (aged 65-100 years) participating in the Cardiovascular Health Study during 1989-1990, the authors performed factor analysis on 10 metabolic risk factors associated with IRS and 11 procoagulation, inflammation, and fibrinolysis variables to examine the clustering of the metabolic and hemostatic risk markers. Factor analysis of the metabolic variables confirmed four uncorrelated factors: body mass, insulin/glucose, lipids, and blood pressure. Adding the hemostatic variables yielded three new factors interpreted as inflammation, vitamin K-dependent proteins, and procoagulant activity. Plasminogen activator inhibitor-1 clustered with the body mass factor, supporting the hypothesis that obesity is related to impaired fibrinolysis. Fibrinogen clustered with the inflammation summary factor rather than procoagulant activity, supporting the position that fibrinogen principally reflects underlying inflammation rather than procoagulant potential. The authors conclude that should hemostatic variables be shown to contribute to IRS-related cardiovascular disease, apart from plasminogen activator inhibitor-1, they may do so independently of the established metabolic abnormalities.

Effect of cholera toxin on rat liver lysosome acidification.

We have shown that cholera toxin and cAMP greatly increase both acidification rates of liver endosomes and the liver and endosome content of fluid-phase endocytosis probes. In this study lysosomes were purified from control and cholera toxin-treated livers that were pulsed with fluorescein conjugated dextran and chased overnight. Cholera toxin-treated livers weighed less, contained less protein and exhibited higher contents of lysosomal marker enzymes, consistent with the catabolic effects of this agent. By contrast to its effects on endosomes, cholera toxin had no consistent or significant effect on lysosome acidification rates, steady-state internal pH or potassium content, proton leak rates or fluorescein-dextran content. We conclude that cholera toxin and cAMP predominantly alter earlier steps of endocytosis but may also increase transfer of probes from lysosomes to bile.

Comparative dosimetry of copper-64 and yttrium-90-labeled somatostatin analogs in a tumor-bearing rat model.

90Y-DOTA-tyrosine3-octreotide (90Y-DOTA-Y3-OC) is currently being evaluated as a radiotherapy agent for trials in patients with somatostatin-receptor positive cancer. In this study, we compared the estimated absorbed doses to human organs, as well as to a CA20948 rat tumor, of 90Y- and 64Cu-labeled DOTA-Y3-OC and DOTA-Y3-octreotate (DOTA-Y3-TATE). Assuming that the radiopharmaceutical biodistributions are the same in rodents and humans, human absorbed dose estimates were obtained from rat biodistribution data. The absorbed doses of 90Y-DOTA-Y3-TATE were determined from the biodistribution of the 88Y-labeled peptide, with and without co-injection of a therapeutic amount of the 90Y-labeled peptide. Additionally, the absorbed doses of 90Y-DOTA-Y3-TATE were determined from data using two different biodistribution endpoints, 48 h and 168 h. Human absorbed dose estimates were calculated using MIRD methodology assuming that rats and humans have the same biodistribution. The biodistribution of the radiolabeled somatostatin analogs was dependent on the peptide and the radiometal. For 90Y-DOTA-Y3-TATE, the tumor dose was dependent on both the administration of therapeutic 90Y-peptide and the biodistribution endpoint. Our data suggested that, for both radionuclides, the TATE derivatives imparted a higher absorbed dose to the tumor than the OC analogs. 90Y-DOTA-Y3-OC and 64Cu-DOTA-Y3-OC were comparable with respect to their tumor-to-normal tissue dose ratios, while 90Y-DOTA-Y3-TATE appeared to have distinct advantages over 64Cu-DOTA-Y3-TATE.

Radiotherapy and dosimetry of 64Cu-TETA-Tyr3-octreotate in a somatostatin receptor-positive, tumor-bearing rat model.

64Cu [T1/2 = 12.8 h; beta+ = 0.655 MeV (19%); beta- = 0.573 MeV (40%)] has shown promise as a radioisotope for targeted radiotherapy. It has been demonstrated previously that the somatostatin analogue 64Cu-TETA-octreotide (64Cu-TETA-OC, where TETA is 1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid) significantly inhibited the growth of somatostatin receptor-positive CA20948 rat pancreatic tumors in Lewis rats (C. J. Anderson et al., J. Nucl. Med., 39: 1944-1951, 1998). In this study, we evaluated the radiotherapeutic efficacy of a new 64Cu-labeled somatostatin analogue, 64Cu-TETA-Tyr3-octreotate (64Cu-TETA-Y3-TATE), in CA20948 tumor-bearing rats. A single dose of 15 mCi (555 MBq) of 64Cu-TETA-Y3-TATE was shown to be more effective in reducing tumor burden than the same dose of 64Cu-TETA-OC. In multiple dose experiments, tumor-bearing rats were administered three doses of either 10 or 20 mCi (370 or 740 MBq) of 64Cu-TETA-Y3-TATE at 48-h intervals. Rats given 3x10 mCi (3x370 MBq) showed extended mean survival times compared with rats given a single dose; however, no complete regressions occurred. Complete regression of tumors was observed for all rats treated with 3x20 mCi (3x740 MBq), with no palpable tumors for approximately 10 days; moreover, the mean survival time of these rats was nearly twice that of controls. Toxicity was determined by physical appearance and hematological and enzyme analysis, which revealed no overt toxicity and only transient changes in blood and liver chemistry. Absorbed dose estimates showed the dose-limiting organ to be the kidneys. The radiotherapy results, along with absorbed dose estimates to target and clearance organs, confirm that 64Cu-labeled somatostatin analogues warrant continued consideration as agents for targeted radiotherapy.

A chimeric anti-CEA antibody with heavy interchain disulfide bonds deleted: molecular characterization and biodistributions in normal and tumor bearing mice.

We have deleted the interchain disulfide bonds in a chimeric anti-CEA antibody (chT84.66) by mutating two cysteines in the heavy chain to glycine residues. The resulting antibody delta SSchT84.66 was expressed in high yield in a bioreactor and purified to homogeneity in a single step on an anti-idiotypic antibody affinity column. The molecular size of the antibody was 150 kDa as judged by gel filtration, SDS gel electrophoresis under non-reducing conditions, and MALDI-TOF/MS. The 150 kDa antibody had nearly identical kinetic (Kon = 1.53 x 10(6) M-1 s-1, .koff = 1.14 x 10(-5) s-1) and affinity constants (Kaff = 1.34 x 10(11) M-1) compared to the parent murine (Kaff = 1.25 x 10(11) M-1) and chimeric (Kaff = 1.16 x 10(11) M-1) antibodies when tested on biosensor chips. When delta SSchT84.66 was conjugated to the isothiocynato derivative of DTPA, radiolabeled with 111In, and injected into either normal or nude mice bearing tumor xenografts, it gave nearly identical biodistributions to chT84.66. delta SSchT84.66 and chT84.66 antibodies gave a maximum tumor uptake of 48 and 74% ID/g, and tumor to blood ratios of 5.3 and 6.2 at 48 h, respectively. We conclude that delta SSchT84.66 irreversibly associates into H2L2 dimers after concentration, that the dimers are stable under both the in vitro and in vivo conditions used in this study, and the properties of the antibody are virtually indistinguishable from the parent chT84.66 antibody.