Evaluation of the feeding value of Dichrostachys cinerea pods for fattening pigs in Cuba.

Dichrostachys cinerea (L.) Wight & Arn. is a tropical leguminous shrub widely regarded as an invasive species in Cuba, after having invaded a significant proportion of its arable land during the past decades. Concurrently, smallholder pig producers are highly constrained by the scarcity of protein feeds. This study aimed to assess the feeding value of D. cinerea pod meal (DCPM) as an alternative protein supplement for pigs in Cuban smallholder production systems. An on-farm feeding trial was carried out with three groups (N = 10) of growing-fattening pigs over 60 days, where DCPM replaced 0, 15, and 30% in DM of a dietary commercial concentrate. Then, in an in vivo digestibility trial with eight growing pigs, apparent digestibilities of DCPM were determined for dry matter (DM), organic matter (OM) and crude protein (CP). Finally, in vitro digestibilities for OM (fecal and ileal) and CP (ileal) were determined. In the feeding trial, pig body weight gains were not affected by increased dietary substitution levels of concentrate for DCPM. Blood parameters, with a few exceptions, did not show significant differences among groups. Values for in vivo OM and CP digestibilities were 40.81 and 50.26%, and substantially higher than in vitro values. In conclusion, our results showed that at least 30% of DM in commercial concentrate could be substituted by DCPM without affecting pig growth performances under Cuban smallholder conditions. The low digestibility of DCPM is, however, not acceptable for intensive pig production systems. In vitro enzyme digestibility methods developed for commercial pig feeds are not suitable for DCPM without further calibration.

Technical note: In vitro total gas and methane production measurements from closed or vented rumen batch culture systems.

This study compared measured gas production (GP) and computed CH4 production values provided by closed or vented bottles connected to gas collection bags. Two forages and 3 concentrates were incubated. Two incubations were conducted, where the 5 feeds were tested in 3 replicates in closed or vented bottles, plus 4 blanks, for a total of 64 bottles. Half of the bottles were not vented, and the others were vented at a fixed pressure (6.8 kPa) and gas was collected into one gas collection bag connected to each bottle. Each bottle (317 mL) was filled with 0.4000 ± 0.0010 g of feed sample and 60 mL of buffered rumen fluid (headspace volume = 257 mL) and incubated at 39.0°C for 24 h. At 24 h, gas samples were collected from the headspace of closed bottles or from headspace and bags of vented bottles and analyzed for CH4 concentration. Volumes of GP at 24 h were corrected for the gas dissolved in the fermentation fluid, according to Henry's law of gas solubility. Methane concentration (mL/100mL of GP) was measured and CH4 production (mL/g of incubated DM) was computed using corrected or uncorrected GP values. Data were analyzed for the effect of venting technique (T), feed (F), interaction between venting technique and feed (T × F), and incubation run as a random factor. Closed bottles provided lower uncorrected GP (-18%) compared with vented bottles, especially for concentrates. Correction for dissolved gas reduced but did not remove differences between techniques, and closed bottles (+25 mL of gas/g of incubated DM) had a greater magnitude of variation than did vented bottles (+1 mL of gas/g of incubated DM). Feeds differed in uncorrected and corrected GP, but the ranking was the same for the 2 techniques. The T × F interaction influenced uncorrected GP values, but this effect disappeared after correction. Closed bottles provided uncorrected CH4 concentrations 23% greater than that of vented bottles. Correction reduced but did not remove this difference. Methane concentration was influenced by feed but not by the T × F interaction. Corrected CH4 production was influenced by feed, but not by venting technique or the T × F interaction. Closed bottles provide good measurements of CH4 production but not of GP. Venting of bottles at low pressure permits a reliable evaluation of total GP and CH4 production.

In Vivo 3D Power Doppler Imaging Using Continuous Translation and Ultrafast Ultrasound.

The introduction of ultrafast ultrasound and spatiotemporal filtering has significantly improved the sensitivity of Doppler ultrasound imaging. This work describes the development of a 3D power Doppler imaging technique which uses a 1D-array ultrasound probe that mechanically translates at a constant speed. The continuous translation allows for a fast scan of a large 3D volume without requiring complex hardware. The technique was realized in a prototype and its feasibility illustrated using phantom and in vivo kidney and breast lesion experiments. Although this 3D Doppler imaging technique is limited in some aspects, it enables power Doppler imaging of a large volume in a short acquisition time with less computational costs.

