Kinetics of murine gammaherpesvirus 68 gene expression following infection of murine cells in culture and in mice.

A model system to study the pathogenesis of gammaherpesvirus infections is the infection of mice with murine gammaherpesvirus 68 (MHV-68). To define the kinetics of infection, we developed an RNase protection assay to quantitate gene expression from lytic (K3, Rta, M8, DNA polymerase [DNA pol], and gB) and candidate latency (M2, M3, M9, M11, ORF73, and ORF74) genes. All candidate latency genes were expressed during lytic infection of 3T3 cells. Four kinetic classes of transcripts were observed following infection of 3T3 cells: immediate-early (K3, Rta, M8, and ORF73), early (DNA pol), early-late (M3, M11, and ORF74), and late (M2, M9, and gB). To assess the kinetics of viral gene expression in vivo, lungs, spleens, and mediastinal lymph nodes (MLN) were harvested from MHV-68-infected mice. All transcripts were expressed between 3 and 6 days postinfection (dpi) in the lungs. In the spleen, K3, M3, M8, and M9 transcripts were expressed between 10 and 16 dpi when latency is established. The K3, M3, M8, M9, and M11 transcripts were detected in the MLN from 2 through 16 dpi. This is the first demonstration of MHV-68 gene expression in the MLN. Importantly, our data showed that MHV-68 has different kinetics of gene expression at different sites of infection. Furthermore, we demonstrated that K3, a gene recently shown to encode a protein that downregulates major histocompatibility complex class I on the surface of cells, is expressed during latency, which argues for a role of K3 in immune evasion during latent infection.

Effect of nitroimidazole sensitizers on in vitro glycolytic metabolism of hypoxic squamous cell carcinoma.

Two nitroimidazole compounds, misonidazole (MISO) and nimorazole (NIMO), were evaluated for their potential to modify uptake of [5,6-3H] 2-fluoro-2-deoxy-D-glucose (3H-FDG) in the human squamous carcinoma cell line UT-SCC-5 exposed to increasing levels of hypoxia. UT-SCC-5 cells were incubated with 0-10 mM of MISO or NIMO under normal or reduced oxygen concentrations of 20%, 1.5%, or 0% with 5% CO2 for 6 h, after which 74 KBq of 3H-FDG was added in media for 1 h. In the presence of normal concentrations of O2, both sensitizers increased 3H-FDG uptake by up to 178% (MISO) or 84% (NIMO) when compared with untreated cells. In anoxia, MISO decreased 3H-FDG uptake to 35% of that of control whereas NIMO-treated cells showed a respective decrease in tracer uptake to 62%. Clonogenic assays clearly indicated that MISO was toxic and NIMO moderately toxic for hypoxic cells, whereas both sensitizers exerted only a very modest effect on survival of fully oxygenated cells. Our findings indicate that nitroimidazole treatment consistently increases 3H-FDG uptake into UT-SCC-5 cells under normal oxygen concentrations. In hypoxia, the observed decrease in tracer uptake is dependent on both the level of ambient oxygen and drug concentration and may reflect both direct toxicity and inhibition of glycolysis. The observations may be useful for further applications of 18F-FDG positron emission tomography (PET) to monitor effects of hypoxic cell radiosensitizers on tumor metabolism in vivo.

[Infection by Pseudomonas aeruginosa in patients with human immunodeficiency virus infection. Descriptive study of 35 cases]. / Infección por Pseudomonas aeruginosa en pacientes infectados por el virus de la inmunodeficiencia humana. Estudio descriptivo de 35 casos.

