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Objectives: The purpose of this study was to determine the relative risk of deep vein thrombosis (DVT) during pregnancy and puerperium in a setting that was not influenced by diagnostic suspicion and referral bias., Study Design: Three hundred ninety-three consecutive females with suspicion of a first episode of venous thrombosis were referred in the same way to diagnostic centers. Medical history was obtained before objective testing for deep vein thrombosis., Results: Seventy-six of the 393 females were diagnosed as having deep vein thrombosis; the others were used as control subjects. The odds ratios for pregnancy, cesarean section, and puerperium after adjustment for age, center, family history, and calendar time were, respectively, 5.7 (95% CI 2.5-12.9), 4.0 (95% CI 1.3-12.6), and 2.9 (95% CI 1.2-6.9)., Conclusion: In this case-control study, in which patients and control patients were subject to the same referral and diagnostic procedures, we found similar risk estimates for pregnancy and puerperium as in previous studies. Our findings show that previous studies were less affected by diagnostic suspicion and referral bias than presumed.

As part of an ongoing study to determine the basis for high prevalences of veno-occlusive disease, glomerulosclerosis, and chronic lymphoplasmacytic gastritis in cheetahs, a retrospective pathology survey of captive cheetahs in the Republic of South Africa (RSA) was conducted. The RSA population was selected because its genetic composition and captive management were similar to those of the cheetah population in U.S. zoos, in which these diseases are common. For this study, archived pathology materials at the University of Pretoria Faculty of Veterinary Sciences in Onderstepoort and the Faculty of Veterinary Science, MEDUNSA, from 69 cheetahs that died between 1975 and 1995 were reviewed, and prevalences of common lesions were compared with those in the U.S. population. Gastritis associated with Helicobacter-like organisms was the most prevalent disease, accounting for close to 40% of the mortalities, including several cheetahs < 3 yr old. Glomerulosclerosis and veno-occlusive disease also were major causes of mortality in RSA cheetahs. RSA cheetahs also had adrenal cortical hyperplasia, cardiac fibrosis, lymphocytic depletion of the spleen, systemic amyloidosis, and splenic myelolipomas. The presence in the captive RSA cheetah population of the same unusual diseases that are common in U.S. cheetahs suggests a species predilection to develop these diseases in captivity.

Objective: To determine the influence of insulin therapy on physical symptoms, emotional and general well-being, and treatment satisfaction in patients with type 2 diabetes., Research Design and Methods: A descriptive prospective 2-year cohort study was performed. The study population consisted of 272 eligible NIDDM patients of Dutch origin > or = 40 years of age who had a known diabetes duration > or = 3 months and who were treated with diet and/or oral hypoglycemic agents. Dependent variables in the logistic regression analysis were scores on the Type 2 Diabetes Symptom Checklist, the Profile of Mood States, and questions regarding general well-being and treatment satisfaction. Potential determinants under study were age, sex, known diabetes duration, insulin dose, duration of insulin therapy, comorbidity, baseline and change in metabolic parameters and cardiovascular risk factors., Results: A baseline and 2-year questionnaire were available for 157 patients (58%). During follow-up, 39 of them (24.8%) were treated with insulin. Initiation of insulin therapy was significantly associated with improved glycemic control (mean HbA1c 8.2 +/- 1.4 [SD] to 7.4 +/- 0.9%, P = 0.001) and weight gain (BMI 27.1 +/- 3.9 to 28.6 +/- 4.3 kg/m2, P = 0.000). Of all symptom and well-being scores, only feelings of emotional fatigue worsened significantly, although modestly (0.4-1.7 on a scale of 0.0-10.0, P = 0.02). Although diabetes management with insulin was experienced as more demanding (P = 0.04), treatment satisfaction scores were not adversely influenced (2.5-1.9, P = 0.39). High insulin doses were significantly and independently associated with high symptom scores (total score, hypoglycemic score) and with low mood (displeasure score, anger, tension, emotional fatigue) and perceived state of health., Conclusions: Initiation of insulin therapy in type 2 diabetes improves glycemic control effectively, has little influence on physical and psychological well-being dimensions, and does not affect treatment satisfaction.