Neurotoxic effects of MDMA (ecstasy) on the developing rodent brain.

The incidence of methamphetamine abuse is particularly high in adolescents and is a common problem among women of childbearing age, leading to an increasing number of children with prenatal exposure. MDMA (3,4-methylenedioxymethamphetamine, ecstasy) is an amphetamine-like stimulant and is known to induce apoptotic damage to fine serotonergic fibers in the adult rat brain. Little is known about toxic effects of MDMA and potential underlying molecular mechanisms in the developing brain. Here, we investigated whether MDMA exposure during the period of rapid brain growth causes neurodegeneration in the developing rat brain. MDMA significantly enhanced neuronal death in the brains of 6-day-old rat pups at a dose of 60 mg/kg, but no significant toxicity was detected at the ages of 14 and 21 days. Brain regions mainly affected were the cortex, septum, thalamus, hypothalamus and the cornu ammonis 1 region. To explore possible molecular mechanisms involved in this neurodegenerative process, we investigated the impact of MDMA on the expression of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor. Neonatal exposure of 6-day-old rats to MDMA triggered a considerable increase in cortical BDNF and NT-3 levels. Moreover, P7 CD1/BDNF knockout mice were noticeably more sensitive to MDMA exposure as compared to their wild-type age-matched littermates. These data suggest that a single injection of MDMA causes neurodegeneration in the neonatal rat brain. The upregulation of BDNF and NT-3 expression may indicate an important compensatory mechanism leading to the survival of neuronal cells in the developing brain.

Effects of alternative feed additives to medicinal zinc oxide on productivity, diarrhoea incidence and gut development in weaned piglets.

The use of medicinal zinc oxide (ZnO) must be phased out by 2022, thus prompting an urgent need for alternative strategies to prevent diarrhoea in weaner piglets. The objectives of this study were to assess the impact on weaner piglet performance, diarrhoea incidence and gut development, when (1) dietary ZnO supplementation was substituted by alternative commercial products based on macroalgae, specific probiotics or synbiotics, or (2) dietary ZnO inclusion was reduced from 2500 to 1500 ppm. A total of 4680 DLY piglets (DanBred, Herlev, Denmark), weaned around 35 days of age, were randomly assigned according to sex and BW to six different dietary treatment groups. A basal diet was supplemented with no ZnO (NC = negative control), 2500 ppm ZnO (PC = positive control), 1500 ppm ZnO (RDZ = reduced dose of ZnO) or commercial macroalgae (OceanFeed™ Swine = OFS), probiotic Miya-Gold or synbiotic GærPlus products. The piglets entered and exited the weaner unit at ~7.0 and 30 kg BW, respectively. In-feed ZnO was provided the first 10 days post-weaning, while the alternative supplements were fed throughout the weaner period. As expected, the average daily feed intake, average daily weight gain (ADG), feed conversion ratio (FCR) and diarrhoea incidence were improved in the PC compared to NC group (P < 0.05) during phase 1 consistent with improved indices of villi development observed in subgroups of piglets sacrificed 11 days post-weaning. Reduction of ZnO to 1500 ppm lowered ADG (P < 0.05) and slightly increased incidence of diarrhoea during the first 10 days after weaning (but not later) without affecting FCR. None of the three alternative dietary additives, including a 10-fold increased dose of GærPlus than recommended, improved piglet performance, gut health and gut development above that of NC piglets. The OFS piglets sacrificed 11 days after weaning had significantly lower weights of hindgut tissue and contents compared to the PC group, consistent with antimicrobial activity of the product, which was detected from anaerobic in vitro fermentation. In conclusion, dietary ZnO supplementation during the first 10 days post-weaning may be reduced from 2500 to 1500 ppm without major negative implications for weaner piglet performance and health in herds under a high management level. However, none of the alternative dietary supplements were able to improve piglet performance or gut health, when ZnO was omitted from the diet.

The selective alpha7 nicotinic acetylcholine receptor agonist A-582941 activates immediate early genes in limbic regions of the forebrain: Differential effects in the juvenile and adult rat.

