Differences between normal and ras-transformed NIH-3T3 cells in expression of the 170kD and 180kD forms of topoisomerase II.

The activity of topoisomerase II and the cellular content of the 170kD and 180kD forms of the enzyme were studied as functions of transformation and growth state by using normal and ras-transformed NIH-3T3 cells. Total topoisomerase II activity, as measured by the unknotting of P4 DNA, was higher in ras-transformed than in normal cells in similar growth states, and was higher in exponentially growing than in plateau cells for both cell lines. Total topoisomerase II levels, as measured by immunoblotting, showed a similar dependence on transformation and growth state. The relative amounts of the 170kD and 180kD forms of the enzyme varied as a function of transformation and growth state. The proportion of 170kD topoisomerase II was higher in ras-transformed than in untransformed cells and depended much less on growth state in the ras-transformed cells. The topoisomerase II activity in extracts of ras-transformed cells was more sensitive to inhibition by teniposide and merbarone, drugs which selectively inhibit the 170kD form of topoisomerase II. The ras-transformed cells were also more sensitive to the cytotoxic effects of these drugs. An increase in the relative cellular content of 170kD topoisomerase II is characteristic of ras-transformed 3T3 cells, and the levels of this form of the enzyme appear to be less dependent on proliferation state than in untransformed cells. The susceptibility of certain tumors to killing by topoisomerase II-directed drugs may be due to a higher proportion of 170kD enzyme as well as a higher level of total topoisomerase II activity.

Population PK-PD Model for Tolerance Evaluation to the p38 MAP Kinase Inhibitor BCT197.

The p38 mitogen-activated protein kinase (p38) is a key signaling pathway involved in regulation of inflammatory cytokines. Unexpectedly, several clinical studies using p38 inhibitors found no convincing clinical efficacy in the treatment of chronic inflammation. It was the objective of this study to characterize the population pharmacokinetics (PK) of BCT197 in healthy volunteers and to examine the relationship between BCT197 exposure and pharmacodynamics (PD) measured as inhibition of ex vivo lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFα), a downstream marker of p38 activity. PK was characterized using a two-compartment model with mixed-order absorption and limited-capacity tissue binding. The PK-PD relationship revealed that suppression of TNFα was partly offset over time, despite continuous drug exposure. This may indicate a mechanism by which the inflammatory response acquires the ability to bypass p38. Simulations of posology dependence in drug effect suggest that an intermittent regimen may offer clinical benefit over continuous dosing and limit the impact of tolerance development.

The effect of lithium on the membrane molecular dynamics of normal human erythrocytes.

Erythrocytes from normal adults with no personal or family history of bipolar affective disorder were analyzed by fluorescence spectroscopy to determine what effect, if any, acute in vitro incubation with lithium had on erythrocyte membrane dynamics. The effects on erythrocyte membrane molecular dynamics of varying concentrations of Li2CO3 (0.25-2.0 meq/liter), varying incubation temperatures (25-40 degrees C), and varying incubation times (5-185 min) were investigated. Following incubation with Li2CO3, the erythrocytes were labeled with either 4-phenylspiro-[furan-2(3H),--1'phthalan]--3,3'-dione (fluorescamine), which binds to membrane surface primary amines, or 12(9)anthroyl stearate [12(9)AS], which inserts deep in the membrane hydrocarbon core. The membrane molecular dynamics were then determined by fluorescence anisotropy measurements. These studies demonstrate that clinically relevant concentrations of Li+ incubated with intact normal human erythrocytes significantly alters molecular dynamics on the erythrocyte membrane surface, with less striking changes in the hydrocarbon core. A possible interpretation of these findings is that hydrated Li+ alters the electrostatic interaction of membrane surface molecules, as well as the surrounding solvent (water) structure, with a resultant increase in the molecular motion of these molecules. Alterations in membrane receptor motion could potentially alter receptor functional activity. If similar motional alterations were to occur in the interior of a membrane channel, such as an ionophore, the functional activity of the channel could also be potentially altered. These findings provide additional insight into possible biological actions of Li+, as well as potential molecular alterations in bipolar affective disorder erythrocytes.