In primary care it is difficult to treat the growing number of non-insulin-dependent diabetic (NIDDM) patients according to (inter)national guidelines. A prospective, controlled cohort study was designed to assess the intermediate term (2 years) effect of structured NIDDM care in general practice with and without 'diabetes service' support on glycaemic control, cardiovascular risk factors, general well-being and treatment satisfaction. The 'diabetes service', supervised by a diabetologist, included a patient registration system, consultation facilities of a dietitian and diabetes nurse educator, and protocolized blood glucose lowering therapy advice which included home blood glucose monitoring and insulin therapy. In the study group (SG; 22 general practices), 350 known NIDDM patients over 40 years of age (206 women; mean age 65.3 +/- SD 11.9; diabetes duration 5.9 +/- 5.4 years) were followed for 2 years. The control group (CG; 6 general practices) consisted of 68 patients (28 women; age 64.6 +/- 10.3; diabetes duration 6.3 +/- 6.4 years). Mean HbA1c (reference 4.3-6.1%) fell from 7.4 to 7.0% in SG and rose from 7.4 to 7.6% in CG during follow-up (p = 0.004). The percentage of patients with poor control (HbA1c > 8.5%) shifted from 21.4 to 11.7% in SG, but from 23.5 to 27.9% in CG (p = 0.008). Good control (HbA1c < 7.0%) was achieved in 54.3% (SG; at entry 43.4%) and 44.1% (CG; at entry 54.4%) (p = 0.013). Insulin therapy was started in 29.7% (SG) and 8.8% (CG) of the patients (p = 0.000) with low risk of severe hypoglycaemia (0.019/patient year). Mean levels of total and HDL-cholesterol (SG), triglycerides (SG) and diastolic blood pressure (SG + CG) and the percentage of smokers (SG) declined significantly, but the prevalence of these risk factors remained high. General well-being (SG) did not change during intensified therapy. Treatment satisfaction (SG) tended to improve. Implementation of structured care, including education and therapeutic advice, results in sustained good glycaemic control in the majority of NIDDM patients in primary care, with low risk of hypoglycaemia. Lowering cardiovascular risk requires more than reporting results and referral to guidelines.

The purpose of the study was to assess the prevalence of foot (pre-)ulcers and their determinants in type II diabetic patients in a primary health care setting. Six hundred and nine patients (246 men, mean age 64.8 (range, 40-94) years, diabetes duration, 4.3 (0-44.9) years) from 22 general practices attended a regional shared care project in Amsterdam. At first visit all patients were examined by a podiatrist. Amputations, active fool ulcers (Wagner stage 1 or 2) and pre-ulcers (Wagner stage 0, hard skin with or without macerating changes) were recorded in 0 (0%), 11 (1.8%) and 79 (12.9%) patients, respectively. In multivariate logistic regression analysis, after adjustment for age and gender, diabetes duration, cigarette smoking, peripheral vascular disease (assessed by calculating ankle/brachial index), sensory neuropathy (by Semmes-Weinstein monofilament 5.07), dry feet and severe hammer toes were independently and significantly associated (pre-)ulceration. In conclusion, one of every seven type II diabetic patients in primary health care has a foot (pre-)ulcer. Patients at risk for foot ulceration can be identified by inspection and the use of simple instruments.

The authors summarise the issues of concern in game ranching, and the challenges involved in maintaining and utilising wild animals. The more intensive farming of ostriches and crocodiles is discussed. An indication of regulatory controls on the movement and possession of wild animals is given. Institutions concerned with aspects of wildlife care, rehabilitation and research are described.