Due to the cognitive-enhancing properties of alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) agonists, they have attracted interest for the treatment of cognitive disturbances in schizophrenia. Schizophrenia typically presents in late adolescence or early adulthood. It is therefore important to study whether alpha7 nAChR stimulation activates brain regions involved in cognition in juvenile as well as adult individuals. Here, we compared the effects of the novel and selective alpha7 nAChR agonist 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) in the juvenile and adult rat forebrain using two markers, activity-regulated cytoskeleton-associated protein (Arc) and c-Fos, to map neuronal activity. Acute administration of A-582941 (1, 3, 10 mg/kg) induced a dose-dependent increase in Arc mRNA expression in the medial prefrontal cortex (mPFC) and the ventral/lateral orbitofrontal (VO/LO) cortex of juvenile, but not adult rats. This effect was mitigated by the alpha7 nAChR antagonist methyllycaconitine. A-582941 also increased c-Fos mRNA expression in the mPFC of juvenile, but not adult rats. Furthermore, A-582941 increased the number of Arc and c-Fos immunopositive cells in the mPFC, VO/LO, and shell of the nucleus accumbens, in both juvenile and adult rats. The A-582941-induced c-Fos protein expression was significantly greater in the mPFC and VO/LO of juvenile compared with adult rats. These data indicate that A-582941-induced alpha7 nAChR stimulation activates brain regions critically involved in working memory and attention. Furthermore, this effect is more pronounced in juvenile than adult rats, indicating that the juvenile forebrain is more responsive to alpha7 nAChR stimulation. This observation may be relevant in the treatment of juvenile-onset schizophrenia.

Effect of feed type and essential oil product on equine chewing activity.

The ingestive and post-digestion effect of a blend of special essential oil compounds (EO) on eating, chewing and faecal parameters were measured in horses. Ingestive effects appear after no adaptation. Post-digestion effects appear after adaptation. Six Icelandic horses were assigned to two groups in a Latin Square subplot design with EO treatments to four different roughage types and four different concentrates. The horses were fed four different roughage meals and two different concentrate meals on each of the four sampling days. Eating time and saliva were observed during meals. Jaw movements (JM) were recorded using a special chewing halter. Eating time was derived from JM and related to DM intake. The size characteristics of faecal particles were measured by using image analysis. All chewing characteristics measured were significantly affected by roughage (p < 0.001) and concentrate type (p < 0.01). EO had a significant ingestive effect on the frequency of observed saliva during concentrate meals. No significant (p < 0.05) post-digestive or ingestive effect of EO was found for any measured chewing characteristic, which was reflected in the absence of effect on faecal particle dimensions. In conclusion, effect of type of roughage and concentrate was more significant than potential effects of EO.

Three-dimensional ultrasound strain imaging of skeletal muscles.

In this study, a multi-dimensional strain estimation method is presented to assess local relative deformation in three orthogonal directions in 3D space of skeletal muscles during voluntary contractions. A rigid translation and compressive deformation of a block phantom, that mimics muscle contraction, is used as experimental validation of the 3D technique and to compare its performance with respect to a 2D based technique. Axial, lateral and (in case of 3D) elevational displacements are estimated using a cross-correlation based displacement estimation algorithm. After transformation of the displacements to a Cartesian coordinate system, strain is derived using a least-squares strain estimator. The performance of both methods is compared by calculating the root-mean-squared error of the estimated displacements with the calculated theoretical displacements of the phantom experiments. We observe that the 3D technique delivers more accurate displacement estimations compared to the 2D technique, especially in the translation experiment where out-of-plane motion hampers the 2D technique. In vivo application of the 3D technique in the musculus vastus intermedius shows good resemblance between measured strain and the force pattern. Similarity of the strain curves of repetitive measurements indicates the reproducibility of voluntary contractions. These results indicate that 3D ultrasound is a valuable imaging tool to quantify complex tissue motion, especially when there is motion in three directions, which results in out-of-plane errors for 2D techniques.

Cell proliferation and histologic classification of bronchogenic carcinoma.

The rate of cell proliferation of 99 bronchogenic carcinomas (94 primary tumors and 5 metastases) was evaluated from the labeling index after in vitro incorporation of [3H]thymidine; the rate was then correlated with the histologic tumor type according to the classification of the World Health Organization (WHO). Cell proliferation was significantly slower in adenocarcinoma (WHO type III) than in squamous cell carcinoma (WHO type I), small cell anaplastic carcinoma (WHO type II), and large cell carcinoma (WHO type IV). Cells proliferated at a significantly higher rate in large cell carcinoma than in the squamous cell type, whereas no significant difference was observed between the other cell types. Dedifferentiated forms of squamous cell carcinomas had a higher rate of cell proliferation than did differentiated forms of the same cell type. Metastases of small cell anaplastic carcinoma did not differ in cell proliferation from primary tumors of the same cell type.

