Thymidine Infusion Research Articles

High-Dose Thymidine Infusions in Patients With Leukemia and Lymphoma

We have recently explored the cytokinetic effects of thymidine given by continuous infusion in an animal model and have demonstrated an arrest of cell cycle traverse in rapidly proliferating tissues. Similar biologic effects have been observed in clinical trials upon infusing thymidine at a dose rate of 75 g/sq m/day. Fifteen courses of at least 5 days in duration have been administered to 11 patients with either leukemia or lymphoma. Steady-state serum thymidine levels were achieved in the range of 1-2 mM and the serum half-life of thymidine was approximately 90 min upon completion of the infusion. The associated toxicity included myelosuppression, headache, anorexia, nausea, vomiting, and diarrhea. The antitumor effect was dependent on the disease being treated and the percentage of blasts in the peripheral blood. Three patients with AML and three patients with T-cell leukemia responded to the thymidine infusions with an abrupt decrease in peripheral blast count. In contrast, no response was observed in two patients with poorly differentiated lymphocytic lymphoma of B-cell origin in a leukemic phase. One patient with T-cell leukemia had a marked clinical response with resolution of hepatosplenomegaly and another patient with the same diagnosis had partial clearing of the bone marrow. The cytokinetic effect on the bone marrow or peripheral blasts was monitored by microfluorimetry and labeling with precursors to monitor DNA synthesis. These data indicate an arrest of DNA synthesis during the period of thymidine infusion. The clinical effects observed with the high-dose thymidine infusions are correlated with measurements of thymidine kinase and phosphorylase levels detected in the blasts obtained from the leukemic patients. The relative ratios of thymidine kinase to phosphorylase are predictive of cellular sensitivity to the cytokinetic effects of thymidine.

Passage of spermatozoa through the epididymis of the boar (Sus scrofa domesticus)

Previous studies of epididymal transit in the pig have had insufficient animal numbers to provide a comprehensive picture of a continuous process. The present study attempts to addresses this issue. Radioactively labeled thymidine was infused into the testicular arteries of 48 sexually rested young adult boars of the Goettingen Miniature Pig breed. Hemicastrations were performed in random order in 2 animals daily on Days 21–24 following thymidine injection and in 4 animals daily on Days 25–41 and 43, 45, 47 and 49. Sperm obtained from 12 epididymal sites between the proximal caput and distal cauda were autoradiographically examined to record the percentage of labeled sperm and labeling intensity at different times after thymidine infusion. An initial surge of labeled spermatozoa emerged in the proximal caput 28 days after thymidine injection. After 2 d labeled sperm had arrived at the distal caput and, after another 2, the bulk of labeled sperm was found in the corpus. From there the sperm advanced to the transition of corpus and cauda, where progress was arrested until Day11. On Day 12, transit was resumed and by Day 13 sperm had passed through the cauda and vacated the epididymis via the ductus deferens.

Does Multicolor Lineage Tracing of Endothelial Cells Provide a Black and White Answer on Clonal Expansion in Post-Natal Angiogenesis?

Postnatal angiogenesis occurs in physiological (ie, blood vessel growth in muscle in response to exercise and with cyclic changes within menstrual tissue) and pathological conditions (eg, diabetic retinopathy). Many attempts to promote postnatal angiogenesis have been attempted to drive revascularization of ischemic tissues. Surprisingly, we continue to lack fundamental information on which endothelial cells possess replicative potential in blood vessels. Prior work established that infusion of H3 thymidine into young mammals revealed endothelial cell proliferation in small and large vessels that is high at birth, varies within and among organ vasculature, and declines rapidly over the first few weeks of life. In adult animals, the rate of homeostatic endothelial cell turnover has been quantified as <1% in rodents. Despite these pieces of information, we do not know specifics about the individual proliferative capacities of endothelial cells, whether this activity is restricted to a small subset or is a feature ubiquitously present within all endothelial cells in a blood vessel.1 Article, see p 670 Manavski et al2 in this issue have used inducible endothelial cell–restricted multicolor reporter (Confetti) transgenic mice to interrogate the clonal expansion of individual endothelial cells within normally developing murine tissue vasculature or within tissues challenged by ischemia and hypoxia. This system has permitted quantification of all the endothelial cells within a fixed sampling volume of the tissue vasculature to detect an increase in local distribution of a singular color of endothelial cells within areas of clonal expansion compared with near equivalent levels of random color distribution in areas lacking clonal expansion. In brief, the authors failed to detect significant color enrichment (<10% clonal expansion) in the developing retinal vasculature at postnatal day 5, consistent with prior work in zebrafish that had identified random integration or cell mixing accounting for endothelial cell distributions in developmental …