The role of the complement system in the pathogenesis of pulmonary leukostasis in myelocytic leukemia was studied in a rat model. Acute myelocytic leukemia was induced in six Brown-Norway rats, and complement levels were assayed during the course of the disease. Whole complement activity (CH50) and hemolytic activity of C1q, C3, and C4 decreased from day 16 after induction of the leukemia, when the rats developed pulmonary leukostasis. In addition, local complement activation was established in the lung vessels by immunofluorescence microscopy in advanced stages of pulmonary leukostasis. Finally, following systemic activation of the complement system by injection of cobra venom factor (CVF), leukemic rats (n = 6) died of pulmonary leukostasis 4.5 days earlier than did leukemic controls (n = 6). These findings suggest that, in acute myelocytic leukemia in Brown-Norway rats, pulmonary leukostasis is induced by activation of the complement system. This finding could lead to new modes of treatment for a life-threatening complication of leukemia.

In this study the hypothesis that inhibition of the de novo pathway results in stimulation of salvage pathway activity was tested. The key enzyme in the balance between these two pathways is ribonucleotide reductase (RR), which can be inhibited by hydroxyurea (HU). The metabolism of 1-beta-D-arabinofuranosylcytosine and 5-Aza-2 deoxycytidine (Aza-dC), which are activated via the salvage pathway, was evaluated in cells from Ara-C-sensitive and -resistant myelocytic leukemia cell line (BNML-Cl/0 and BNML-Cl/Ara-C). The combination of HU and Ara-C caused as much as a threefold increase of Ara-CTP; it significantly increased the incorporation of Ara-C into DNA and induced synergistic cytotoxicity, as evaluated in a colony assay. Even in the deoxycytidine (CdR) kinase-deficient Ara-C-resistant cell line, HU was partially able to restore sensitivity to Ara-C and Aza-dC. dCTP levels are reduced during the first 10 h after incubation with HU, but this effect vanishes at the time when phosphorylation is maximal. Increased CdR kinase activity in cell-free extracts could explain the enhanced synthetic salvage pathway activity, which is likely due to the fact that more enzyme is present (Vmax has increased by Km unchanged). RR inhibition combined with Ara-C might provide a means of eliminating leukemic cells with suboptimal anabolic salvage pathway activity, which otherwise survive Ara-C chemotherapy.

We studied the usefulness of the determination of plasma D-dimer levels (using an ELISA) in combination with non-invasive testing with impedance plethysmography (IPG) or real-time ultrasonography (US) for the diagnosis of deep-vein thrombosis (DVT), in outpatients with clinically suspected DVT. This combined approach was compared to serial non-invasive testing alone in these patients. The sensitivity of a positive D-dimer test (greater than 300 micrograms/l) for the presence of DVT was 100% (70/70 patients; 95% C.I.: 95-100%), whereas the specificity was 29% (69/239 patients; 95% C.I.: 23-34%). The proportion of patients in which a definitive decision about the presence or absence of DVT could be made on the day of referral, was calculated for both approaches. When applying the combined approach, in 42% of all referred patients the diagnosis of DVT could either be established or refuted on entry, as opposed to only 19% of patients using serial non-invasive testing alone. Also, the costs per DVT diagnosed were calculated for the two diagnostic approaches. For the diagnosis of DVT the costs using serial IPG were comparable to the costs using the combination of IPG and the D-dimer test. The same conclusion holds for the comparison of serial US with the combination of US and D-dimer testing. We conclude that for the diagnosis of DVT in symptomatic outpatients the combination of non-invasive testing with the D-dimer test might be preferred over serial non-invasive testing alone, although the safety of such an approach remains to be established in future management studies.

