Regulation of B cell lymphomagenesis by a malignant Qal+ inducer T cell clone.

A series of Thy-1.2+ Ly-1+ Qa-1+ malignant T cell clones have been isolated from murine sarcoma virus-murine leukemia-Moloney (MSV-MuLV-M)-induced B cell lymphomas or from MSV-MuLV-M-infected B6 mice. These T cell clones enhance both antigen-independent and -dependent lymphocyte differentiation and function. They also induce the differentiation of granulocytes and erythrocytes in the stem cell compartment, a function that parallels the immunopathology of the disease in vivo. The malignant T cell appears to sustain B lymphoma growth in vivo by releasing a factor (BCGF) that promotes B cell proliferation.

Amplification of granulopoiesis by T cell subpopulations.

Regulation of granulopoiesis by activated splenic T lymphocyte subpopulations was investigated. The addition of syngeneic T cells activated by the mitogen concanavalin A or by mixed lymphocyte response (MLR) to normal bone marrow cultures stimulated granulopoiesis, while, in contrast, resting T cells did not. Functional analyses of Fluorescence Activated Cell Sorter (FACS) purified subpopulations of MLR activated T cells showed that both subsets enhanced granulocyte colony forming cells (CFUc) differentiation. Further studies using Ly 23+ T cells stimulated in vitro to suppress the primary generation of plaque forming cells (PFC) showed that these "suppressor cells" simultaneously enhanced CFUc differentiation. These results show that T cell "helper" function in hematopoietic regulation is not restricted to the inducer subset of T cells.

T-cell regulation of erythropoiesis during acute lymphoblastic leukemia.

How and where erythropoiesis is maintained during advanced leukemic disease is an important and, as yet, unresolved question in hematology. To address the potential role of T-lymphocytes as cells that regulate CFU-E differentiation during leukemogenesis, an experimental model of disease has been developed in inbred Balb/c mice. Specifically, three-week-old Balb/c By mice were injected with murine sarcoma virus-murine leukemia virus-Moloney (MSV-MuLV-M), which resulted 6-8 months later in the development of immunoblastic T-cell sarcomas with a leukemic phase. Splenic T cells from either normal or tumor-bearing mice were assessed for their relative ability to modulate erythroid differentiation. Quantitatively, T cells, Ly1 or Ly 2,3 T-cell subsets isolated from tumor-bearing animals significantly enhanced erythropoiesis when compared with comparable normal T-cell subsets. These data suggest that the compensatory shift of erythropoiesis from the bone marrow to the spleen observed during leukemogenesis was facilitated by splenic T cells. In this circumstance, the enhanced erythropoietic function may be mediated by splenic T cells, which are selectively activated by virus.

3-Quinolinecarboxamides. A series of novel orally-active antiherpetic agents.

A series of novel 3-quinolinecarboxamides that are structurally similar to the quinolone class of antibacterial agents possess excellent antiherpetic properties. By modifying the quinoline ring at the 1-, 2-, 3-, and 7-positions, analogues were identified that have up to 5-fold increased HSV-2 plaque-reduction potency relative to acyclovir. In a single-dose mouse model of infection, one of the most potent derivatives in vitro, 1-(4-fluorophenyl)-1,4-dihydro-4-oxo-7-(4-pyridinyl)-3-quinolinecarbo xamide (97), displayed comparable oral antiherpetic efficacy to acyclovir at 1/16 the dose; in a multiple-dose regimen, however, 97 was 2-fold less potent. In mice dosed orally with 97, sustained plasma drug levels were evident that may account for the high efficacy observed. The molecular mechanism of action of these agents is not known; however, based on in vitro studies with acyclovir resistant mutants, it is likely that the mechanism differs from that of acyclovir. In vitro plaque-reduction potency was not generally predictive of oral efficacy in mice. An X-ray crystal structure of 97 corroborated the assignment of structure and provided useful insights as to the effect of conformation on plaque-reduction potency.