A phase II trial of thymidine and carboplatin for recurrent malignant glioma: A North American Brain Tumor Consortium Study

This phase II study in recurrent high-grade glioma evaluated the response rate, toxicities, and time to treatment failure of high-dose carboplatin modulated by a 24-h infusion of thymidine (75 g/m(2)). The trial was based on preclinical data and a prior phase I study ( J. Clin. Oncol. 17, 2922-2931, 1999); a phase II recurrent high-grade glioma study was initiated in July of 1998. Thymidine was given over 24 h; carboplatin was given over 20 min at hour 20 of the thymidine infusion. The starting dose of carboplatin had a value of 7 for the area under the curve (AUC), with allowance for dose escalation of 1 AUC unit per cycle if grade 2 toxicity was observed. Treatment cycles were repeated every 4 weeks. Accrual as of September 1999 was 45 patients [4 were unevaluable]: 76% with glioblastoma multiforme (GBM), 20% with anaplastic oligodendroglioma, 2% with mixed type, and 2% with anaplastic astrocytoma. Most patients had prior chemotherapy (78%). As observed in the earlier phase I study (in which carboplatin pharmacokinetics were unaltered by thymidine or antiseizure medications), thymidine was myeloprotective, resulting in a minimal need for dose reduction for patients having a >2 grade toxicity (in only 4% of the courses of treatment). Of 101 total courses, the number of courses (at the AUCs) was 3 (5), 4 (6), 58 (7), 20 (8), 11 (9), and 5 (10). Grade 3 nonhematologic toxicities included headache (4%), altered consciousness (3%), fatigue (1%), and nausea (3%). Responses included 2 partial (1 oligodendroglioma, 1 GBM; 5%); 3 minor (1 anaplastic astrocytoma, 2 GBM; 7.3%); 6 stable disease (14.6%); and 30 progressive disease (73.2%). For GBM patients, median survival was 23 weeks (with a 95% confidence interval of 20 to 50 weeks), and progression-free survival was 8 weeks (with a 95% confidence interval of 7-16 weeks). These results in GBM were comparable to other phase II GBM trials and thus do not represent a therapeutic advance in the treatment of GBM. Taken collectively, however, results are consistent with continued investigation of thymidine in combination with chemotherapeutic agents for high-grade glioma and other malignant diseases. The significant myeloprotection afforded by thymidine may have particular relevance to polychemotherapeutic regimens.

Transport of alovudine (3′-fluorothymidine) into the brain and the cerebrospinal fluid of the rat, studied by microdialysis

Extracellular unbound concentrations of alovudine were sampled by microdialysis in order to study the transport of alovudine between the blood and the brain and the cerebrospinal fluid (CSF) in the rat. The AUC (area under the curve) ratio CSF/blood was higher than the brain/blood ratio after i.v. infusion of alovudine 25 mg/kg/hr after a loading dose of 25 mg/kg in 5 minutes (n = 4). Neither i.v. infusion of thymidine (25 mg/kg/hr, n = 5; 100 mg/kg/hr, n = 2) nor acetazolamide (50 mg/kg i.p. Bolus followed by 25 mg/kg i.p. every second hour, n = 3) influenced the brain/blood AUC ratio after alovudine 25 mg kg s.c. injection compared to controls (n = 5). Finally, perfusion through the microdialysis probe with thymidine (1000 μM, n = 3) had also no effect on the brain/blood AUC ratio after alovudine 25 mg kg s.c. Because neither thymidine nor acetazolamide has significant influence on the ability of alovudine to penetrate the blood-brain barrier in the rat, neither thymidine transport nor carboanhydrase dependent CSF production appear to be major determinants of the blood-brain concentration gradient. Thus, it is concluded that alovudine reaches the extracellular fluid of the brain not by cerebrospinal fluid, but via the cerebral capillaries and that the existence of a concentration gradient over both blood-brain and CSF-brain barrier can probably be explained by the presence of an active process pumping alovudine out from the brain.

Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells

Previous studies demonstrated that intermittent treatment with PTH increases osteoblast number and bone formation in growing and adult rats. The cellular mechanism for this increase in osteoblast number was investigated in 16-month-old female rats. Continuous [3H]thymidine infusion over a 1-week intermittent PTH [human PTH-(1-34)] treatment period was performed to determine the percentage of newly formed osteoblasts that originate from progenitor cells. To verify increases in bone formation, we performed histomorphometry and Northern blot analysis of selected bone matrix proteins. PTH treatment resulted in dramatic increases in fluorochrome-labeled perimeter (727%), osteoid perimeter (735%), osteoblast number (626%), and steady state mRNA levels of osteocalcin (946%) and type 1 collagen (> 1000%). Autoradiographic analysis of metaphyseal sections revealed no difference in the percentage of [3H]thymidine-labeled osteoblasts between PTH- and vehicle-treated groups (4.3 +/- 1.3% vs. 5.7 +/- 2.7%, respectively). Similar changes were observed in PTH-treated ovariectomized rats. As the PTH-induced increase in osteoblast number did not require proliferation of progenitor cells we carried out an additional experiment in adult ovariectomized rats to determine the onset of PTH action. Incorporation of [3H]proline in the distal femoral epiphysis of PTH-treated adult ovariectomized rats was increased within 24 h. We conclude that the rapid PTH-induced rise in bone formation did not require cell proliferation and was most likely due to activation of preexisting bone lining cells to osteoblasts.

Increased Proliferative Capacity of CD4 + and CD8 + T Lymphocytes from Mutant sph ha/sph ha Mice Is Associated with Increased IL-2 Receptor Expression

We previously discovered that mutant anemic mice (sph ha/sph ha) show increased numbers of cycling lymph node T lymphocytes when analyzed by pulse and continuous infusion of tritiated thymidine. We have now further analyzed this in vivo phenomenon by evaluating the in vitro proliferative response of anti-CD3 activated lymphocytes from anemic mice using flow cytometric cell cycle analysis with 5′-bromodeoxyuridine and Hoechst dye. We determined that sorted CD4 + and CD8 + T lymphocytes from anemic mice have significantly greater proliferative capacity when compared with syngeneic control (+/+) mice ( P < 0.001). In order to explain this increased growth capacity, we examined whether these cells exhibit differences in cell-surface phenotype (Pgp-1 and IL-2 receptor expression), activation state, or transmembrane signaling, or alterations in accessory cells or cytokines. Increased proliferation of T cells from anemic mice was associated with a larger percentage of T cells expressing IL-2R (p55 or CD25) at 24 and 48 hr after activation. Increased proliferative capacity was not associated with differences in activation state, Pgp-1 phenotype, transmembrane signaling, accessory cells, or cytokines. The mechanism for the abnormally high proliferative rate of T cells from anemic mice remains unclear, but we suggest that this mutant mouse may provide an important model for further studies on the molecular basis of T-cell replication.

Prevention of stenosis after vascular reconstruction: Pharmacologic control of intimal hyperplasia — A review

Prevention of stenosis after vascular reconstruction: Pharmacologic control of intimal hyperplasia — A review

Formation of 5-hydroxymethyl-2′-deoxyuridine in hepatic DNA of rats treated with γ-irradiation, diethylnitrosamine, 2-acetylaminofluorene or the peroxisome proliferator ciprofibrate