Clinical examination of a 75-day-old captive juvenile wild dog suffering from lassitude revealed pale mucous membranes, icterus, laboured respiration, a "water-hammer" pulse and splenomegaly. A peripheral blood smear containing numerous Babesia-infected erythrocytes confirmed the diagnosis of babesiosis. Treatment was unsuccessful and the animal died shortly after receiving a blood transfusion. The findings at necropsy were typical for acute babesiosis and included anaemia, icterus, splenomegaly and haemoglobinuria. In addition, marked atrophy of the thymus and lymph nodes was evident. Microscopic and electron microscopic examination of selected tissues disclosed high parasitaemia with vascular stasis and injury to both endothelial and parenchymal components. It is speculated that vaccination-induced immune incompetence predisposed to development of clinical babesiosis.

The pathogenesis of pulmonary leukostasis in leukemia was studied in a rat model by investigating the course of its development. Leukemia was induced by inoculating rats with leukemic cells. The earliest stage of leukostasis was found from day 14 onward, when leukemic cells appeared in the peripheral blood, and was characterized by accumulation of leukemic cells at the capillary level. Simultaneous with the increase of leukemic cell concentrations in the peripheral blood, accumulation in capillaries increased gradually over a period of several days. This was accompanied by increasing severity of tachypnea. Shortly before death, aggregates consisting almost solely of leukemic cells were found in medium-sized blood vessels. This stage was rapidly followed by the end-stage, characterized by complete obstruction of the lung vasculature--including the largest arteries and veins--by leukemic cell aggregates, giving rise to extensive hemorrhages and edema. The end-stage was considered to be the cause of death, which occurred 18-26 days after the inoculation. The histological and ultrastructural findings in this study suggest that besides the size and stiffness of individual leukemic cells, interactions not only between leukemic cells, but also between leukemic cells and the endothelium play a role in the pathogenesis of pulmonary leukostasis.

In this study we describe the establishment of a leukemic cell line (BNML-CL/ara-C), originating from the 1-beta-D-arabinofuranosylcytosine (ara-C)-resistant brown Norway rat myelocytic leukemia model (BNML/ara-C), that retains the in vivo generated ara-C resistance. Its biological and biochemical characteristics have been compared with a cell line, derived from the ara-C-sensitive BNML model (BNML-CL/O). Resistance to ara-C was attributed to a decrease in phosphorylation of ara-C. Deoxycytidine (dCyd) kinase activity in crude cell extracts with dCyd as substrate showed similar enzyme activities in both cell lines, whereas with ara-C as substrate no dCyd kinase activity was detectable in the ara-C-resistant cell line. Two isoenzymes of dCyd kinase with different substrate specificities have been described (Cheng, Y.C., Domin, B., and Lee, L.S. Biochim. Biophys. Acta, 481: 481-492, 1977), cytoplasmic (dCyd kinase I, substrates: dCyd and ara-C) and mitochondrial (dCyd kinase II, substrates: dCyd and thymidine). In the ara-C-sensitive BNML model, thymidine induced a reduction of dCyd kinase activity when dCyd was used as substrate. However, thymidine did not affect kinase activity with ara-C was used as substrate. In the BNML-CL/ara-C, thymidine even induces a dCyd kinase inhibition of 85% with dCyd as substrate. It is likely that the ara-C-specific dCyd kinase deficiency in BNML-CL/ara-C cells was due to a selective loss of dCyd kinase I, whereas dCyd kinase II activity remained intact.

In this study 10 patients with acute myelocytic leukemia (AML) each received a rapid intravenous injection of high dose cytosine arabinoside (HD Ara-C; 1 g/m2). Bone marrow aspirates were obtained before and after Ara-C administration to determine the percentage of cells in S-phase measured by flow cytometry. In 5 out of 10 cases synchronization of the leukemic cells in S-phase of the cell cycle was observed. However, the time of maximum synchronization turned out to be difficult to predict. Therefore, the strong correlation between percentage of cells in S-phase at diagnosis and the time of maximal accumulation of S-phase cells after Ara-C administration, as observed by others in childhood AML, could not be confirmed for adult AML patients. Although synchronization of AML cells after in vivo Ara-C administration could be demonstrated in at least half of the patients, the practical consequences are such that clinical application was hampered.

