Complex challenges of estimating the age and vitality of muscle wounds: a study with matrix metalloproteinases and their inhibitors on animal and human tissue samples.

The estimation of wound age and wound vitality is a recurring task in forensic routine work and has been subject of forensic research for a long time. By now, an unrestrictedly reliable marker or set of markers has not been found. In a study on myocardial infarctions, matrix metalloproteinases (MMP) 2 and 9 as well as tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) were detected immunohistochemically in mechanically wounded myocardium (ECG electrodes, vessel ligations). Against this background, the potency of MMP-9, MMP-2, and TIMP-1 as markers for the estimation of wound age and wound vitality was tested in a broad approach with human tissue samples drawn during autopsies and with an animal model, the isolated perfused Langendorff heart. The study comprised samples of injured human skeletal muscle, injured human myocardium, rats' hearts with vital wounds, and rats' hearts with postmortem-inflicted wounds that were all stained immunohistochemically. The results showed great scattering, leading to the conclusion that MMP-2, MMP-9, and TIMP-1 are not suitable for wound age estimation. Merely the results for TIMP-1 suggested that this marker might be able to differentiate between vital and postmortem-inflicted wounds. With a view to the promising results of the preceding study, the results underline the necessity to test possible markers of wound age/wound vitality on a large and diverse sample set.

Effects of cocaine and levamisole (as adulterant) on the isolated perfused Langendorff heart.

Cocaine-related deaths occur regularly in forensic routine work. In cases in which the detected concentration of cocaine is rather low and other causes of death apart from intoxication can be ruled out, the question arises if adulterants of cocaine might have played a crucial role. In the present study, cardiac effects of cocaine, of the adulterant levamisole and of mixtures of both were evaluated using the isolated perfused Langendorff heart. While exposed to the substances, functional parameters heart rate, left ventricular pressure and coronary flow were documented. Relevant alterations of these parameters were found for cocaine as well as for levamisole. Exposing the hearts to a mixture of both resulted in a combination of these effects; the emergence of new alterations or an obvious aggravation were not detected. Nevertheless, the results imply that the consumption of cocaine adulterated with levamisole bares an increased risk for cardiac complications, especially in the presence of preexisting cardiac pathologies.

The impact of sex and myocardial ischemic preconditioning on immunohistochemical markers of acute myocardial infarction.

The immunohistochemical detection of dityrosine, troponins I (cTNI) and T (cTnT), and connexin 43 has been proposed as a tool for the diagnosis of myocardial infarction with short survival times. Results of clinical and experimental studies reveal that gender and/or ischemic preconditioning of the heart may have an influence on severity and magnitude of myocardial infarction. To clarify the question, if the above-mentioned markers are influenced by sex or ischemic preconditioning, experiments on isolated rat hearts using the Langendorff technique were performed. Using the hearts of 12 male and 12 female Wistar rats a local ischemia was induced through ligation of the left coronary artery. Furthermore, 12 male rat hearts underwent ischemic preconditioning of the heart by stopping the perfusion of the whole heart for 30 min and subsequently reperfusing the heart for another 60 min, before inducing local ischemia. The perfusion time after ligation varied from 10 to 60 min. A control group was comprised out of 6 male and 2 female rat hearts. These were placed in the Langendorff system for 60 min without further manipulation or received ischemic preconditioning without subsequent local ischemia or were excised without being mounted on the Langendorff system at all. All hearts were fixed in formalin and stained immunohistochemically. Depletion of the marker cTnT appeared to be less in females when compared to male hearts, for all other markers tested, no apparent difference in staining results were seen when comparing male and female rat hearts. Male rat hearts with ischemic preconditioning showed no difference compared to male rat hearts without ischemic preconditioning when stained fort dityrosine. Connexin 43 staining was less pronounced in hearts with ischemic preconditioning, whereas cTnI as well as cTnT depletion was more pronounced in preconditioned hearts. The presented findings indicate to some extent the vulnerability of the investigated markers for the influencing factors tested.

Biomechanical stress in myocardial infarctions: can endothelin-1 and growth differentiation factor 15 serve as immunohistochemical markers?