The peroxisome proliferator ciprofibrate was tested for its ability to induce DNA damage in the form of 5-hydroxymethyl-2'-deoxyuridine (HMdU), an adduct that results from the reaction of thymine in DNA with hydroxyl radicals. In order to quantify HMdU, DNA containing [3H]thymidine of high specific activity had to be obtained. Since hepatocytes normally have a very low rate of DNA synthesis, rats were subjected to partial hepatectomy to stimulate DNA synthesis and then were administered [methyl-3H]thymidine by three p.o., i.p. or i.v. injections 20, 22 and 24 h after partial hepatectomy; or by slow infusion through the portal vein, starting 20 h after partial hepatectomy for 4 h. The specific activity of DNA in rats receiving [3H]thymidine through the portal vein was considerably higher than in rats receiving p.o., i.p. or i.v. injections. Rats were then exposed to various doses of gamma-irradiation after partial hepatectomy and infusion of [6-3H]thymidine through the portal vein. DNA from the liver was extracted, enzymatically hydrolyzed and analyzed by HPLC. The percentage of HMdU in DNA increased in a dose-dependent manner. Rats were then treated with the carcinogens 2-acetylaminofluorene (AAF) or diethylnitrosamine (DEN) in conjunction with partial hepatectomy and infusion of [methyl-3H]thymidine. There was an increase in HMdU formation after a single administration of DEN or AAF. Another group of rats was fed a diet containing the peroxisome proliferator ciprofibrate for 3 weeks. After partial hepatectomy and infusion of [6-3H]thymidine, these rats were fed the same ciprofibrate-containing diet for 2-4 more weeks. HMdU was detected in DNA at 2-4 weeks after [6-3H]thymidine infusion, but the level at 4 weeks was nearly 50% less than at 2 weeks. This study shows that oxidative DNA damage in the form of HMdU is induced in the liver by gamma-irradiation, DEN, AAF and peroxisome proliferation.

Granulosa cell proliferation in very small follicles of cycling rats studied by long-term continuous tritiated-thymidine infusion.

It is generally believed that in adult rodents the vast majority of very small follicles (less than 20 granulosa cells in cross section) are not growing. However, the only data to support this assumption were collected with methods that may not be sufficiently sensitive to distinguish between nongrowing follicles and slowly growing follicles. In this study, the method of long-term continuous [3H]thymidine (3H-TdR) infusion was used to examine growth of very small follicles. Young adult rats were given continuous infusions of 3H-TdR for up to 7 days. Autoradiographs were prepared from 2-microns-thick sections of the ovaries. The proportion of labeled very small follicles increased as the duration of the infusion increased. After 7 days of continuous 3H-TdR infusion, 37% of follicles with 4 or fewer granulosa cells had at least one labeled granulosa cell, as did 68% of follicles with 5 8 granulosa cells, 86% of follicles with 9 12 granulosa cells, and 100% of follicles with 13 or more granulosa cells. The number of labeled cells per labeled follicle (the labeling index) also increased with increasing infusion time. However, few follicles had labeling indices of 100%, even after 7 days of continuous 3H-TdR infusion. These results suggest that a substantial fraction of very small follicles is growing in adult cycling rats; however, the rate of growth appears to be quite slow.

Maintenance of the normal rat alveolar macrophage cell population. The roles of monocyte influx and alveolar macrophage proliferation in situ.

We investigated the relative importance of monocyte influx and alveolar macrophage proliferation in maintenance of the alveolar macrophage population in normal rats. Two experimental approaches were used. To detect possible monocyte influx, we radiolabeled blood monocytes by a continuous infusion of tritiated thymidine for 7 days and assayed lavaged alveolar macrophages for radiolabel by autoradiography. Although heavily labeled monocytes occurred in the blood, no heavily labeled cells were detectable among lavaged alveolar macrophages by cohort analysis. To evaluate macrophage proliferation in vivo, we injected normal unlabeled rats with colchicine and examined lavaged alveolar macrophages for arrested mitoses. Mitotic figures were observed among lavaged alveolar macrophages, and calculations at serial times after colchicine injection indicated that 1.83% of alveolar macrophages entered mitosis each day. On the basis of these 2 lines of evidence, we conclude that the alveolar macrophage cell population in normal rats is supported mainly by cellular proliferation in situ rather than direct monocyte influx from the blood compartment.

Kinetics of cellular proliferation after arterial injury. IV. Heparin inhibits rat smooth muscle mitogenesis and migration.