Novel cAMP PDE III inhibitors: imidazo[4,5-b]pyridin-2(3H)-ones and thiazolo[4,5-b]pyridin-2(3H)-ones and their analogs.

The transformation of milrinone to 1,3-dihydro-5-methyl-6-(4-pyridinyl)-2H-imidazo[4,5-b]pyridin-2-one (13a), 5-methyl-6-(4-pyridinyl)thiazolo[4,5-b]pyridin-2(3H)-one (51), and 7-methyl-6-(4-pyridinyl)-1,8-naphthyridin-2(1H)-one (22) resulted in very potent cAMP PDE III inhibitors with in vitro activity in the nanomolar range. 1,3-Dihydro-2H-imidazo[4,5-b]pyridin-2-ones 13 were prepared from 2-aminopyridine-3-carboxylic acids (7, 10) via Curtius rearrangement. 1,8-Naphthyridin-2(1H)-one 22 and the corresponding 3,4-dihydro derivative 28 were prepared from 5-bromo-2-methyl[3,4'-bipyridin]-6-amine (21) and 5-bromo-2-methyl[3,4-bipyridin]-6(1H)-one (24), respectively, via Heck reaction. Thiazolo[4,5-b]pyridin-2(3H)-ones 35 were prepared from 6-bromo[3,4'-bipyridin]-6-amines 30 and 32 via a four-step sequence. Treatment of 6-amino-2-methyl[3,4'-bipyridine]-5-thiol (59) with ethyl bromoacetate and ethyl bromodifluoroacetate gave pyridothiazinones 60 and 61, respectively.

Novel and potent adenosine 3',5'-cyclic phosphate phosphodiesterase III inhibitors: thiazolo[4,5-b][1,6]naphthyridin-2-ones.

The transformation of 3-bromo-1,6-naphthyridin-2(1H)-ones 8 to thiazolo[4,5-b][1,6]naphthyridin-2(1H)-ones 12 resulted in a 2-9-fold increase in cAMP phosphodiesterase (PDE) III inhibitory potency. Unlike the secondary binding sites on the cAMP PDE III isozyme which interact with the methyl group of milrinone (2) and CI-930 (4), the site which interacts with the 5-substituents of 1,6-naphthyridin-2(1H)-ones and the 8-substituents of thiazolo[4,5-b][1,6]naphthyridin-2(1H)-ones 12 is able to accommodate a diverse group of substituents which have different steric and electronic requirements.

Synthesis and structure-activity relationships of 6-heterocyclic-substituted purines as inactivation modifiers of cardiac sodium channels.

Purine-based analogs of SDZ 211-500 (5) were prepared and evaluated as inactivation modifiers of guinea pig or human cardiac sodium (Na) channels expressed in Xenopus oocytes. Substances which remove or slow the Na channel inactivation process in cardiac tissue are anticipated to prolong the effective refractory period and increase inotropy and thus have potential utility as antiarrhythmic agents. Heterocyclic substitution at the 6-position of the purine ring resulted in compounds with increased Na activity and potency, with 5-membered heterocycles being optimal. Only minor modifications to the benzhydrylpiperazine side chain were tolerated. Selected compounds which delayed the inactivation of Na channels were found to increase refractoriness and contractility in a rabbit Langendorff heart model, consistent with the cellular mechanism. Activity in both the oocyte and rabbit heart assays was specific to the S enantiomers. Preliminary in vivo activity has been demonstrated following intravenous infusion. The most promising compound on the basis of in vitro data is the formylpyrrole (S)-74, which is 25-fold more potent than DPI 201-106 (1) in the human heart Na channel assay.

1,3,5-Trialkyl-2,4,6-triiodobenzenes: novel X-ray contrast agents for gastrointestinal imaging.