OBJECTIVE: Analysis of the spectrum of diseases attributed to Pseudomonas aeruginosa in patients with coinfection with the immunodeficiency virus (HIV). METHODS: Retrospective study of 35 cases of coinfection with P. aeruginosa-HIV, attended from 1985 until 1995. Analysis of putative factors implicated in mortality secondary to P. aeruginosa infection. RESULTS: The spectrum of infection due to P. aeruginosa was: ORL infection (4 cases), infection of upper respiratory tract (4 cases), pneumonia (21 cases), infected bronchiectasias (one case), endocarditis (2 cases) and primary bacteremia (3 cases). Most of these infections were community-acquired ones (30 cases [85.7%]). Degree of immunodepression was variable, with 12 cases (34.3%) affecting to patients with more than 200 CD4+lymphocytes x 10(-9)/l. Radiological pattern of pneumonias consisted in alveolar consolidation (18 cases [85.7%]), necrotizing pneumonia (2 cases [9.5%]) and interstitial pattern (one case [4.8%]). More than a 80% of isolates of P. aeruginosa was sensible to ceftazidime, ciprofloxacin, aminoglycosides, ureidopenicillins and imipenem. Recidives of the P. aeruginosa infection were detected in 7 cases (20%): 4 cases of ORL infection (100%) and 3 cases of lower respiratory tract infection (13.6%). Overall mortality was a 20% (7 cases), being directly attributed to P. aeruginosa infection in every one of the cases, all of them pneumonias. Secondary bacteremia was associated to a higher mortality (odds ratio [OR] 18.67; p = 0.0207). CONCLUSIONS: P. aeruginosa affect to the HIV-infected patients, independently of their immunodepression degree, affecting to different localizations. This bacteria continues to be sensible to conventional anti-Pseudmonas treatment. Pneumonia with secondary bacteremia is associated to a higher mortality.

Magnetically-enhanced radionuclide therapy (MERiT): in vitro evaluation.

PURPOSE: Radionuclide therapy is a promising method for delivering radiation dose selectively to tumors. In situations where electron -emitters are used and the tumor is small relative to the maximum range of therapeutic electrons, these particles exit the tumor before delivering the maximum amounts of radiation dose. In this study, the method of magnetically constraining electrons to small tumors, known as magnetically -enhanced radionuclide therapy (MERiT), is explored using in vitro experiments. METHODS AND MATERIALS: The potential utility of MERiT was investigated by first measuring the reduction of number of electrons exiting a small sphere containing 90Y embedded in a block of plastic scintillator. Measurements of total energy deposited in the plastic scintillator made inside and outside a 7 Tesla magnetic field were compared. Furthermore, an experiment utilizing lymphoma cells of human origin was performed. Groups of cells were added to wells containing 90Y-labeled bovine serum albumin (and control groups containing no radioactivity) were placed either inside a 7 Tesla magnet or at a position where the magnetic field was minimal (essentially zero) for 18 hr. RESULTS: The presence of a 7 Tesla magnetic field reduced the amount of energy deposited in the scintillator by 16.63 +/- 1.05%. This demonstrates that the magnetic field constrains a large fraction of the emissions to the sphere and implies that normal tissues adjacent to radiotracer-avid tumors can be protected from radiation dose. Results from the cell culture experiment showed that the presence of a 7 Tesla magnetic field significantly (p < 0.005) reduced the number of viable cells remaining after treatment with non-specific 90Y-labeled bovine serum albumin by 11.7% compared to the appropriate control group (90Y treated, not exposed to magnetic field). CONCLUSIONS: These initial physical and biological studies indicate that magnetically-enhanced radionuclide therapy can be effective in increasing radiation absorbed dose to small tumors, consequently reducing radiation dose to surrounding normal structures.

Effect of hyperglycemia on in vitro tumor uptake of tritiated FDG, thymidine, L-methionine and L-leucine.