Heparin inhibits the development of intimal thickening after carotid injury in the rat; however, the specific cellular events responsible for this effect have not been defined. In this study, smooth muscle cell growth fraction and migration into the intima were quantitatively measured in heparin-treated animals. All rats were subjected to left carotid balloon injury and received continuous intraperitoneal infusion of tritiated thymidine; they were given either heparin or lactated Ringer's solution intravenously for periods of time up to 7 days. Both smooth muscle cell growth fraction and migration of nondividing smooth muscle cells were markedly reduced in heparin-treated rats. If heparin was administered for only the first 3 days after carotid injury, both smooth muscle growth fraction and migration were reduced at 7 days; on the other hand, heparin had no effect on growth fraction and migration if given from day 4 to day 7. Finally, heparin given for a period of 1 week produced marked reduction in smooth muscle cell accumulation in injured arteries at 2 and 4 weeks. These results suggest that the effect of heparin on injury-induced intimal thickening might be due to inhibition of both smooth muscle cell entry into the growth fraction and migration of medial smooth muscle cells into the intima. These effects are long lasting, and are not reversed even if heparin is stopped after a short course of administration.

Cell proliferation and DNA repair in the liver during early stages of chemical carcinogenesis

X-chromosomal phosphoglycerate kinase mocaicisms in organ samples of female heterozygous mice provided a means to prove, because of a selective expression of one of the two allozymes, the clonal origin of carcinogen-induced preneoplastic hepatocellular populations (see also ref. 61). The occurrence of these clonal preneoplastic foci was used in rats to determine cell cycle dependent differences of transformation sensitivity and DNA repair. The highest transformation rate was found after carcinogen exposure in late G 1/early S phase of the cell cycle. Experimental disturbance of DNA precursor pools by continuous thymidine infusion during carcinogen exposure results in an increased formation of preneoplastic clones. This is a further argument in favor of an essential role of base-mispairing during initiation. Cell cycle dependent fluctuations of O 6-methylguanine DNA transferase with an increasing enzyme activity in late G 1 and a maximum in early S phase indicate that cells possess an increased potential for eliminating promutagenic O 6-methylguanine during the most transformation sensitive parts of the cell cycle to prevent base-mispairing during DNA replication or transcription. However, this putatively protective effect is limited because the enzyme is rapidly expended in the reaction and drops again in later stages of the cell cycle.

Influence of concomitant infusion of thymidine and inosine on methotrexate activity in normal and P388-bearing mice

Combinations of thymidine and inosine (ranging from 0 to 7.5 mg/hr) were co-administered during a 72-hr continuous i.v. infusion of 3 μg/hr methotrexate in normal and P388 solid tumor-bearing DBA/2 mice. Methotrexate alone was lethal to all normal mice. Inosine at 1.0–7.5 mg/hr could reverse toxicity up to 100% while thymidine at 0.5–7.5 mg/hr was less effective (⩽86% survival). Combinations of the nucleosides averted toxicity more effectively than either compound alone and in a synergistic manner. From P388 tumor-bearing mice 27% survived methotrexate and eventually died of tumor. Co-infusion of thymidine or inosine decreased the percentage of toxic deaths and caused an increase in life span of more than 50% as compared to untreated tumor-bearing mice. However, the diminishing effect on survival with thymidine and inosine doses above 0.5 mg/hr indicated loss of the antitumor effect of methotrexate. This was also observed with combinations of the nucleosides. The influence of thymidine on the antitumor effect of methotrexate was compared in L1210- and P388-bearing mice (both in ascites) with 13 μ g/hr methotrexate for 48 hr and 4 mg/hr thymidine for 96 hr. The increase in life span for L1210-bearing mice (8.6 days, 96%) was significantly longer than that with P388-bearing mice (6.1 days, 56%), probably due to biochemical differences between these tumors. It is concluded that co-administration of inosine and/or thymidine allows the use of methotrexate doses otherwise not tolerated, though with loss of anti-tumor effect. The choice of the tumor model may greatly influence the outcome of in-vivo studies on the modulation of methotrexate action by nucleosides.

Methotrexate-induced renal impairment: clinical studies and rescue from systemic toxicity with high-dose leucovorin and thymidine.