Examination of the gastrointestinal (GI) tract has been performed for decades using barium sulfate. Although this agent has many recognized limitations including extreme radiopacity, poor intrinsic affinity for the GI mucosa, and very high density, no alternative contrast agents have emerged which produce comparable or better contrast visualization. In fact, the various techniques of the GI radiologic examination (i.e., single contrast, double contrast, biphasic) were developed to compensate for its limitations. Each of these techniques requires complex patient manipulation to achieve adequate mucosal coating or compression to overcome the marked radiopacity of barium sulfate in order to obtain a diagnostically useful examination. A series of novel radiopaque oils, the 1,3, 5-trialkyl-2,4,6-triiodobenzenes, was designed to improve the efficacy, stability, and safety of barium formulations. These substances were prepared in two steps from 1,3,5-trichlorobenzene. Compound 17 (1,3,5-tri-n-hexyl-2,4,6-triiodobenzene), formulated as an oil-in-water emulsion, was found to be well-tolerated in rodents (mice, hamsters, rats) following acute oral and/or intraperitoneal administrations at 4 times the anticipated human clinical dose. No metabolism of 17 was detected in rat, hamster, dog, monkey, or human hepatic microsomes, suggesting the lack of oral toxicity was a consequence of poor absorption. In imaging experiments in dogs, emulsions of 17 have demonstrated excellent mucosal coating and improved radiodensity relative to barium sulfate suspensions. On the basis of the preliminary imaging and toxicity data, compound 17 was selected as a potential development candidate.

Time course of nodal enhancement with CT X-ray nanoparticle contrast agents: effect of particle size and chemical structure.

RATIONALE AND OBJECTIVES: Levels of CT enhancement in rabbit lymph nodes were followed with time after subcutaneous injection of four iodinated, insoluble nanoparticle contrast agents to provide experimental support for the hypothesis that clearance of these agents is related to the chemical structure of the agent itself. The impact of particle size was also studied. METHODS: Subcutaneous injections (2 x 0.25 mL) were made in the dorsum of rabbit paws with 15% suspensions of four nanoparticle contrast agents. Images were obtained at 4, 10, 24, 48, and 72 hours and 5, 7, and 14 days after injection. Average attenuation (in Hounsfield units [HU]), node volume, and total iodine uptake were estimated from the CT scans for each lymph node at each time point. RESULTS: All the agents provided adequate enhancement of both the popliteal and axillary lymph nodes of the rabbit (ie, > delta100 HU). Lymph node volume appears to be related to the persistence of enhancement, with long-lived agents demonstrating the greatest increase in size. The rate of clearance from the lymph nodes is related to the structure of the agent. CONCLUSIONS: Clearance of insoluble, iodinated nanoparticle contrast agents from lymph nodes can be modulated by changes in the structure of the agent itself. Using the same agent, smaller particles deliver material to the lymph nodes more quickly and clear more quickly.

Cardiovascular activity of WIN 65579, a novel inhibitor of cyclic GMP phosphodiesterase 5.

This study describes the phosphodiesterase inhibitory potency and cardiovascular actions of WIN 65579 (1-cyclopentyl-3-ethyl-6-(3-ethoxy-4-pyrridyl)-1H-pyrazolo[3,4-d]p yrimidin-4-one), a potent, new cGMP phosphodiesterase 5 inhibitor. WIN 65579 is a competitive inhibitor of phosphodiesterase 5, with IC50 values of 2-3 nM for phosphodiesterase 5 from human or canine vascular sources. WIN 65579 has low affinity for phosphodiesterases 1, 2 and 3 (IC50 > 3-10 microM), and is somewhat selective for phosphodiesterase 4 (IC50 approximately 100 nM). WIN 65579 is an endothelial-dependent relaxant of rat aortic smooth muscle (EC50 = 60 nM) and lowers mean arterial blood pressure in conscious spontaneous hypertensive rats following intravenous or oral dosing. WIN 65579 also increases plasma cGMP levels, and reinstates vascular responsiveness to nitroglycerin in conscious rats that are nitroglycerin-tolerant. These data show that WIN 65579 is one of the more potent phosphodiesterase 5 inhibitors, and that WIN 65579 possesses cardiovascular activities consistent with vascular phosphodiesterase 5 inhibition in vivo.