UNLABELLED: We have previously demonstrated in vitro and in vivo that tumor uptake of FDG is markedly diminished by acute hyperglycemia. This in vitro study was designed to determine if tumor uptake of PET tracers (FDG, thymidine, L-methionine and L-leucine) is affected by acute or chronic hyperglycemia. METHODS: Human ovarian adenocarcinoma (HTB 77 IP3) cells were grown in media containing 100 or 300 mg/dl of glucose. At 7, 20, 38, 51 and 72 days after initial culture, uptake of 3H-labeled FDG, thymidine, L-methionine and L-leucine into the cells was determined in the presence of 100 or 300 mg/dl of glucose. RESULTS: With acute hyperglycemia (300 mg/dl of glucose), the percent decreases in uptake of FDG, thymidine, methionine and leucine were 76.7%, 22.4%, 7.4% and 11.1%, respectively, as compared to assay at 100 mg/dl of glucose (mean day 51 and day 72 data). Significant decreases were observed in FDG and thymidine uptake with acute hyperglycemia (p < 0.0005). When cells grown at 300 mg/dl of glucose for 51 and 72 days were assayed at 100 mg/dl of glucose, the mean percent decreases in uptake of these tracers were 10.4%, 7.8%, 8.0% and 16.8%, respectively, as compared to cells grown and assayed at 100 mg/dl of glucose. No significant decrease was observed in tumor uptake of these tracers, except for leucine (p < 0.05). CONCLUSION: These human adenocarcinoma cells do not significantly change FDG uptake with chronic hyperglycemia while acute hyperglycemia markedly reduces uptake of FDG and thymidine. Neither methionine nor leucine uptake is significantly affected by acute hyperglycemia. To optimally evaluate tumor biology by PET, the fasting state seems necessary for FDG and thymidine studies, while methionine or leucine appears more suitable for hyperglycemic patients.

Pleural effusion in patients infected with the human immunodeficiency virus.

In order to analyze the etiology, cytological and biochemical characteristics, and outcome of pleural disease in patients infected with HIV, the medical records of 86 HIV-positive patients with pleural effusion were reviewed. Controls were 106 HIV-negative patients with parapneumonic or tuberculous effusion. Most HIV-positive patients were intravenous drug abusers (95.3%). Pleural effusions in HIV-positive patients were caused by infections in 76 (89.4%) cases. Parapneumonic effusion was diagnosed in 59 patients and tuberculous pleuritis in 15 patients. Staphylococcus aureus was the most frequently isolated bacteria. Parameters for differentiating complicated cases of parapneumonic exudate from uncomplicated cases, such as pleural fluid pH < 7.20 (sensitivity 80% vs. 84.3%), pleural fluid glucose < 35 mg/dl (sensitivity 45% vs. 56.25%) pleural fluid LDH > 1600 UI/l (sensitivity 85% vs. 62.50%), showed similar sensitivity in HIV-positive and HIV-negative patients. Monocytes in pleural fluid were significantly decreased in tuberculous pleuritis in HIV-positive patients (506 +/- 425 vs. 1014 +/- 1196 monocytes/ml, p < 0.05). No significant differences were detected in the outcome of HIV-positive and HIV-negative patients with pleural disease. It can be concluded that the pleural effusion was of predominantly infectious etiology in HIV-positive patients from populations with a high prevalence of intravenous drug abuse. Neither the biochemical parameters in pleural fluid nor the outcome differed significantly between HIV-positive and HIV-negative patients.

Influence of hypoxia on tracer accumulation in squamous-cell carcinoma: in vitro evaluation for PET imaging.

Hypoxic accumulation of 2-[5,6-3H]fluoro-2-deoxy-D-glucose ([3H]FDG),L-[methyl-3H] methionine ([3H]MET), and L-[1-3H]leucine ([3H]LEU) was evaluated in two cell lines (UT-SCC-5 and UT-SCC-20) obtained from patients with squamous-cell carcinoma of the head and neck. Both cell lines were exposed to decreasing oxygen atmosphere (20%, 1.5%, or 0% O2) for 6 h, after which they were incubated for a further 1 h with tritiated FDG, MET, or LEU. An anoxic atmosphere resulted in a mean increase of [3H]FDG uptake of 120% and 46% over a baseline 20% oxygen atmosphere for UT-SCC-5 and UT-SCC-20A, respectively. Both total and acid-precipitable [3H]MET uptake remained unchanged at 0% versus baseline, whereas acid-precipitable [3H]LEU uptake decreased by 46% for UT-SCC-5 and by 34% for UT-SCC-20A at 0% O2. Our findings demonstrate that [3H]FDG accumulation is increased in hypoxic UT-SCC cell lines probably through activation of the metabolic steps associated with the glycolytic pathway. The decrease in acid-precipitable [3H]LEU uptake in hypoxia may indicate a decline in protein synthesis, whereas the unchanged [3H]MET uptake probably reflects the unaffected amino acid transport and slow incorporation of radiolabeled methyl group of MET in tumor proteins and nucleic acids. FDG and LEU, but probably not MET, warrant additional study as hypoxia-avid or hypoxia-reduced tracers for assessment of treatment effects designed to modify hypoxia.