Myocardial infarctions go along with biomechanical stress, i.e. stretching of muscle fibres, and the expression of certain marker molecules. We tested if two of those markers, endothelin-1 (ET-1) and growth differentiation factor 15 (GDF-15), can be used as immunohistochemical markers for myocardial ischaemia/infarctions. The study included experiments with an animal model, the isolated perfused Langendorff heart, as well as the investigation of human tissue samples drawn during autopsies. The overall picture of our results showed that GDF-15 is very sensitive and expressed very fast, not only as a consequence of ischaemia/infarctions, but also under other circumstances. Even an expression only caused by agony had to be discussed. ET-1, on the other hand, was less sensitive but only positive in those human cases with ischaemia/infarction that also showed typical alterations in conventional histology. Therefore, both markers did not proof to be a suitable diagnostic tool for myocardial infarctions. However, positive staining for ET-1 was also seen in rats' hearts that suffered from arrhythmias after electric shock and in the myocardium of the right ventricle in human control cases in which a right heart failure has to be discussed. Thus, especially ET-1 should be subject of further studies that focus on these pathologies.

Dityrosine as a marker of acute myocardial infarction? Experiments with the isolated Langendorff heart.

The isolated Langendorff heart was used to evaluate dityrosine as a marker of acute myocardial infarctions. The animal model allowed the generation of local infarctions with defined survival times, as well as infarctions with and without reperfusion. The results showed that dityrosine, at least under the conditions of the animal model, occurs very shortly after early ischemia and infarctions, since positive staining results were already obtained after a survival time of only 5 min. Furthermore, it could be proved that the occurrence of dityrosine does not depend on a reperfusion of the ischemic muscle area and that there are no differences in the staining patterns of infarctions with and without reperfusion. Positive staining results for dityrosine in control hearts without infarctions had to be considered when evaluating the tissue samples of the study hearts. In part, the positive staining results of the control hearts seemed to be an artefact of the Langendorff system, easily identifiable by a distinctive staining pattern. Positive staining results in tissue samples of hearts that suffered from arrhythmia on the other hand implied that the occurrence of dityrosine is not specific for myocardial infarctions. Taking into account the results of previous works on human tissue samples, however, these findings did not question the use of dityrosine as a diagnostic tool; they simply showed that myocardial damage due to oxidative stress might occur under various pathologic conditions.

Ischaemic and morphine-induced post-conditioning: impact of mK(Ca) channels.

BACKGROUND: Mitochondrial calcium-sensitive potassium (mK(Ca)) channels are involved in cardiac preconditioning. In the present study, we investigated whether also ischaemic-, morphine-induced post-conditioning, or both is mediated by the activation of mK(Ca) channels in the rat heart in vitro. METHODS: Animals were treated in compliance with institutional and national guidelines. Male Wistar rats were randomly assigned to one of seven groups (each n = 7). Control animals were not further treated. Post-conditioning was induced either by 3 × 30 s of ischaemia/reperfusion (I-PostC) or by administration of morphine (M-PostC, 1 µM) for 15 min at the onset of reperfusion. The mK(Ca)-channel inhibitor paxilline (1 µM) was given with and without post-conditioning interventions (M-PostC+Pax, I-PostC+Pax, and Pax). As a positive control, we determined whether direct activation of mK(Ca) channels with NS1619 (10 µM) induced cardiac post-conditioning (NS1619). Isolated hearts underwent 35 min ischaemia followed by 120 min reperfusion. At the end of reperfusion, infarct sizes were measured by triphenyltetrazolium chloride staining. RESULTS: In the control group, infarct size was 53 (5)% of the area at risk. Morphine- and ischaemic post-conditioning reduced infarct size in the same range [M-PostC: 37 (4)%, I-PostC: 35 (5)%; each P<0.05 vs control]. The mK(Ca)-channel inhibitor paxilline completely blocked post-conditioning [M-PostC+Pax: 47 (7)%, I-PostC+Pax: 51 (3)%; each P<0.05 vs M-PostC and I-PostC, respectively]. Paxilline itself had no effect on infarct size (NS vs control). NS1619 reduced infarct size to 33 (4)% (P < 0.05 vs control). CONCLUSIONS: Ischaemic- and morphine-induced post-conditioning is mediated by the activation of mK(Ca) channels.

Cyclosporine A administered during reperfusion fails to restore cardioprotection in prediabetic Zucker obese rats in vivo.