Pulmonary delivery of nanoparticles of insoluble, iodinated CT X-ray contrast agents to lung draining lymph nodes in dogs.

Lung cancer continues to be a leading cause of death around the world. Staging of this disease is critically dependent upon the involvement or noninvolvement of the lymph nodes which drain the region of lung containing the lesion/tumor. Palpation, unenhanced CT, and lymph node excision (i.e., mediastinectomy) are currently used to ascertain the status of these regional draining lymph nodes. The work reported herein details the first efforts toward the pulmonary instillation of iodinated nanoparticles for contrast-enhanced CT of lung draining lymph nodes. The data reflect the impact of dose, time post instillation, and formulation (surfactant) upon the observed CT enhancement of the tracheobronchial lymph nodes of beagle dogs. In addition, initial safety is discussed with both macroscopic and microscopic observations. The results indicate that pulmonary instillation of small volumes of iodinated nanoparticles could be successfully used to aid staging of lung cancer by CT imaging.

Percutaneous computed tomography lymphography in the rabbit by subcutaneously injected nanoparticulates.

RATIONALE AND OBJECTIVES: Surgical lymphangiography is infrequently used in staging cancer because of its inherent limitations. Radiopaque nanoparticulates target lymph nodes draining interstitial tissues and could make percutaneous lymphography feasible. METHODS: Experimental nanoparticulate contrast agent formulations were injected subcutaneously in the forepaw or hindpaw of normal rabbits or rabbits with induced reactive nodal hyperplasia. Axillary and popliteal nodes were imaged with thin-section computed tomography (CT) using quantitative methods to measure node enhancement. Dose-response (0.1-2.0 ml) and time course (4 hr to 10 weeks) of enhancement were assessed. RESULTS: Nodal enhancement above 100 Hounsfield units was consistently obtained. Enhancement was significantly related to dose and peaked at 10 hr with slow washout over the observation period. Nodes with reactive hyperplasia were larger and had heterogeneous enhancement patterns distinctly different from normal nodes. CONCLUSION: Percutaneous CT lymphography effectively depicts the macroscopic intranodal architecture in rabbits.

Time-lapse quantitative computed tomography lymphography: assessing lymphatic function in vivo.

RATIONALE AND OBJECTIVES: Immobility and massage produce different local limb lymph flow rates. We studied their influence on accumulation of radiopaque nanoparticulates in regional lymph nodes of normal rabbits. METHODS: Quantitative lymphography at 10-min intervals was used to follow the transport of subcutaneous (s.c.) nanoparticulates produced from insoluble esters of diatrizoic acid. In one design, both hindpaws received 0.5 ml of nanoparticulate s.c., and one hindpaw was massaged. In a second design, one hindpaw was injected and massaged while imaging the popliteal, presacral, and paraaortic nodes every 10 min. RESULTS: Gentle massage rapidly increased popliteal node accumulation in comparison with the immobile limb. On the massaged side, mean Hounsfield (HU) units, maximum Hounsfield units, and calculated iodine were significantly greater at 10 min and all subsequent times. In the node transfer experiments, it took 12, 30, and 45 min, respectively, to obtain 100-HU mean attenuation; 200-HU maximum attenuation thresholds were achieved at 20, 47, and 69 min, respectively. CONCLUSION: Quantitative computed tomography lymphography reflects local lymph physiology. Gentle massage of the s.c. injection site is a powerful lymphotropic stimulus.

Nanoparticulate computed tomography contrast agents for blood pool and liver-spleen imaging.