Four separate groups of patients have been studied: (1) The effect of high-dose methotrexate (MTX) administration on glomerular filtration rate was determined by pre- and posttreatment inulin and creatinine clearances in nine patients. Measurements were made prior to and 24-40 hr after drug administration. Inulin and creatinine clearances both decreased a mean of 43%. No signs of systemic toxicity occurred. (2) Three other patients given high-dose courses of MTX developed MTX toxicity. Their creatinine clearance decreased an average of 61%. (3) In a separate group of five patients undergoing weekly MTX treatment, comparison of serum MTX pharmacokinetics with and without alkalinization of the urine demonstrated no significant difference in peak serum MTX levels or serum MTX decay. (4) Eight additional patients with severe renal dysfunction secondary to MTX were treated with increased doses of leucovorin and a continuous infusion of thymidine (8 g/m2/day) once renal failure was recognized. When high-dose leucovorin and thymidine were begun 48-72 hr after the MTX infusion, severe toxicity in the form of leukopenia, thrombocytopenia, diffuse mucositis, stomatitis, or skin rash was averted. We concluded the following: (1) high-dose MTX causes a subclinical decrease in glomerular filtration rate with each administration, even in nontoxic courses; (2) alkalinization of the urine with sodium bicarbonate does not alter plasma MTX decay, while volume expansion (hydration) is maintained constant; and (3) rigorous monitoring of serum creatinine and serum MTX levels 24-48 hr after MTX administration allows for the institution of rescue measures, including leucovorin and thymidine, which will abort the systemic toxicity that accompanies MTX-induced renal failure.

Purine and pyrimidine activities in acute and chronic lymphocytic leukaemia: Relation to cellular proliferative status

The cellular levels of the purine catabolic enzymes adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) and those for the pyrimidine activities thymidine phosphorylase and thymidine kinase isozymes have been measured concurrently in peripheral blood nucleated cells of patients with acute lymphoblastic leukaemia, chronic lymphocytic or prolymphocytic leukaemia and correlated with the spontaneous tritiated thymidine uptake of the isolated cells. Highest ADA levels occurred in T-ALL cells but considerable overlap of individual activities occurred for non-T, non-B ALL, B-CLL and T-CLL cells. The levels of PNP showed no distinct discriminatory trend in cells of the lymphoid proliferative disorders examined. Thymidine phosphorylase activity was markedly reduced in T-ALL and T-CLL cells with a stepwise increase in the level of mean activities for non-T, non-B ALL, B-CLL and B-PLL cells to that of isolated normal peripheral blood lymphocytes. Spontaneous tritiated thymidine uptake of the abnormal lymphoid cells exhibited a correlation between cellular thymidine kinase isozyme 1 and elevated ADA levels. The use of ADA inhibitors together with thymidine infusion for the treatment of lymphoproliferative disorders is discussed.

Cells of the marginal zone of the spleen are lymphocytes derived from recirculating precursors.

The origin of the B cells of splenic marginal zones was studied using transfer experiments in rats depleted of marginal-zone cells. Cyclophosphamide given as a single dose of 500 mg/m2 was used to deplete the marginal zones. Approximately 90% depletion was still apparent 10 days after treatment. Fetal liver cells did not induce rapid repopulation of the marginal zone. Also bone marrow cells from rats depleted of recirculating lymphocytes were inefficient in this respect. Conversely, thoracic duct lymphocytes and bone marrow cells from normal rats were efficient at restoring marginal-zone cell numbers in cyclophosphamide-treated rats. Thoracic duct cells passaged through an irradiated intermediate host and collected from that host's thoracic duct were also efficient at achieving marginal-zone reconstitution. In rats receiving 1000 rd whole body irradiation, which were protected with fetal liver cell transfer, marginal zones did repopulate at about 3 weeks. It is concluded that marginal-zone B cells, after leaving primary lymphoid organs, enter the recirculating pool for a period of at least several days before settling in the marginal zone. The turnover rate of marginal-zone cells was assessed using tritiated thymidine infusion. Most marginal-zone cells were not labeled after 5 days continuous labeling suggesting that the marginal-zone B cells are not rapidly dividing.

The graft-versus-host reaction in the rabbit spleen. Ultrastructural evidence for two differentiated T-cell lines.

Lethally irradiated (900 rads), bone-marrow-protected rabbits were given allogeneic thymocytes intravenously. Autoradiography by means of local splenic infusion of (H3) thymidine revealed progeny of two differentiated T-cell lines. Electron microscopy confirmed these observations in detail. An analogy with cellular immunity reactions in an "isolated T-cell system", performed in draining lymph nodes after a variety of antigenic stimuli is presented. The functional properties of these T-blasts and their lymphoid descendants are discussed.