BACKGROUND AND AIMS: Hyperglycaemia blocks sevoflurane-induced postconditioning, and cardioprotection in hyperglycaemic myocardium can be restored by inhibition of the mitochondrial permeability transition pore (mPTP). We investigated whether sevoflurane-induced postconditioning is also blocked in the prediabetic heart and if so, whether cardioprotection could be restored by inhibiting mPTP. METHODS AND RESULTS: Zucker lean (ZL) and Zucker obese (ZO) rats were assigned to one of seven groups. Animals underwent 25 min of ischaemia and 120 min of reperfusion. Control (ZL-/ZO Con) animals were not further treated. postconditioning groups (ZL-/ZO Sevo-post) received sevoflurane for 5 min starting 1min prior to the onset of reperfusion. The mPTP inhibitor cyclosporine A (CsA) was administered intravenously in a concentration of 5 (ZO CsA and ZO CsA+Sevo-post) or 10 mg/kg (ZO CsA10+Sevo-post) 5 min before the onset of reperfusion. At the end of reperfusion, infarct sizes were measured by TTC staining. Blood samples were collected to measure plasma levels of insulin, cholesterol and triglycerides. Sevoflurane postconditioning reduced infarct size in ZL rats to 35±12% (p<0.05 vs. ZL Con: 60±6%). In ZO rats sevoflurane postconditioning was abolished (ZO Sevo-post: 59±12%, n.s. vs. ZO Con: 58±6%). 5 mg and 10 mg CsA could not restore cardioprotection (ZO CsA+Sevo-post: 59±7%, ZO CsA10+Sevo-post: 57±14%; n.s. vs. ZO Con). In ZO rats insulin, cholesterol and triglyceride levels were significant higher than in ZL rats (all p<0.05). CONCLUSION: Inhibition of mPTP with CsA failed to restore cardioprotection in the prediabetic but normoglycaemic heart of Zucker obese rats in vivo.

Helium-induced early preconditioning and postconditioning are abolished in obese Zucker rats in vivo.

Preconditioning is abolished in the prediabetic Zucker obese rat. It has been shown that prevention of mitochondrial permeability transition pore (mPTP) opening is involved in preconditioning by the noble gas helium. Here, we investigated: 1) whether helium induces pre- and postconditioning in Zucker rats and 2) whether possible regulators of the mPTP [i.e., mitochondrial respiration or the extracellular signal-regulated kinase (Erk) 1/2, Akt/glycogen synthase kinase (GSK)-3beta signaling pathway] are influenced. Anesthetized Zucker lean (ZL) and Zucker obese (ZO) rats were randomized to seven groups. Control animals were not treated (ZL-/ZO-Con). Preconditioning groups (ZL-/ZO-He-PC) inhaled 70% helium for 3 x 5 or 6 x 5 min, and postconditioning groups (ZL-/ZO-He-PostC) inhaled 70% helium for 15 min at the onset of reperfusion. Animals underwent 25 min of ischemia and 120 min of reperfusion. In additional experiments, hearts were excised after the third helium exposure for analysis of mitochondrial respiration and for Western blot analysis of Erk1/2, Akt, and GSK-3beta phosphorylation. Helium reduced infarct size from 52 +/- 3% (mean +/- S.E.) to 32 +/- 2% and 37 +/- 2% in ZL rats (ZL-HE-PC, ZL-He-PostC), respectively, but not in ZO rats [ZO-He-PC, 56 +/- 3%; ZO-He-PC (6x), 57 +/- 4%; and ZO-He-PostC, 51 +/- 3% versus ZO-Con, 54 +/- 3%]. Mitochondrial respiration analysis showed that helium causes mild uncoupling in ZL rats (2.27 +/- 0.03 versus 2.51 +/- 0.03) but not in ZO rats (2.52 +/- 0.04 versus 2.52 +/- 0.03). Helium had no effect on Erk1/2 and Akt phosphorylation. GSK-3beta phosphorylation during ischemia was reduced after helium application in ZL but not in ZO rats. Helium-induced preconditioning is abolished in obese Zucker rats in vivo, probably caused by a diminished effect of helium on mitochondrial respiration.

Helium-induced late preconditioning in the rat heart in vivo.