RATIONALE AND OBJECTIVES: We investigated the properties of a group of iodine-containing, insoluble compounds formulated as nanoparticles for use as potential blood pool and liver-spleen contrast agents. METHODS: High-resolution, quantitative computed tomography (CT) was performed prior to and at intervals following the intravenous administration of the contrast agents to rabbits. Time-density characteristics for three organs were evaluated. RESULTS: Excellent enhancement of blood (< or = 232 Hounsfield units [HU]), liver (< or = 263 HU), and spleen (< or = 350 HU) was achieved at the administered dose of 3.0 ml/kg. The composition of the agents influenced the biodistribution, as well as the residence time in blood, and time to peak enhancement in liver. CONCLUSION: Iodinated nanoparticulate compounds are promising CT contrast agents. Development of agents with desirable pharmacokinetic and biodistribution profiles may permit application-specific contrast enhancement.

Hepatic imaging with iodinated nanoparticles: a comparison with iohexol in rabbits.

RATIONALE AND OBJECTIVES: We evaluated the efficacy of a particulate computed tomography (CT) contrast agent in an animal model of focal liver disease. METHODS: Ethyl ester of diatrizoic acid (EEDA) is an iodinated (89 mg I/ml) nanoparticulate (200 nm) contrast agent intended for intravenous use that is currently undergoing preclinical testing in our laboratory. Focal liver abscesses were created in 11 New Zealand White rabbits. Iohexol and EEDA were administered to each animal on different days. CT scanning was performed at intervals following contrast agent administration. Liver and abscess enhancement were measured and compared. Dynamic imaging experiments in normal animals were also performed using both agents. RESULTS: EEDA resulted in significantly greater enhancement of the liver and liver-to-abscess contrast than did iohexol at all time points beyond 5 min at approximately 25% of the total iodine load. During dynamic imaging, liver and aortic enhancement were greater with EEDA than with iohexol, except during a 20- to 40-sec period immediately following contrast agent administration. CONCLUSION: EEDA is superior to iohexol for imaging liver abscesses. Our results suggest that liver-directed agents such as EEDA may prove to be more efficacious than currently available extracellular agents designed for liver CT scanning.

Interstitial MR and CT lymphography with Gd-dTPA-co-alpha, omega-diaminoPEG(1450) and Gd-dTPA-co-1,6-diaminohexane polymers: preliminary experience.

RATIONALE AND OBJECTIVES: The authors assessed the efficacy of two gadolinium-based polymers used as lymphotrophic contrast media for computed tomography (CT) and magnetic resonance (MR) imaging. MATERIALS AND METHODS: Two gadolinium-based polymers, gadolinium diethylenetriaminepentaacetic acid (DTPA)-co-1,6-diaminohexane (NC 22181) and Gd-DTPA-co-alpha, omega-diamino-polyethylene glycol(1450) (NC-66368), were formulated at a concentration of 80 mmol/L gadolinium. Doses of 0.1, 0.25, 1.0, or 2.0 mL per paw were administered subcutaneously into the hindpaws of normal rabbits. Spin-echo T1-weighted MR imaging (1.5 T) of rabbit popliteal and iliac nodes was performed before and immediately, 10 minutes, 2-3 hours, and 24 hours after injection. CT was performed 2-3 hours after injection of the high doses only. RESULTS: MR imaging revealed prompt enhancement of the popliteal nodes with both polymers at doses of 0.25 mL and above. For doses of 1.0 mL or less per paw, nodal percentage enhancement was maximal at 2 hours and then declined at 24 hours. At the highest doses, however, a reservoir of subcutaneous contrast material remained at the injection site and resulted in peak enhancement at 24 hours. At CT, popliteal node enhancement was faintly visible 2-3 hours after the administration of NC 22181. At lower doses, no enhancement was appreciable at CT. CONCLUSION: At 80 mmol/L formulations, the two gadolinium-based polymers provide excellent popliteal nodal enhancement on MR images. In addition, high doses of one polymer (NC 22181) were sufficiently concentrated in popliteal nodes to be visible on CT scans. Thus, this agent may be useful for both CT and MR lymphography.

Blood pool and liver enhancement in CT with liposomal lodixanol: comparison with lohexol.