Determinants of complete remission induction and maintenance in chemotherapy with or without irradiation of small cell lung cancer.

The possible influence of pretreatment patient characteristics upon the probabilities of complete remission (CR) induction and maintenance was investigated in a series of 815 nonresected patients with small cell lung cancer. All patients underwent pretreatment staging which enabled allocation of 391 patients to trials for limited stage disease and 424 patients to trials for extensive disease. Three controlled trials for each disease stage were conducted between 1973 and 1981. All therapeutic regimens consisted of combinations of between three and six agents (lomustine, cyclophosphamide, methotrexate, vincristine, doxorubicin, etoposide) with or without irradiation. Thirty-five % of the limited stage patients and 18% of the extensive stage patients were alive and had achieved a complete remission 16 weeks after initiation of the treatment, i.e., after four cycles of chemotherapy. Relationships between pretreatment characteristics and the probability to pass this benchmark were examined by logistic regression analysis. The probability of CR was negatively related to increased serum lactate dehydrogenase and male sex in both disease stages. Pretreatment anemia (less than 12 g/liter) and poor performance status were associated with a reduced CR rate in limited and extensive stage disease, respectively. Factors related to the maintenance of complete remission were subsequently examined in the 211 complete responders by use of Cox's regression analysis. Complete responders with extensive disease prior to treatment had greater cumulative risk of relapse than those with limited disease (P less than 0.01). Hyponatremia had a significant negative influence on the remission duration in limited disease while age greater than 60 years and bone marrow metastases had significantly negative influence in extensive disease. Using the models it was possible to identify subgroups of patients with CR rates ranging from 5 to 55% and to stratify complete responders according to estimated risks of subsequent relapse.

Continuous versus alternating combination chemotherapy for advanced small cell carcinoma of the lung.

In a 2-year period, 146 patients with small cell carcinoma of the lung, staged as having extensive disease, were randomized to receive either continuous chemotherapy consisting of (a) 1-(2-chloroethyl-3-cyclohexyl-1-nitrosourea, cyclophosphamide, methotrexate, and vincristine followed by (b) 4'-demethylepipodophyllotoxin 9-[4,6-O-(R) ethylidene-beta-D-glucopyranoside] and doxorubicin at progression of disease or a regimen of (a) alternating with (b). Seventy-six patients received the continuous regimen; 70 patients received alternating treatment. Response rates were 68 and 72%, respectively. The median duration of response was 16 weeks in patients receiving continuous treatment compared to 28 weeks in patients receiving alternating treatment (p less than 0.05). No survival time difference was observed between the groups, median survival being 36 and 38 weeks, respectively. Four patients became long-term survivors (5.6 +, 5.5 +, 5.1, and 4.7 + years). All received alternating therapy. Six toxic deaths were observed among patients receiving continuous therapy compared to only one death among those in the alternating regimen. In conclusion, alternating combination chemotherapy leads to prolonged duration of remission. Duration of survival is not prolonged in uncured patients, but an increased possibility of long-term disease-free survival cannot be precluded.

Targeting the cytotoxicity of topoisomerase II-directed epipodophyllotoxins to tumor cells in acidic environments.

The epipodophyllotoxins etoposide and teniposide are probably the most important drugs in the treatment of small cell lung cancer. The drugs are used in maximally tolerated doses, and the toxicity of the drugs precludes significant dose increments. The cellular target is the nuclear enzyme topoisomerase II which, in the presence of these drugs, causes an extensive fragmentation of DNA. The cell kill can be antagonized by distinct drug types. We have demonstrated previously that the intercalating drug aclarubicin and the cardioprotecting agent ICRF-187 antagonize the cytotoxicity of etoposide in vitro. We have studied possible ways of using this antagonism as a means of differentially protecting normal tissue. Here we demonstrate that the intercalating agent chloroquine prevents the introduction of topoisomerase II-mediated DNA breaks and thereby antagonizes the cytotoxicity of etoposide. This interaction depends on the extracellular pH. Chloroquine, in contrast to etoposide, is a weak base and therefore does not enter the cell if the extracellular fluid is acidic, as is the case in most solid tumors. We propose that such a pH-dependent drug interaction may be useful in directing topoisomerase II drug effects toward solid tumors. Thus, by lowering the extracellular pH (pHe) from neutral (pHe = 7.4) to acidic (pHe = 6.0), [3H]chloroquine accumulation was decreased 5-fold in a human small cell lung cancer cell line, OC-NYH, and in murine leukemia L1210 cells. In parallel, the antagonism exhibited by chloroquine on etoposide cytotoxicity was pHe dependent. Thus, no protection by chloroquine was observed at pHe = 6.5, whereas at pHe = 7.4, etoposide cytotoxicity was almost completely antagonized with a 460-fold protection or more than eight doublings of the cell population. This exploitation of antagonist extracellular trapping by acidic pH is a novel model for regulation of anticancer drug effects.