BACKGROUND: A recent study showed that the noble gas helium induces early myocardial preconditioning. Cyclooxygenase-2 (COX-2) has been shown to be an important mediator in the signal transduction of late preconditioning. In the present study, we investigated whether helium induces late preconditioning in a concentration-dependent, time-dependent, or in both manner and whether COX-2 activity, mitochondrial function, or both are involved. METHODS: The study was performed in male Wistar rats and consisted of two parts. In part 1, late preconditioning was achieved by administration of 70%, 50%, 30%, and 10% helium for 15 min 24 h before ischaemia/reperfusion (I/R). Based on the findings of part 1, in additional experiments 30% helium was administered subsequently three and two days before I/R. Furthermore, additional rats were pretreated with the COX-2 inhibitor NS-398 (5 mg kg(-1)) with and without 30% helium. Additional experiments were performed for mitochondrial analysis. RESULTS: Helium concentrations of 70%, 50%, and 30% but not 10% reduced infarct size [He-LPC 70: 37(13)%, He-LPC 50: 34(16)%, He-LPC 30: 40(9)%; each P<0.05 vs CONTROL: 55(8)%, He-LPC 10: 53(4)%; P>0.05 vs CONTROL]. Repeated administration of helium did not further enhance cardioprotection. NS-398 completely abolished cardioprotection by 30% helium [He-LPC 30+NS-398: 57(9)%; P<0.05 vs He-LPC 30] but had itself no effect on infarct size [NS-398: 55(9)%; P>0.05 vs CONTROL]. There were no differences in mitochondrial function after helium preconditioning. CONCLUSIONS: Helium induces late preconditioning. Cardioprotection is already maximal with administration of one cycle of 30% helium and is abolished by functional blockade of COX-2 activity.

Hyperglycaemia blocks sevoflurane-induced postconditioning in the rat heart in vivo: cardioprotection can be restored by blocking the mitochondrial permeability transition pore.

BACKGROUND: Recent studies showed that hyperglycaemia (HG) blocks anaesthetic-induced preconditioning. The influence of HG on anaesthetic-induced postconditioning (post) has not yet been determined. We investigated whether sevoflurane (Sevo)-induced postconditioning is blocked by HG and whether the blockade could be reversed by inhibiting the mitochondrial permeability transition pore (mPTP) with cyclosporine A (CsA). METHODS: Chloralose-anaesthetized rats (n=7-11 per group) were subjected to 25 min coronary artery occlusion followed by 120 min reperfusion. Postconditioning was achieved by administration of 1 or 2 MAC sevoflurane for the first 5 min of early reperfusion. HG was induced by infusion of glucose 50% (G 50) for 35 min, starting 5 min before ischaemia up to 5 min of reperfusion. CsA (5 or 10 mg kg(-1)) was administered i.v. 5 min before the onset of reperfusion. At the end of the experiments, hearts were excised for infarct size measurements. RESULTS: Infarct size (% of area at risk) was reduced from 51.4 (5.0)% [mean (sd)] in controls to 32.7 (12.8)% after sevoflurane postconditioning (Sevo-post) (P<0.05). This infarct size reduction was completely abolished by HG [51.1 (13.2)%, P<0.05 vs Sevo-post], but was restored by administration of sevoflurane with CsA [35.2 (5.2)%, P<0.05 vs HG+Sevo-post]. Increased concentrations of sevoflurane or CsA alone could not restore cardioprotection in a state of HG [Sevo-post2, 54.1 (12.6)%, P>0.05 vs HG+Sevo-post; CsA10, 58.8 (11.3)%, P>0.05 vs HG+CsA]. CONCLUSIONS: Sevoflurane-induced postconditioning is blocked by HG. Inhibition of the mPTP with CsA is able to reverse this loss of cardioprotection.

Effects of 1 alpha, 25-dihydroxyvitamin D3 on the human chronic myelogenous leukemia cell line RWLeu-4.