Inhibition of endothelin-mediated topoisomerase I activation by pertussis toxin.

Cultured rat mesangial cells contain high affinity endothelin (ET) receptors at high densities. Exposure of these cells to ET resulted in a transient activation of topoisomerase I extractable activity, which reached its maximum value at approximately 2 min and returned to basal value after approximately 10 min of treatment. The activation of this enzyme was dependent upon the concentration of ET added. Incubation of the cells with pertussis toxin inhibited ET-induced increases in topoisomerase I activity in a concentration-dependent manner, suggesting that involvement of pertussis toxin-sensitive GTP-binding protein in ET-mediated action. Endothelin had no detectable effect upon extractable topoisomerase II activity.

Clinical and serologic follow-up in patients with neuroborreliosis.

The authors performed a clinical and serologic follow-up study after 4.2 +/- 1.2 years in 44 patients with clinical signs of neuroborreliosis and specific intrathecal antibody production. All patients had been treated with ceftriaxone 2 g/day for 10 days. Although neurologic deficits decreased significantly, more than half the patients had unspecific complaints resembling a chronic fatigue syndrome and showed persisting positive immunoglobulin M serum titers for Borrelia in the Western blot analysis.

Treatment of stroke on an intensive stroke unit: a novel concept.

In industrialised nations stroke ranks as number three among causes of death and is the most frequent cause of disability in old age. Demographic changes will result in stroke gaining increasing importance for individuals as well as for society as a whole. Stroke is already a major cost factor for healthcare and social security systems because of its high long-term costs. Therapeutic nihilism, although still widespread among patients and some physicians, is no longer justified. Long-term outcome after stroke can be significantly improved by providing therapy in wards specialised in early rehabilitation, so-called 'stroke units'. Recent magnetic resonance imaging (MRI) and positron emission tomography (PET) studies, as well as lysis therapy studies have shown that the first 3-6 h are crucially important. For this reason, the concept of "intensive stroke units" also called "intensive care stroke units" has been implemented in Germany. The goal of an intensive stroke unit is the optimal care of stroke patients under intensive care conditions for the first 3-4 days with the aim of improving outcome, long-term morbidity, and reducing long-term healthcare costs. Another important objective is the development and research of new therapeutic concepts and approaches that are based on pathophysiological considerations. A further goal is the initiation of specific therapies depending on the suspected underlying pathophysiology, for example, local or systemic thrombolysis, full-dose heparinisation, platelet aggregation inhibitors, oral anticoagulants, neuroprotective agents, decompression craniotomy, sympathomimetically supported volume therapy and hypothermia. A final objective is to minimise the number of complications through intensive monitoring. Basic acute management includes optimal oxygen supply, rapid normalisation of blood glucose and body temperature, volume therapy, maintaining a high blood pressure and cardiac output to improve remaining cerebral perfusion in the presence of ischaemically impaired autoregulation, treating cerebral oedema, prophylaxis of thrombosis, and early mobilisation. Rapid and easy access to computerised tomography (CT), MRI, Doppler and duplex scanning of the brain-supplying blood vessels, and echocardiography is essential. The ready availability of intensive care monitoring (blood pressure, electrocardiography, central venous pressure, transcranial Doppler (TCD), TCD embolism detection, cerebral pressure, electroencephalography and cardiac output is also imperative. We would like to stress at this point that this manuscript is a personal view describing stroke care in Germany. Many of the principles described have not been widely adopted elsewhere, perhaps in part due to a lack of available facilities. However, many of our recommendations are based on logical principles and thus, we feel, bear further scrutiny.

Camptothecin hyper-resistant P388 cells: drug-dependent reduction in topoisomerase I content.