Urokinase and plasminogen activator inhibitor type 1 in pulmonary adenocarcinoma.

The urokinase pathway of plasminogen activation is involved in proteolytic degradation of various tissues, including dissolution of the extracellular matrix and basement membranes during the process of cancer cell invasion. We have studied the prognostic value of urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAI-1) in tumor extracts from 106 patients with adenocarcinoma of the lung. uPA and PAI-1 levels were determined by sandwich enzyme-linked immunosorbent assays. No correlation was found between uPA and PAI-1 (r = 0.23). High PAI-1 levels were significantly associated with short-duration overall survival (P = 0.017), while uPA levels showed no significant association with overall survival. Relating the levels of PAI-1 to other prognostic factors such as stage and age, no significant correlations were found. The prognostic impact of uPA and PAI-1 were investigated together with other prognostic factors in Cox multivariate analysis. PAI-1 was found to be an independent prognostic variable for survival, the relative risks being 1.5 (low versus medium PAI-1 values (95% confidence interval, 1.2-1.8) and 2.2 (low versus high (95% confidence interval, 1.8-2.6)). In patients with stage I disease (69 patients) high levels of PAI-1 were significantly associated with poor prognosis compared to low levels (P = 0.037). These data indicate that PAI-1 is a potentially important prognostic factor in pulmonary adenocarcinoma and may as such be used to select patients with low stage and poor prognosis for adjuvant therapy subsequent to complete surgical resection.

Prognostic impact of urokinase, urokinase receptor, and type 1 plasminogen activator inhibitor in squamous and large cell lung cancer tissue.

We have studied the prognostic value of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and type 1 plasminogen activator inhibitor (PAI-1) in tumor extracts from 84 patients with squamous cell lung carcinoma and 38 patients with large cell lung carcinoma, measuring each molecule with sandwich enzyme-linked immunosorbent assays. High uPAR levels were significantly associated with short overall survival in patients with squamous cell lung carcinomas when the median value was used as a cutoff point (P = 0.038), while no statistically significant prognostic impact of uPA and PAI-1 levels was found in this group of patients. The combination of high uPAR and high PAI-1 levels did, however, have a particular significant association with short overall survival (P = 0.008). None of the 3 components was found to have a statistically significant prognostic impact in the group of 38 large cell lung cancer patients. There was a positive correlation between uPAR and PAI-1 levels in both groups and between uPA and uPAR levels in the large cell carcinoma patients. In a multivariate analysis, high uPAR was found to be an independent prognostic variable in squamous cell carcinoma patients, with a relative risk of 2.1 (95% confidence interval, 1.1-4.0) and tumor size the only other significant prognostic parameter. These data suggest that uPAR is an important prognostic factor in squamous cell lung carcinoma. In addition to the potential direct clinical usefulness, this information could be helpful in understanding the biology of this disease and developing new therapeutic approaches.

Clonal heterogeneity of small-cell anaplastic carcinoma of the lung demonstrated by flow-cytometric DNA analysis.

Flow-cytometric DNA analysis yields information on ploidy and proliferative characteristics of a cell population. The analysis was implemented on small-cell anaplastic carcinoma of the lung using a rapid detergent technique for the preparation of fine-needle aspirates for DNA determination and a newly developed procedure for storing aspirates at -80 degrees. Thirty-eight different metastases in 30 consecutive patients with small-cell anaplastic carcinoma of the lung were examined with a total of 273 fine-needle aspirations. The results on ploidy are reported in this paper. The degree of contamination of the aspirates with normal cells was determined by differential counts. The ratio of the peak channel numbers for the G1 phase of the tumor cells to that of the diploid standard (DNA index) was calculated and used for ploidy identification. Twenty-nine patients were evaluable with respect to DNA index determination. The coefficient of variation of the DNA index determinations was estimated as 0.039. In 23 (79%) patients, only one cell line could be detected. Evidence of the presence of 2 tumor cell clones with different ploidy was obtained in the remaining 6 (21%) patients. Of the 35 malignant clones thus demonstrated, 26 (74%) were significantly different from diploid (p less than or equal to 0.01). Four (11%) were hypodiploid, 3 (9%) were hypotriploid, and 19 (54%) were hypo- or near-tetraploid. Clonal heterogeneity in the tumors of 21% of the patients is a conservative estimate. Assessment of the detection limit set by the methodology used and the restricted number of samples studied in each patient indicate that the true occurrence of clonal heterogeneity in small-cell carcinoma of the lung may be much higher.