In this study we investigated the Ara-CTP-forming capacity of leukemic cells in different phases of the cell cycle. Cells from two leukemic cell lines and leukemic bone marrow cells from patients and rats (BNML model) with acute myelocytic leukemia were separated according to cell cycle phase by means of an albumin density gradient in a specially designed sedimentation chamber. We found that the activity of CdR kinase and Cyt deaminase is much less influenced by cell-cycle phase progression than TdR kinase activity. For the leukemic cell lines HL-60 and BNML-CL/O CdR kinase activity is even independent of cell-cycle phase. In addition, Ara-CTP formation is not restricted to cells in S-phase. Cell cycle phase-independent Ara-CTP formation creates a situation in which cells which are not in S-phase during exposure to Ara-C might undergo the cytotoxic effects of Ara-C as soon as they enter S-phase.

The assays for the detection of unlabeled 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside, Ara-C) incorporation into DNA was simplified. The procedure includes DNA isolation from leukemic cells, quantification of DNA concentrations, breakdown by enzymatic digestion of DNA to nucleosides and a radioimmunoassay (RIA) using an antibody against Ara-C. Different techniques for quantification of DNA concentrations are compared. A fluorimetric technique using Hoechst 33258 is preferred because it is the most specific method. Comparison of this RIA assay with measurement of [3H]-Ara-C/DNA formation under similar conditions in HL-60 cells showed a correlation of 0.99. Ara-C incorporation into DNA of leukemic cells was studied using two rat-leukemia cell lines, one of which is sensitive to Ara-C and the other is an Ara-C-resistant wild type: BNML-Cl/0 and BNML-Cl/Ara-C, respectively. The results showed that Ara-C is incorporated when the cells are incubated at concentrations equal to or higher than the Ara-C concentration that induces 50% growth inhibition after 48 h incubation (IC50). This implies that at lower Ara-C concentration, i.e. levels that do not induce cytotoxicity, Ara-C is not incorporated into DNA. Similar results were obtained with human HL-60 myeloid leukemia cells. The detection limit of this assay is 2 pmol/ml Ara-C; therefore, the assay is more sensitive than measurement of Ara-C triphosphate (Ara-CTP), the only metabolite that can be measured in leukemic cells from patients after in vivo Ara-C administration. On the basis of in vitro studies, the finding of detectable Ara-C/DNA levels in vivo is expected to correlate with cytotoxicity; whether or not the Ara-C/DNA level itself is informative remains to be evaluated.

A pilot study was undertaken to determine the efficacy of remission induction therapy with 1000 mg/m2 high-dose cytosine-arabinoside (one i.v. push injection every 24 h, days 1-12), 65 mg/m2 6-thioguanine (orally, days 1-12), and 40 mg/m2 doxorubicin (i.v., days 13 and 14) without maintenance or consolidation therapy as a possible cure of childhood acute nonlymphocytic leukemia. Children in first remission with a suitable donor were offered the opportunity of allogeneic bone marrow transplantation (BMT). Between March 1980 and June 1981, 24 of 27 consecutive, newly diagnosed children entered the pilot study. Complete remission was achieved in 15 (71.4%) of 21 treated patients. Eleven children received no further therapy: 9 relapsed within 5 months (all hematologically), 2 have been in longstanding continuous complete remission (CCR) for 70+ and 77+ months. Four children underwent allogeneic BMT: 2 have been in CCR for 68+ and 75+ months, 1 child died of graft-versus-host disease, and 1 child relapsed 60 months after BMT. Although the remission rate (71.4%) was satisfactory, only a minority (18%) of the patients who received no further therapy seem to be cured.