The effects of 1 alpha, 25-dihydroxyvitamin D3 (VD3) on proliferation, differentiation, and macromolecular synthesis in the new Philadelphia chromosome-positive chronic myelogenous leukemia cell line, RWLeu-4, were investigated. Binding of [3H]VD3 was saturable, with approximately 2000-3000 sites/cell, and half-maximal binding occurring at 0.21-0.33 nM. Treatment of RWLeu-4 cells with VD3 induced 24R-hydroxylase activity, a marker of vitamin D3 responsiveness in many tissues. Exposure of RWLeu-4 cells to VD3 also inhibited proliferation and DNA synthesis with a 50% effective dose of 3.5-10 nM within 72 h; in addition, protein and RNA synthesis were inhibited by VD3 treatment. Exposure of RWLeu-4 cells to 5 nM VD3 for 72 h caused 50% of the cells to differentiate into macrophage/monocyte type cells as judged by nitroblue tetrazolium staining and adherence to plastic. Progressive expression of cell surface maturation-specific antigens of the monocyte/macrophage lineage was induced by treatment of RWLeu-4 cells with VD3 for 24 to 72 h at doses that inhibited cellular proliferation. c-myc RNA, which is constitutively expressed in RWLeu-4 cells, increased after 0.5 h of treatment with 50 nM VD3 and then rapidly decreased to barely detectable levels after 4 h of treatment. Finally, the in vitro tyrosine kinase activity associated with the p210bcr-abl oncogene product was decreased approximately 50% by VD3 treatment. Because of the presence of a functional receptor-effector system for VD3 and multiple biological responses to the hormone, these cells provide a unique model system with which to probe the specific effects of VD3 on cell growth and differentiation in chronic myelogenous leukemia.

Therapeutic effects and pharmacokinetics of recombinant human granulocyte-macrophage colony-stimulating factor in childhood cancer patients receiving myelosuppressive chemotherapy.

Twenty-five children with refractory solid tumors were given recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) in escalated doses of 60 to 1,500 micrograms/m2 as 2-hour intravenous infusions, beginning 24 hours after myelosuppressive treatment with cisplatin and etoposide. Tolerance to rhGM-CSF was exceptional even at dose levels that exceeded the maximum-tolerated dosage (MTD) reported for adults. The agent produced dose-related increases in platelet and neutrophil counts, resulting in significantly shorter durations of severe neutropenia and thrombocytopenia (P less than .01 for each analysis). At the higher dosages (greater than or equal to 750 micrograms/m2), treatment with rhGM-CSF reduced the median number of days of antibiotic therapy for fever and neutropenia by approximately one half. We conclude that rhGM-CSF is well tolerated by leukopenic children in doses as high as 1,500 micrograms/m2. An MTD was not reached in this study. The ability of the growth factor to reduce severe neutropenia and thrombocytopenia suggests it will have an important role in the management of childhood solid tumors.

Recombinant human interleukin-3 (rhIL-3) enhances the mobilization of peripheral blood progenitor cells by recombinant human granulocyte colony-stimulating factor (rhG-CSF) in normal volunteers.

To identify a precisely timed and safe protocol for progenitor cell mobilization, we studied the effects of rhIL-3 and rhG-CSF administration to normal volunteers. rhG-CSF 5 micrograms/kg/d was administered subcutaneously (s.c.) for 7 consecutive days either alone or preceded by rhIL-3 5 micrograms/kg/d s.c. for 4 consecutive days in sequential or partially overlapping schedules. The combined cytokines were well-tolerated--adverse effects were similar to those of the individual agents. Total white blood cell (WBC) and neutrophil counts rose briskly in response to rhG-CSF, and peak mean values were similar between treatment cohorts. Mean platelet counts were modestly elevated during rhG-CSF treatment only in the cohorts receiving rhIL-3 and rhG-CSF. Mean circulating CD34+ cells peaked on day 5 in the rhG-CSF group (38.9+/-14.3/microliter), day 6 in the sequential rhIL-3/rhG-CSF group (56.4+/-12.4/microliter), and day 6 in the partial overlap group (46.1+/-10.9/microliter). On day 3, mean CD34+ cell counts of the subjects who received sequential treatment were markedly higher than observed in the other groups (p<0.05) and were estimated to have been sufficient for collection of adequate grafts by single 10-L leukapheresis procedures in 60% of subjects. Circulating clonogenic cells (CFU-GM and/or BFU-E) were substantially higher in the sequential group than the rhG-CSF group on days 3-6 but were only minimally elevated above baseline in the partial overlap group. The numbers of circulating CD34+/Lin-/Thy-1+ cells (putative stem cells) were increased substantially, especially in the sequential group. On the basis of this pilot trial, we conclude that priming with rhIL-3 is a safe and well-tolerated method for enhancing the mobilization of human blood progenitors and stem cells by rhG-CSF.

[Prevention of bicycle accidents]. / Prävention von Fahrradfahrerunfällen.