Sequence comparisons of A/AA/6/60 influenza viruses: mutations which may contribute to attenuation.

Influenza virus infection is a worldwide public health threat. Cold-adaptation was used to develop a vaccine line (ca A/AA/6/60 H2N2) which promised to reduce the morbidity and mortality associated with influenza and to serve as a model for other live virus vaccines. This study establishes that two distinct lines of wt A/AA/6/60 viruses exist with different phenotypic and genotypic characteristics. The two virus lines have the same parent but different passage histories. The first line is both temperature sensitive (ts) and attenuated in ferrets and the second line (after multiple passages in chick kidney cells, eggs and mice) is non-ts and virulent in ferrets. Both lines of viruses have been further differentiated by sequence analysis. We have identified point mutations common to all virulent viruses but absent from the attenuated viruses. This was accomplished by comparing the nucleotide sequences of the six internal genes in three different attenuated passages of A/AA/6/60 with those of five different virulent passages of the same virus. The corresponding nucleotides of the attenuated viruses, therefore, represent candidate attenuating lesions: 6 in the basic polymerase genes (5 in PB1, 1 in PB2), 2 in the acidic polymerase gene (PA), 1 in the matrix (M) gene, 2 in the non-structural (NS) gene, and none in the nucleoprotein (NP) gene. Two of the 5 attenuating lesions in PB1 are silent; 1/2 in PA is silent; and 1/2 in NS is silent. Further changes which might be identified by comparing nucleotide and amino acid sequences of the A/AA/6/60 viruses with those of other influenza viruses may also contribute to the attenuation of the ca virus. Our study identifies nucleotides which more precisely define virulence for this virus and suggests that growth of the virus at low temperature may have preserved a non-virulent virus population rather than attenuating a virulent one.

Effects of hypoxia on the uptake of tritiated thymidine, L-leucine, L-methionine and FDG in cultured cancer cells.

UNLABELLED: We previously demonstrated in vitro that FDG uptake into viable cancer cells increases in the presence of hypoxic versus normoxic conditions. Since position-emitter labeled thymidine and amino acids are being used for PET, we evaluated uptake into tumor cells of several tracers (thymidine, L-leucine, L-methionine and FDG) in the presence of either normoxic or hypoxic atmospheres in vitro. METHODS: Uptake of tritiated thymidine, L-leucine, L-methionine and FDG into two human tumor cell lines (HTB 63 melanoma and HTB 77 IP3 ovarian carcinoma) was determined after a 4-hr exposure to each of three different oxygen atmospheres (0, 1.5 and 20% O2) in vitro. RESULTS: Under moderately hypoxic conditions (1.5% O2), thymidine uptake decreased significantly from the 20% O2 baseline for both melanoma and ovarian carcinoma cell lines (33% and 15%, respectively) and with anoxia, thymidine uptake declined from baseline by 43% and 21%, respectively. Leucine uptake decreased substantially in the melanoma cells, by 23% when exposed to 1.5% O2 and 36% in the presence of 0% O2, but only modestly or not at all in the IP3 cells. After 1.5% or 0% O2 exposure, methionine uptake was not significantly different from 20% O2 levels in either cell line. In contrast, FDG uptake in both cell lines increased significantly (23% and 38%, respectively) over normoxic (20% O2) conditions when cells were exposed to moderate hypoxia. FDG uptake also increased over basel conditions after anoxia, by 11% and 30% for melanoma and ovarian carcinoma cells, respectively. CONCLUSION: Hypoxia decreases cellular uptake of thymidine and increases FDG uptake in two different malignant human cell lines. Leucine uptake decreases with hypoxia in the melanoma cell line but not markedly in the IP3 cell line, while hypoxia does not alter methionine uptake in either cell line significantly. Hypoxia has varying effects on metabolic tracers used for PET. The use of paired hypoxia-sensitive PET tracers has potential for noninvasively characterizing tissue oxygenation levels.