The in vivo development of an ara-C-resistant leukemic cell line is reported in a rat leukemia model (BNML) that is generally accepted as a relevant model for human acute myelocytic leukemia. It took 32 continuous leukemia transplant generations, performed over 20 months, and a total dose of 28.5 g ara-C/kg to induce complete resistance. Preliminary data indicate that the development of ara-C resistance is related with decreased intracellular levels of deoxycytidine kinase. Deoxycytidine deaminase levels were not increased. Thus this enzyme does not seem to be involved with induction of resistance. This preclinical rat model for human AML provides a solid basis for studies in depth on the mechanism(s) and possible prevention and effective treatment of resistance to ara-C.

For 14 patients with acute leukemia flow cytometry was used to determine the percentage of cells in S-phase flushed out of a bone marrow biopsy compared with the percentage in a bone marrow aspirate; there was a statistically significant difference (p less than 0.01) between the two. The percentage dilution of the bone marrow aspirate by peripheral blood was then calculated, according to Holdrinet et al. [1], in order to correct the percentage S-phase cells in the aspirate. The data presented show that when the percentage S-phase cells in the aspirate is corrected for blood dilution, it closely approaches the percentage S-phase cells in the biopsy (p greater than 0.10).

Seventy-two adults were treated for acute myelogenous leukaemia (AML). Forty-two had previously untreated AML and 30 had AML after a preleukaemic phase, refractory AML or relapsed AML. The previously untreated patients received a 7-day course of cytosine arabinoside (100 or 200 mg/m2 daily), daunorubicin and vincristine while the remaining patients received a 7-day course of cytosine-arabinoside (1 g/m2 q 12h for 6 days) and amsacrine (on day 7). The percentage of malignant cells and the reduction in the percentage of malignant cells were determined by means of bone marrow aspirates taken on day 6 of the chemotherapy course and at the time of diagnosis. Both variables correlated significantly with the ultimate treatment outcome; the reduction in the percentage of malignant cells correlated even more significantly than the absolute percentage malignant cells in the day-6 bone marrow. By means of multiple regression analysis it became possible to calculate the probability of achieving complete remission for the individual patient; this is given by the equation: probability = 1.9-0.009X (% malignant cell reduction). In addition, the mean percentage of malignant cells in the day-6 bone marrow was significantly higher for patients who failed to achieve than those who entered complete remission. Eighty-six per cent of the patients with less than 20% malignant cells on day 6 entered remission, while 75% of the patients with more than 21% malignant cells failed to achieve complete remission (p less than 0.001). Although all of these calculations support the predictive value of the day-6 bone marrow aspirate, the 95% confidence intervals are too large to allow reliable and safe predictions; therefore more patients must be studied to demonstrate the reliability of this test.

In this study, it has been shown that in 21 patients with AML the dCyd kinase and dCyd deaminase activities correspond closely to the clinical response to ara-C remission induction therapy. Patients with primary disease were treated with a conventional-dose ara-C regimen whereas nonresponders and relapsed patients followed an ID ara-C regimen (1 g/m2 X 12). Of these 21 patients (11 with primary disease and ten relapsed), seven had ara-C resistant disease (three primary and four relapsed patients). Five of the seven patients had a very low dCyd kinase and normal dCyd deaminase activity, whereas the other two had a normal dCyd kinase and an increased dCyd deaminase activity.

Increased dosages of cytosine arabinoside (Ara-C) have been shown to be active in remission induction and consolidation treatment of patients with primary refractory acute lymphoblastic leukemia (ALL) and lymphoblastic non-Hodgkin's lymphoma (lyNHL). From August 1983 to December 1986 we treated 25 patients with ALL (9) and lyNHL, stage III and IV (16), median age 22 (range 15-48 yr) with a protocol consisting of remission induction with vincristine, prednisone, adriamycine and Ara-C (1 g/m2 twice daily as 2-h infusion d1-6) and intrathecal methotrexate, followed by consolidation courses with vincristine, prednisone, adriamycine and Ara-C (3 g/m2, twice daily as 2-h infusion d1-4) and intrathecal methotrexate. Some patients received CNS and/or mediastinal irradiation. No maintenance was given. 18 patients (72%) achieved complete remission (5 of the 11 previously treated and 13 of the 14 previously untreated patients). Consolidation courses were given to 17 patients. 5 of them relapsed in the bone marrow (3), skin (1) and CNS plus bone marrow (1) at 5, 5, 6, 6 and 24 months. The duration of complete remission ranged from 5 to 51+ months; the median could not yet be calculated. Short-term intensive treatment might be a worthwhile approach for ALL and lyNHL.