For a very precise analysis of all injured bicyclists in Germany it would be important to have definitions for "severely injured", "seriously injured" and "critically injured". By this, e.g., two-thirds of surgically treated bicyclists who are not registered by the police could become available for a general analysis. Elderly bicyclists (> 60 years) are a minority (10 %) but represent a majority (50 %) of all fatalities. They profit most by wearing a helmet and would be less injured by using special bicycle bags, switching on their hearing aids and following all traffic rules. E-bikes are used more and more (145 % more in 2012 vs. 2011) with 600,000 at the end of 2011 and are increasingly involved in accidents but still have a lack of legislation. So even for pedelecs 45 with 500 W and a possible speed of 45 km/h there is still no legislative demand for the use of a protecting helmet. 96 % of all injured cyclists in Germany had more than 0.5 ‰ alcohol in their blood, 86 % more than 1.1 ‰ and 59 % more than 1.7 ‰. Fatalities are seen in 24.2 % of cases without any collision partner. Therefore the ADFC calls for a limit of 1.1 ‰. Some virtual studies conclude that integrated sensors in bicycle helmets which would interact with sensors in cars could prevent collisions or reduce the severity of injury by stopping the cars automatically. Integrated sensors in cars with opening angles of 180° enable about 93 % of all bicyclists to be detected leading to a high rate of injury avoidance and/or mitigation. Hanging lamps reduce with 35 % significantly bicycle accidents for children, traffic education for children and special trainings for elderly bicyclists are also recommended as prevention tools. As long as helmet use for bicyclists in Germany rates only 9 % on average and legislative orders for using a helmet will not be in force in the near future, coming up campaigns seem to be necessary to be promoted by the Deutscher Verkehrssicherheitsrat as, e.g., "Helmets are cool". Also, spots in TV should be broadcasted like "The 7th sense" or "Traffic compass", which were warning car drivers many years ago of moments of danger but now they could be used to warn bicyclists of life-threatening situations in traffic.

Retroviral marking and transplantation of rhesus hematopoietic cells by nonmyeloablative conditioning.

The ability to engraft significant numbers of genetically modified hematopoietic stem and progenitor cells without the requirement for fully myeloablative conditioning therapy is a highly desirable goal for the treatment of many nonmalignant hematologic disorders. The aims of this study were to examine, in nonhuman primates (rhesus), (1) the effects of pretreatment of host animals with cytokines (G-CSF and SCF), i.e., before nonmyeloablative irradiation, on the degree and duration of neo gene marking of circulating leukocytes after autologous cell reinfusion and (2) to compare transduction of primitive hematopoietic target cells in the presence of our standard transduction cytokine combination of IL-3, IL-6, and stem cell factor (SCF) and in the presence of an alternative combination containing SCF, G-CSF, and the thrombopoietin analog MGDF. Cytokine-mobilized rhesus peripheral blood progenitor/stem cells (PBSCs) were enriched for CD34+ cells and transduced with neo vectors (either G1Na or LNL6) for 96 hr in cultures containing rhIL-3, rhIL-6, and rhSCF or MGDF, rhSCF, and rhG-CSF and cryopreserved. Four animals underwent minimal myeloablative conditioning with 500 cGy irradiation with or without pretreatment with SCF and G-CSF, followed by reinfusion of the cryopreserved cells on the subsequent day. Neutrophil nadirs (< or =500/mm3) were 0-3 days in duration; there were no significant periods of severe thrombocytopenia. Marking of circulating granulocytes and mononuclear cells was extensive and durable in all animals (exceeding 12% in the mononuclear cells of one animal) and persisted beyond the final sampling time in all animals (up to 33 weeks). No difference in extent or duration of marking was attributable to either cytokine presensitization of recipients prior to irradiation, or to the substitution of MDGF and G-CSF for IL-3 and IL-6 during transduction.

Pharmacodynamics of daily subcutaneous recombinant human interleukin-3 in normal volunteers.