Exposure to strong static magnetic field slows the growth of human cancer cells in vitro.

Proposals to enhance the amount of radiation dose delivered to small tumors with radioimmunotherapy by constraining emitted electrons with very strong homogeneous static magnetic fields has renewed interest in the cellular effects of prolonged exposures to such fields. Past investigations have not studied the effects on tumor cell growth of lengthy exposures to very high magnetic fields. Three malignant human cell lines, HTB 63 (melanoma), HTB 77 IP3 (ovarian carcinoma), and CCL 86 (lymphoma: Raji cells), were exposed to a 7 Tesla uniform static magnetic field for 64 hours. Following exposure, the number of viable cells in each group was determined. In addition, multicycle flow cytometry was performed on all cell lines, and pulsed-field electrophoresis was performed solely on Raji cells to investigate changes in cell cycle patterns and the possibility of DNA fragmentation induced by the magnetic field. A 64 h exposure to the magnetic field produced a reduction in viable cell number in each of the three cell lines. Reductions of 19.04 +/- 7.32%, 22.06 +/- 6.19%, and 40.68 +/- 8.31% were measured for the melanoma, ovarian carcinoma, and lymphoma cell lines, respectively, vs. control groups not exposed to the magnetic field. Multicycle flow cytometry revealed that the cell cycle was largely unaltered. Pulsed-field electrophoresis analysis revealed no increase in DNA breaks related to magnetic field exposure. In conclusion, prolonged exposure to a very strong magnetic field appeared to inhibit the growth of three human tumor cell lines in vitro. The mechanism underlying this effect has not, as yet, been identified, although alteration of cell growth cycle and gross fragmentation of DNA have been excluded as possible contributory factors. Future investigations of this phenomenon may have a significant impact on the future understanding and treatment of cancer.

Fluorodeoxyglucose uptake in human cancer cell lines is increased by hypoxia.

UNLABELLED: Malignant neoplasms commonly have increased rates of glucose utilization, poor perfusion and areas of low oxygenation. Autoradiographic studies of excised tumors have shown increased FDG uptake in viable cells near necrotic portions of tumor. We evaluated in vitro whether tumor cell FDG uptake increased with hypoxia. METHODS: The uptake of 3H-FDG into two human tumor cell lines (HTB 63 melanoma and HTB 77 IP3 ovarian carcinoma) was determined after exposure to differing oxygen atmospheres ranging from 0% to 20% O2 for varying time periods. Glucose transport was independently determined as well as estimates of the level of Glut-1 glucose transporter membrane protein. RESULTS: FDG uptake in both the melanoma and the ovarian carcinoma cell lines increased significantly (39.6% +/- 6.7% and 36.7% +/- 9%, respectively) over basal (20% O2) conditions when cells were exposed to a mild hypoxic environment (5% O2) for 1.5 hr. With a 4-hr exposure to 1.5% O2, the increase in FDG uptake was greater at 52.3% +/- 8.9% and 43.5% +/- 19%, respectively. With 4 hr of anoxia, the increase in FDG uptake over basal conditions was 42.7% +/- 10% and 63.3% +/- 13.7% for melanoma and ovarian carcinoma cells, respectively. Membrane transport of 3-O-methylglucose (3-OMG) was increased by hypoxia for melanoma and ovarian carcinoma. Immunochemical assays for Glut-1 showed an increase in the membrane expression of the Glut-1 transporter in cells exposed to hypoxia. CONCLUSION: Hypoxia increases cellular uptake of FDG in two different malignant human cell lines. Increased glucose transport, in part due to increased membrane expression of the Glut-1 glucose transporter, contributes to this phenomenon. Increased FDG uptake in tumors visualized during PET imaging may be partly reflective of tumor hypoxia.