High-dose Ara-C consolidation therapy for patients with primary refractory or relapsed acute leukemia (AML and ALL) or relapsed lymphoblastic non-Hodgkin's lymphoma (LNHL) was investigated. Between January 1983 and January 1986, 47 adult patients with primary refractory or relapsed AML, ALL or lymphoblastic NHL received a remission induction regimen that included intermediate-dose Ara-C (lg/m2/2hr q 12hr X 12). Of the twenty-nine (61.7%) patients who achieved complete remission sixteen (AML 9, ALL 5, LNHL 2) received 1-3 consolidation courses that included high-dose Ara-C (3g/m2/2hr q 12hr X 8). Three patients died as a result of major infections during the pancytopenic phase that followed the first consolidation course and 6 relapsed at 4, 4, 6, 8, 9 and 16 months; at the moment of this report the remaining 7 patients have been in continued remission for 8 to 28 months (6 have been in continued complete remission for greater than or equal to 11 months). The predicted median disease-free interval for patients who survived consolidation therapy is 16 months. Of the 13 patients who did not undergo consolidation chemotherapy 2 subsequently underwent allogeneic bone marrow transplantation and 3 died as a result of major infectious complications while in complete remission. Eight patients received no further treatment because they refused or had previously experienced severe toxicity. The median disease-free interval for this group was only 3 months. Our preliminary data on brief intensive consolidation therapy for patients with relapsed or primary refractory leukemia or non-Hodgkin's lymphoma suggest that this kind of treatment prolongs disease-free interval.

The cell cycle perturbation effect on leukemic cells of the Brown Norway myelocytic leukemia (BNML) after high dose Ara-C injection was used as the rationale for chemotherapy studies. A one log leukemic cell load reduction as determined by means of the leukemic colony forming units-spleen (LCFU-S) assay was observed when the second Ara-C injection was administered during a period of induced accumulation of cells in S phase. evidence was obtained that Ara-C was also cytotoxic for G1 phase cells. By comparing a continuous Ara-C infusion during 24 h with two Ara-C injections at the same total dosage given 12 h apart, it was found that the tumor load was significantly more reduced in the latter group. For the drug combination Ara-C/Adriamycin, maximal LCFU-S reduction of one log was observed when Adriamycin (7.7 mg/kg) was given 12 h after Ara-C. From the increase in survival time after the schedule of 6 X Ara-C plus 1 X Adriamycin (all injections given at 12 h intervals), it could be extrapolated that the tumor load was reduced by 6.7 logs; this was in agreement with the theoretically expected reduction of 6.2 logs based on the LCFU-S experiments. The schedule of 6 X Ara-C plus 1 X Adriamycin reduced the normal haemopoietic stem cell compartment by 2.5 logs. This therapeutic gain is attributed to the specific recruitment-synchronization inducing effect of Ara-C on leukemic cells in the G0 phase.