Antagonistic effect of aclarubicin on daunorubicin-induced cytotoxicity in human small cell lung cancer cells: relationship to DNA integrity and topoisomerase II.

The effect of combinations of the anthracyclines aclarubicin and daunorubicin was investigated in a clonogenic assay using the human small cell lung cancer cell line OC-NYH and a multidrug-resistant (MDR) murine subline of Ehrlich ascites tumor (EHR2/DNR+). It was found that the cytotoxicity of daunorubicin in OC-NYH cells was antagonized by simultaneous exposure to nontoxic concentrations of aclarubicin. Coordinately, aclarubicin inhibited the formation of daunorubicin-induced protein-concealed DNA single-strand breaks and DNA-protein cross-links in OC-NYH cells when assayed by the alkaline elution technique. Aclarubicin had no influence on the accumulation of daunorubicin in these cells. In contrast, the accumulation of daunorubicin in EHR2/DNR+ cells was enhanced by more than 300% when the cells were simultaneously incubated with the MDR modulator verapamil, aclarubicin, or the two agents combined. Yet the cytotoxicity of daunorubicin was potentiated significantly only by verapamil. The increased cytotoxicity of daunorubicin in the presence of verapamil was completely antagonized when aclarubicin was used together with the MDR modulator. Finally, the effect of daunorubicin on the DNA cleavage activity of purified topoisomerase II in the presence and absence of aclarubicin was examined. It was found that daunorubicin stimulated DNA cleavage by topoisomerase II at specific DNA sites. The addition of aclarubicin completely inhibited the daunorubicin-induced stimulation of DNA cleavage. Taken together, these data indicate that aclarubicin-mediated inhibition of daunorubicin-induced cytotoxicity is due mainly to a drug interaction with the nuclear enzyme topoisomerase II. This antagonism at the nuclear level explains why aclarubicin is a poor modulator of daunorubicin resistance even though aclarubicin is able to increase the intracellular accumulation of daunorubicin in a MDR cell line.

Antagonistic effect of aclarubicin on the cytotoxicity of etoposide and 4'-(9-acridinylamino)methanesulfon-m-anisidide in human small cell lung cancer cell lines and on topoisomerase II-mediated DNA cleavage.

The effect of combinations of the anthracycline aclarubicin and the topoisomerase II targeting drugs 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (VP-16) and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) was investigated in a clonogenic assay. The cytotoxicity of VP-16 was almost completely antagonized by preincubating cells with nontoxic concentrations of aclarubicin. The inhibition of cytotoxicity was not seen when the cells were exposed to aclarubicin after exposure to VP-16. The inhibition was significant over a wide range of aclarubicin concentrations (3 nM to 0.4 microM), above which the toxicity of aclarubicin became apparent. A similar effect was seen on the toxicity of m-AMSA. In contrast to aclarubicin, preincubation with Adriamycin did not antagonize the effect of VP-16. With purified topoisomerase II and naked DNA, aclarubicin did not stimulate the formation of cleavable complexes between topoisomerase II and DNA. Aclarubicin concentrations above 1 microM inhibited the baseline formation of cleavable complexes elicited with the enzyme alone. Low (1 to 10 nM) aclarubicin concentrations increased the formation of cleavable complexes obtained with VP-16 and m-AMSA; however, at aclarubicin concentrations above 1 microM an antagonistic effect was obtained. In cells, the m-AMSA- and VP-16-induced, protein-concealed DNA strand breaks were completely inhibitable by aclarubicin preincubation with no synergic dose levels. Our results suggest that aclarubicin inhibits topoisomerase II-mediated DNA cleavage. This inhibition could represent the mechanism of action of the drug and explain the lack of cross-resistance to the classical anthracyclines. The observed antagonism could have consequences for scheduling of aclarubicin with topoisomerase II-active anticancer drugs.