In vitro comparison of cell proliferation kinetics and uptake of tritiated fluorodeoxyglucose and L-methionine in squamous-cell carcinoma of the head and neck.

UNLABELLED: Estimation of tumor proliferative activity is important for the optimal management of head and neck cancer. Noninvasive metabolic imaging with PET may complement currently used cytopathologic methods to study tumor proliferation. METHODS: Fluorodeoxyglucose (FDG) and L-methionine (MET) were studied for their potential to assess in vitro the growth characteristics of three squamous-cell carcinoma (SCC) cell lines established recently in patients with head and neck cancer. A time-course uptake of tritiated FDG and MET was measured over the complete growth cycle of one rapidly growing (UT-SCC-5) and two relatively slower growing (UT-SCC-1A and UT-SCC-9) cell lines. DNA flow cytometry was used for assessment of proliferative activity. RESULTS: There was a strong linear relationship for both FDG and MET uptake versus the viable cell number although absolute MET uptake was consistently lower than that of FDG in the exponential and plateau phases of growth (p < 0.01), leading MET to underestimate cell number. The pattern of MET uptake followed the flow cytometric changes in the proliferation index (% of S + G2/M cells) in two of three cases (UT-SCC-1A and UT-SCC-5) while a similar, although clearly weaker, relationship was seen with FDG and flow cytometry findings in only one case (UT-SCC-5). CONCLUSION: In these three human SCC cell lines assessed in vitro, FDG is a better marker of cell viability than MET, whereas MET is superior for estimating proliferative activity. Extrapolations of these in vitro data to the interpretation of PET images should be made with caution since tumors may have confounding factors that may affect in vivo tracer uptake.

A persistent infection in MDCK cells by an influenza type B virus.

A persistent infection in Madin Darby Canine Kidney (MDCK) cells by an influenza B virus (B/Tecumseh/63/80) has been established and characterized. Virus recovered from the persistent state titrated lower in relation to the parental wild-type (wt) that initiated the infection as measured by hemagglutination and egg and tissue culture infectious dose, suggesting that the virus is a less cytopathic variant of the original wt virus. The persistent virus (pv) has decreased cytopathology for both MDCK and primary chick kidney (PCK) cell lines, and exhibits different RNA and protein electrophoretic migrations. Plaques of the persistent virus are smaller and take longer to appear, indicating that the pv is a slower growing variant of the wt. The small plaque mutant phenotype may play a role in the maintenance of the persistent infection in MDCK cells. The pv differs from the wt antigenically and in its ability to form deposits of uric acid-like crystals beneath the culture monolayers.

In vitro assessment of 2-fluoro-2-deoxy-D-glucose, L-methionine and thymidine as agents to monitor the early response of a human adenocarcinoma cell line to radiotherapy.

The tumor cell uptake of three tracers that can be labeled with isotopes suitable for PET imaging--FDG, L-methionine and thymidine--were examined in vitro in a human ovarian carcinoma cell line (HTB77IP3) at varying times following 30 Gy 60Co irradiation and were compared to a nonirradiated control group. FDG, methionine and thymidine uptake per tissue culture well all increased following irradiation when compared to basal values, although to a much lower extent than the increases in uptake seen in a nonirradiated group. This increase in tracer uptake occurred despite a 6.25-fold decline in viable cell numbers. When examined per cell, FDG uptake per cell increased 9.77-fold, methionine 7.82-fold and thymidine 9.48-fold over basal levels from Day 0 to Day 12 following irradiation. Part of these increases may be due to giant cell formation and/or radiation repair processes that require energy, protein and DNA substrates. While the in vitro system differs from in vivo systems due to the absence of a blood supply in vitro, a lack of infiltrating leukocytes and other factors, our data suggest that early assessment of human adenocarcinoma response to radiotherapy by PET with these tracers may be complicated by this normal increase in tracer uptake postirradiation. Clearly, in this human cancer cell line, early radiation-induced cell death is not associated with an early decline in tumor cell uptake of FDG, methionine or thymidine.