Technique for Preclinical Evaluation of Continuous Infusion Chemotherapy With the Use of WF Rat Acute Myelogenous&lt;xref ref-type="fn" rid="FN1"&gt;2&lt;/xref&gt;

A technique was developed for continuous iv infusion chemotherapy in an inbred rat model of acute myelogenous leukemia. Polyethylene tubing was inserted surgically into the internal jugular vein of adult WF rats, burrowed sc to the base of the tail, and connected to an infusion pump. A flexible spring was sutured at the base of the tail and fastened to the cage wall; it protected the infusion catheter and allowed movement of the rat within the cage. This technique was used to compare bolus with continuous infusion therapy with adriamycin, cytosine arabinoside, and neocarzinostatin. Only small differences were noted in host toxicity and in antitumor effect against tumor grown as a subcutaneous myeloblastoma. Nearly three times more neocarzinostatin was required by continuous infusion for an effect equivalent to that of bolus injection. In contrast, continuous infusion of methotrexate with concurrent thymidine infusion prevented toxicity, enhanced the antitumor effect, and prolonged survival. This infusion system should facilitate rapid preclinical evaluation of drugs considered for constant iv infusion therapy.

Maturation of bone marrow lymphocytes. II. Development of Fc and complement receptors and surface immunoglobulin studied by rosetting and radioautography.

Radioautographic DNA labeling and rosetting techniques were combined to study the development of surface IgM, Fc, and complement receptors (FcR, CR) on small lymphocyte populations in mouse bone marrow. [3H]thymidine was either infused continuously to label newly formed cells for periods up to 4 days, or injected daily, 21--35 days before use, to label a sample of long-lived cells. Bone marrow cells were incubated with sensitized sheep erythrocytes to detect surface IgM, FcR, and CR, respectively, and examined radioautographically after cytocentrifugation. During [3H]thymidine infusion, marrow small lymphocytes lacking surface markers were the first to show [3H]thymidine labeling. Most of these cells became labeled by 4 days (IgM--ve, 89%; FcR--ve, 92%; Cr--ve, 88%). Labeling of small lymphocytes bearing surface IgM, FcR, and Cr began after an initial lag and increased to high values by 4 days (IgM + ve, 73%; FcR + ve, 82%; CR + ve, 83%). Labeled IgM + ve small lymphocytes formed progressively larger rosettes as cell age increased. Some proliferating large lymphoid cells formed rosettes for IgM, FcR, and CR. Labeled long-lived small lymphocytes expressed surface IgM, FcR, and CR, the incidence of each receptor being uniformly high (38--43%) and the rosettes tending to be larger than those formed by newly formed lymphocytes. In double-surface marker studies, FcR and CR rosettes were formed by some IgM--ve small lymphocytes as well as IgM + ve cells in the marrow. After transfusion of marrow cells from donor mice infused with [3H]thymidine for 24 h, many labeled newly formed lymphocytes homed into the splenic red pulp of unlabeled syngeneic recipients. Subsequently, these cells showed a rapid increase in the incidence of rosettes for surface IgM, FcR, and CR, together with a progressive enlargement of each type of rosette. Although all the labeled small lymphocytes recovered from the spleen developed both surface IgM and FcR by 3 days, only approximately one-half developed CR. The results demonstrate that most of the small lymphocytes bearing FcR, CR, and surface IgM in mouse bone marrow are newly formed indigenous cells. Each receptor becomes detectable by rosetting soon after the small lymphocytes are first produced. The newly formed, marrow-derived small lymphocytes are able to continue their development of surface IgM, FcR, and CR after migrating into the spleen, consistent with a maturation of primary B lymphocytes. In addition, the data indicate the genesis in mouse marrow of a non-B lineage of lymphocytes (notably, IgM--ve FcR + ve cells.). A minority of small lymphocytes bearing IgM, FcR, and CR in mouse marrow are long-lived cells, presumptive recirculating immigrants, differing in receptor status from the newly formed cells. The results are discussed with regard to the heterogeneity of marrow lymphocytes and proposed models of primary B lymphocyte and null lymphocyte production.