Long-term disease-free survival in small-cell carcinoma of the lung: a study of clinical determinants.

The influence of treatment and of pretreatment patient characteristics on the probability of long-term disease-free survival in small-cell lung cancer (SCLC) was investigated in a consecutive series of 874 patients. The patients were included in six controlled treatment trials from 1973 to 1981, using different combinations of chemotherapy with or without irradiation. All patients underwent pretreatment staging, including bronchoscopy, peritoneoscopy with liver biopsy, and bone marrow examination. The same procedures were repeated in patients without overt signs of disease 18 months from initiation of treatment, and patients without evidence of SCLC were regarded as long-term survivors. Seventy-two patients were disease-free at restaging, corresponding to 13% of 443 patients with limited-stage disease and 3% of 431 patients with extensive-stage disease. The possible relationship between different pretreatment variables and the probability of 18 months' disease-free survival was investigated by multiple regression analysis. Disease extent was the most important determinant of long-term survival. Being a woman was a positive factor and hypouricemia had negative influence on the long-term results, while features such as performance status and serum lactate dehydrogenase (LDH) did not have significant influence in the regression model. Differences between the efficacy of the applied treatment regimens were less in limited disease than they were in extensive disease, in which six-agent regimens of alternating chemotherapy was significantly better than treatment with three- or four-agent regimens. Accordingly, disease extent seems to be the most pivotal determinant of long-term survival in SCLC, but influence of the patient's sex and serum urate concentration should also be considered.

Mortality and morbidity in long-term surviving patients treated with chemotherapy with or without irradiation for small-cell lung cancer.

Mortality and morbidity was investigated in a consecutive series of 72 patients with small-cell lung cancer (SCLC) who were found to be disease-free at restaging after 18 months of treatment. These patients were all the long-term survivors among 874 patients included in one of six trials between 1973 and 1981. All studies used combination chemotherapy with or without irradiation. Follow-up of the patients varied between 4 and 11 years. The estimated 5-year survival rate subsequent to discontinuation of therapy was 0.24, corresponding to a death rate of 0.25 per year or ten times greater than the expected mortality for persons of the same age group. This high mortality was primarily related to recurrent SCLC, the estimated cumulative risk of relapse reaching 46% at the time of the latest recurrence 5 years from diagnosis. The risk of relapse was generally independent of the pretreatment disease stage although it was reduced in patients with resectable disease and was greater in those with pretreatment liver or bone marrow metastases. Equal risks of relapse were related to the use of regimens with and without radiotherapy. The cumulative risk of relapse in patients surviving 3 years from initiation of the treatment was less than 15% and accordingly, 3 years of follow-up seems sufficient for comparison of long-term results obtained in different trials. The second factor resulting in death or disease was second cancer, for which the cumulated risk increased to 32%, the latest occurring 5.4 years from the diagnosis of SCLC. Five of these cases were non-small-cell lung cancers and three were secondary leukemias. The estimated mortality related to non-neoplastic conditions was just significantly greater than expected. In spite of the increased mortality in this series, 38 of 54 2-year disease-free survivors and 20 of 22 5-year survivors resumed a lifestyle similar to that before diagnosis of SCLC.

Systemic chemotherapy of brain metastases from small-cell lung cancer: a review.

PURPOSE: For decades the treatment of choice in small-cell lung cancer (SCLC) with brain metastases has been corticosteroids and radiotherapy (RT) because of a presumed lack of penetrance of cytostatic agents into parenchymatous brain metastases. In recent years, several reports have appeared on radiologic and clinical responses to systemic chemotherapy without additional RT in patients with metastatic SCLC in the brain. We reviewed the literature and focused on the methodologic aspects in comparison with RT data. DESIGN: We reviewed 12 patient series that included 116 patients and were published between 1981 and 1990. RESULTS: The overall brain response to chemotherapy without irradiation in patients with intracranial metastases at diagnosis was 76%, whereas the response rate of brain relapses was 43%. CONCLUSIONS: We conclude that intracranial metastases from SCLC seem to respond to chemotherapy as readily as other metastatic locations of SCLC do. Thus first-line cranial irradiation probably should be applied routinely only in cases of delayed brain metastases. Whether consolidating cranial RT should be given after a few courses of initial chemotherapy in SCLC patients with brain metastases at diagnosis is unclear and warrants a randomized evaluation.