Does FDG uptake measure proliferative activity of human cancer cells? In vitro comparison with DNA flow cytometry and tritiated thymidine uptake.

The relationship between 3H-2-fluoro-2-deoxy-D-glucose (FDG) uptake and the proliferative rate of a human ovarian adenocarcinoma cell line (HTB77IP3) was examined in vitro. HTB77IP3 cells were plated and allowed to grow through lag, exponential and plateau phases. Proliferative rate assessed by DNA flow cytometry and 3H-thymidine incorporation was highest in the lag phase and fell significantly as the cells progressed from the exponential through plateau phases. By DNA flow cytometry, the proliferation index (% of S+G2/M phase cells) fell from 65% to 23%. Thymidine uptake per cell also declined, by 82%, from lag to plateau phase. By contrast, 3H-FDG uptake per cell was largely unchanged as the cells progressed through the cell growth cycle. Total 3H-FDG uptake was strongly correlated with the number of viable cancer cells present (r = 0.957). Total thymidine uptake, however, substantially underestimated the number of viable cancer cells present. These in vitro differences in tracer uptake suggest that in this adenocarcinoma cell line, FDG measures a substantially different parameter (viable cell number) than thymidine (proliferative rate) and that these differences may result in disparate findings on PET imaging of cancers using these two tracers. Our data for this in vitro system indicate that FDG uptake does not relate to the proliferative activity of cancer cells. However, FDG uptake is strongly related to the number of viable tumor cells.

Quantitative autoradiographic evaluation of the influence of protein dose on monoclonal antibody distribution in human ovarian adenocarcinoma xenografts.

We studied the effect of monoclonal antibody protein dose on the uniformity of radioiodinated antibody distribution within tumor masses using quantitative autoradiography. Groups (n = 11-13/group) of athymic nude mice with subcutaneous HTB77 human ovarian carcinoma xenografts were injected intraperitoneally with an 125I-labeled anticarcinoma-associated antigen murine monoclonal antibody, 5G6.4 using a high or a low protein dose (500 micrograms or 5 micrograms). At 6 days post-injection the macroscopic and microscopic intratumoral biodistribution of radiolabeled antibody was determined. The degree of heterogeneity of the labeled antibody distribution within each tumor was quantified and expressed as the coefficient of variation (CV) of the activity levels in serial histological sections. Tumors from mice given the 500-micrograms protein doses had substantially lower CV values, 0.327 +/- 0.027, than did tumors from animals given 5-micrograms protein doses, 0.458 +/- 0.041, (P = 0.0078), indicating that the higher protein dose resulted in more homogeneous distribution of radioactivity in tumors than did the lower dose. While the percentage of the injected dose reaching the tumor was comparable between groups, injecting the higher dose of protein resulted in significantly lower tumor to non-tumor uptake ratios than those obtained for the lower protein dose. These data indicate, in this system, that to achieve more uniform intratumoral antibody (and radiation for radioimmunotherapy) delivery, a relatively high protein dose must be administered. However, to obtain this increased uniformity, a substantial drop in tumor/background uptake ratios was seen. Quantitative autoradiographic evaluation of human tumor xenografts is a useful method to assess the intratumoral distribution of antibodies.

Improved monoclonal antibody tumor/background ratios with exchange transfusions.

Blood exchange transfusions were performed in nude rats with subcutaneous HTB77 human ovarian carcinoma xenografts in an attempt to improve specific monoclonal antibody (MoAb) tumor/non-tumor uptake ratios. Animals were injected intravenously with both 131I-5G6.4 specific and 125I-UPC-10 non-specific MoAb. Twenty-four hours later 65-80% of the original blood was exchanged with normal heparinized rat blood and then these rodents were sacrificed. Exchange transfusion significantly (P less than 0.05) decreased normal tissue activities of 131I (except for muscle) by 63-85%, while tumor activity decreased only 5%. Tumor to background ratios increased from 0.1-0.8 to 2.3-6.3. Exchange transfusions substantially enhance tumor/normal tissue antibody uptake ratios and, along with plasmapheresis, may be useful in enhancing antibody localization in vivo, particularly for therapy.