RATIONALE AND OBJECTIVES: The authors compared the time course and blood pool and hepatic enhancement of three different doses of liposomal iodixanol with those of iohexol. MATERIALS AND METHODS: A liposomal iodixanol formulation was prepared with 200 mg of iodine per milliliter total and 80 mg of iodine per milliliter encapsulated. Twelve normal New Zealand white rabbits divided into four groups received 75-, 100-, or 150-mg encapsulated iodine per kilogram doses of liposomal iodixanol or 2 mL/kg iohexol with 300 mg of iodine per milliliter. A liver section was scanned with serial computed tomography (CT) before the injection, immediately afterward, and at 1-minute intervals for 10 minutes. Region-of-interest measurements of the aorta and liver were plotted at each time point, and contrast enhancement was plotted as a function of time and iodine dose. RESULTS: All liposomal iodixanol doses produced greater liver enhancement than iohexol. Results were significant (P < .05) for 100 mg and 150 mg iodine per kilogram dose groups at time points beyond 2 minutes. Peak hepatic enhancement (change in attenuation) was 54.9 HU +/- 7.6 with iohexol, compared with 59.6 HU +/- 6.1, 73.3 HU +/- 3.6, and 104.1 HU +/- 8.8 for 75, 100, and 150 mg encapsulated iodine per kilogram doses, respectively. Hepatic enhancement increased rapidly after injection of liposomal iodixanol, plateauing 2-3 minutes later. Blood pool enhancement decreased rapidly. Steady-state liver enhancement with liposomal iodixanol increased linearly with dose. Aortic enhancement was greater with iohexol. CONCLUSION: Liposomal iodixanol yielded greater hepatic enhancement at lower total iodine doses than iohexol. Although liver enhancement occurred rapidly after injection, blood pool enhancement was brief.

CT imaging of intrathoracic lymph nodes in dogs with bronchoscopically administered iodinated nanoparticles.

RATIONALE AND OBJECTIVES: The purpose of the study was to determine if airway instillation of iodinated nanoparticles results in contrast material enhancement of tracheobronchial lymph nodes in dogs. MATERIALS AND METHODS: Eight dogs underwent intrabronchial instillation of iodinated nanoparticles; six dogs received 900 mg each, and two dogs received 450 mg each. Spiral computed tomography (CT) was then performed 2-34 days later. RESULTS: CT scans obtained 2 days after instillation showed the presence of contrast material within the lung parenchyma but no nodal enhancement. Scans obtained 6-34 days after instillation showed enhancement of the right, left, and middle tracheobronchial lymph nodes (analogous to the mediastinal nodes in humans). Mean nodal attenuation on CT images was 117 HU +/- 43, and the mean nodal volume was 129 mm3 +/- 113. Histologic specimens of the nodes showed macrophage hyperplasia. CONCLUSION: Iodinated nanoparticles instilled into small airways are transported to the tracheobronchial lymph nodes, where they result in contrast enhancement.

Lymph node extraction of radiopaque nanoparticulates in the rabbit as measured in vivo with CT.

RATIONALE AND OBJECTIVES: The purpose of this study was to estimate in vivo extraction of lymphographic material in the popliteal node of the rabbit. MATERIALS AND METHODS: Serial quantitative computed tomography (CT) of target tissues in four legs of two rabbits was performed after subcutaneous injection of an improved lymphographic contrast agent. Massage was used as a lymphotrophic intervention. RESULTS: At 15 minutes, the mean change in Hounsfield units measured 815 in the popliteal node, 219 in afferent lymphatic vessels, and 127 in efferent lymphatic vessels. The nodal extraction of nanoparticulates from the lymph was approximately 55%. Nodal massage allowed the amount of nanoparticulate remaining in sinusoidal lymph to be estimated. CONCLUSION: Functional CT performed with timed studies, proper radiopaque materials, and physiologic interventions can depict in vivo lymphatic physiology under minimally invasive conditions.