One injection of 1-beta-D-arabinofuranosylcytosine (ara-C) in BN rats bearing myelocytic leukemia induces recruitment and synchronization of the leukemic cells. A second ara-C injection, given when the largest fraction of cells is in S phase, causes the largest reduction in leukemic clonogenic cells. The relevance of recruitment and synchronization of leukemic cells after high dose ara-C (200 mg/kg) by rapid i.v. injection (comparable with 1 g/m2 in patients) has been tested in rats with respect to survival time and toxicity. Several groups of leukemic rats have been treated with seven injections of ara-C; the intervals between the injections per group were 4, 6, 9, 12, 15, 18, and 24 h, respectively. The longest mean survival time is observed in the group treated every 9 h which is 70.8 days compared to 22.6 days in nontreated leukemic controls. This 9-h interval of ara-C administration corresponds with the moment when DNA synthesis of the leukemic cells resumes after its inhibition by the ara-C. The most severe toxic side effects on the gastrointestinal system are observed in the group that received ara-C every 6 h; no toxic death has occurred in the animals treated with 15-h or longer intervals. The effect of the increasing interval between two ara-C injections on the normal hematopoietic stem cells has been measured with the colony forming unit spleen assay. This study showed that the reduction of normal stem cells due to ara-C is independent of the interval of administration. This differential effect of ara-C on leukemic and normal hematopoietic stem cell kinetics might in part explain the mechanisms of achieving a complete remission in acute leukemia.

50 patients (aged 18-58 years) with acute myelogenous leukaemia after a preleukaemic phase (n = 14), acute myelogenous leukaemia that previously failed to respond to conventional chemotherapy (n = 9) or relapsed disease (n = 27) were given remission induction therapy consisting of cytosine arabinoside (1 g/m2 q 12 h x 12) and m-Amsa (115 mg/m2 for 1 or 3 days). Overall, 27 patients (54%) achieved complete remission. The complete remission rate for patients with acute myelogenous leukaemia after a preleukaemic phase (7/14) and those with primary refractory or relapsed leukaemia (20/36) seems superior to that obtained with conventional remission-induction therapy. 12 patients received 1-3 courses of consolidation chemotherapy with cytosine arabinoside (3 g/m2 q 12 h x 8) and m-Amsa (115 mg/m2 for 1 d). 3 of them subsequently underwent autologous bone marrow transplantation. The median duration of remission for the remaining 9 patients was 8 months. 11 patients did not receive consolidation therapy; their median duration of remission was 3 months. The difference between the two groups was not significant. 4 patients underwent allogeneic bone marrow transplantation after achievement of complete remission. The impact of high-dose cytosine arabinoside consolidation chemotherapy on poor-risk acute myelogenous leukaemia remains unclear.

The effect of high-dose cytosine arabinoside (Ara-C; 1-beta-D-arabinofuranosyl cytosine) injections (200 mg/kg i.v.) on cell cycle perturbation was investigated in a slowly growing rat leukemia (BNML) which is a realistic model for human acute myelocytic leukemia. Flow cytometric analysis showed an initial decrease of cells in S phase from 26 to 13% and a subsequent accumulation of up to 50% at 10-14 h after injection. The low number of S phase cells during the first 8 h might be due to a combination of cell kill in S phase and a block at the G1/S boundary. The results make it very likely that the origin of the accumulated S phase cells is the resting compartment and that these recruited cells enter the proliferation phase as a synchronized cell population. By repeating the Ara-C injection at the time of accumulation of cells in S phase, a similar synchronized wave of recruited cells to that after the first Ara-C injection was observed. Flow cytometric analysis after Adriamycin (7.7 mg/kg i.v.) treatment, which has been shown to be cytotoxic for BNML cells, showed no changes in cell cycle distribution. It was concluded that Adriamycin might have the same toxicity for cells in all of the different cell cycle phases. The application of these data with respect to effective tumor load reduction is discussed in a second report.

From the current studies it can be concluded that comparing the ara-C catabolism to ara-U in leukemic rats and leukemic patients, this process is about 100 times more pronounced in human leukemic cells. The low deaminase activity in leukemic rats probably explains the slow plasma ara-C disappearance curve in the BNML. No cytotoxic effect of ara-U with respect to LCFU-S reduction could be observed, nor did ara-U enhance the cytotoxic effect ara-C in the BNML. These studies have increased the understanding of the relation between ara-C, ara-U plasma levels and deaminase activity.