Normal volunteers received subcutaneous injections of recombinant human interleukin-3 (rhIL-3) on 4 consecutive days to characterize toxicity, pharmacokinetics, and hematopoietic effects. Dosages were 2.5, 5.0, and 7.5 micrograms/kg/day (n = 6 subjects per group). Adverse effects consisted predominantly of flu-like symptoms such as fever and headache. Mean area under the serum concentration-time curve and maximum serum concentration were linearly related to dose. Serum clearance was not apparently related to dose. Clearance increased slightly but significantly between days 1 and 4. Rapid but modest elevations in neutrophil and eosinophil counts were observed during treatment. Mean platelet counts rose modestly, peaking on day 10. Increases of CD34+ cell counts were correlated with increases of colony-forming unit-granulocyte macrophage (peak, day 7).

Pharmacodynamics of subcutaneous recombinant human interleukin-10 in healthy volunteers.

Interleukin-10 inhibits T-lymphocyte activation and proliferation and lipopolysaccharide-induced monocyte production of proinflammatory cytokines. Fifty-four healthy volunteers received single doses of recombinant human interleukin-10 (1.0, 2.5, 5.0, 10, 25, or 50 micrograms/kg) or placebo by subcutaneous injection (randomized double-blind assignment). Clinical adverse events were infrequent at doses below 50 micrograms/kg (five of six subjects had mild flu-like syndrome). Mean serum interleukin-10 concentrations were dose related. The mean terminal-phase half-life ranged from 2.7 to 4.5 hours, and the apparent volume of distribution ranged from 0.70 to 1.35 L/kg. Hematologic changes included transient mild to moderate increases of neutrophil counts, decreases of lymphocyte counts, and a delayed decrease of platelet counts. Recombinant human interleukin-10 significantly suppressed production of the proinflammatory cytokines interleukin-1 beta and tumor necrosis factor-alpha by whole blood stimulated ex vivo with Escherichia coli lipopolysaccharide.

Epirubicin and ifosfamide in metastatic breast cancer.

In a randomized, phase II trial, we evaluated the effectiveness of continued chemotherapy with epirubicin/ ifosfamide versus unmaintained treatment interruption in advanced metastatic breast cancer. Three hundred fifty-seven patients were enrolled and 331 were evaluable for response. Complete response was achieved in 25 patients (8%) and partial response in 121 patients (37%). Pretreatment status correlated significantly with response (complete and partial response). While 54% of unpretreated patients responded, only 42% of the patients responded who had been pretreated with adjuvant chemotherapy and 33% who had been pretreated in the metastatic stage of disease; 69 patients (21%) had disease progression. Of 11 patients pretreated in both the adjuvant and metastatic setting, only two responded. Toxicity of treatment was mild, with leukopenia being the treatment-limiting factor. Thrombocyte levels were not altered significantly by treatment. Thus, there seems to be room for dose escalation using granulocyte colony-stimulating factor. There was no considerable cardiotoxicity, central nervous system toxicity, or cystitis observed. The low rate of cardiotoxicity appeared to be related to dose fractionation of epirubicin. After randomization of patients to treatment interruption versus continuation of chemotherapy, a longer relapse-free survival was observed for patients who continued chemotherapy (mean relapse-free survival, 2+ months); however, this did not translate into prolonged survival. The cumulative scores of toxicity and quality of life parameters showed increasing superiority for treatment interruption. Therefore, a strategy of treatment until maximum response and subsequent treatment interruption seems to be superior to treatment continuation.

Detection of aneuploid cells in acute lymphoblastic leukemia with flow cytometry before and after therapy.

We have evaluated the proliferative activity and DNA content of immunophenotyped hematopoietic cells applying flow cytometry. After indirect immunofluorescence the cell membrane was permeabilized for propidium iodide staining of DNA. Compared with single parameter detection of DNA content this method has more certainty in the determination of aneuploidies in lymphatic leukemia cells. Immunophenotyped residual normal hematopoietic cells were used as an internal standard. If this method was tested for evaluation of therapeutic effects after chemotherapy greater sensitivity in detection of minimal residual disease was observed than when using microscopic evaluation or single parameter DNA analysis in cases of aneuploid lymphoblastic leukemias.

Malignant pheochromocytoma of urinary bladder.

Herein is reported a fifth case of malignant pheochromocytoma of the urinary bladder. Pheochromocytoma represents only 0.4 per cent of bladder tumors with 5 to 10 per cent being malignant. A review of the 4 previously reported cases plus our own suggest that iliac-hypogastric lymphadenectomy should be considered in all cases of pheochromocytoma of the bladder. Early establishment of the malignant potential of these tumors coupled with aggressive surgical treatment improves prognosis.