A subline of P388 leukemia made 10-fold resistant to camptothecin (CPT) by serial passage in drug-treated mice was adapted to growth in tissue culture and made hyper-resistant to CPT by passage in the presence of increasing concentrations of the drug. Cells were obtained that were 1,000-fold resistant to CPT, compared to wild-type P388 cells. Neither topoisomerase I mRNA nor 100 kDa topoisomerase I enzyme was detectable in these cells, and topoisomerase I activity extracted from nuclei was less than 4% of that extracted from nuclei of wild-type cells. An immunoreactive 130 kDa protein that could be an altered, inactive form of topoisomerase I was evident in the hyper-resistant cells. In addition, the cells deficient in topoisomerase I contained enhanced topoisomerase II activity. Maintenance of the hyper-resistant phenotype required continued exposure to CPT; growth in its absence led to loss of hyper-resistance, increased topoisomerase I content and activity, and decreased topoisomerase II activity. The sensitivity of the cells to killing by a number of inhibitors of topoisomerases I and II was consistent with these observations. Thus, P388 cells have the potential to become highly resistant to CPT by severely curtailing topoisomerase I expression; in these circumstances, topoisomerase I and II activities are regulated coordinately.

Decreased vascular endothelial growth factor expression associated with tumor regression induced by (E)-2'-deoxy-2'-(fluoromethylene)cytidine (MDL 101,731).

The effect of the antitumor drug MDL 101,731 [(E)-2'-deoxy-2'-(fluoromethylene)cytidine] on tumor growth and on steady-state vascular endothelial growth factor (VEGF) mRNA levels in MDA-MB-231, PC-3, MCF-7, and HT-29 human tumor xenografts grown in nude mice was examined, using quantitative in situ hybridization. MDL 101,731 caused regression of MDA-MB-231 and PC-3 tumor xenografts, but only inhibition of growth (without regression) of MCF-7 xenografts. The drug caused inhibition of growth of HT-29 xenografts at low doses, and regression at high doses. When treatment with MDL 101,731 led to tumor regression, VEGF mRNA levels were decreased. When treatment led only to inhibition of growth, there was no significant change in VEGF mRNA. Further examination of the tumor xenografts revealed that elevated VEGF mRNA was associated with hypoxic zones surrounding areas of necrosis in the tumors, and that the drop in VEGF mRNA observed in tumors from mice treated with MDL 101,731 correlated with a loss of zones of necrosis. In contrast, treatment with cisplatin led to either an increase (PC-3) or no change (MDA-MB-231) in VEGF mRNA levels, and no loss of necrotic zones. Quantitative analysis of changes in VEGF mRNA levels was supported by immunohistochemical analysis of VEGF protein in the same tumor specimens. In vitro, MDL 101,731 was a potent inhibitor of VEGF secretion in cells exposed to hypoxia, whereas there was no effect of cisplatin on VEGF secretion by three of the four cell lines tested. These findings suggest that inhibition of VEGF expression by MDL 101,731 may distinguish this compound from other classes of cytotoxic agents, such as cisplatin.

Comparison of three approaches to doxorubicin therapy: free doxorubicin, liposomal doxorubicin, and beta-glucuronidase-activated prodrug (HMR 1826).

BACKGROUND: Three approaches to doxorubicin therapy are directly compared: free doxorubicin, liposomal doxorubicin and beta-glucuronidase-activated prodrug (HMR 1826). MATERIALS AND METHODS: The optimal dose of HMR 1826 was determined to be 200 mg/kg once a week and subsequent studies were carried out comparing HMR 1826 at 200 mg/kg 1x/wk, liposomal doxorubicin (Doxil) at 9 mg/kg 1x/wk and free doxorubicin at 7 mg/kg 1x/wk in seven different human tumor xenograft models. RESULTS: All three forms of doxorubicin inhibited tumor growth with similar efficacy in each of the tumor models with the exception of MDA-MB-231 tumor xenografts, which were resistant to free doxorubicin but sensitive to Doxil and HMR 1826. Overall less weight loss was observed with HMR 1826 treatment. CONCLUSIONS: The efficacy of HMR 1826 is equal to or better than that of doxorubicin and Doxil at a safe dose and schedule, indicating that the beta-glucuronidase activated prodrug approach is safe and effective.

Clinical value of specific intrathecal production of antibodies.

The production of intrathecal antibodies is considered a highly specific marker for an infection of the central nervous system (CNS), e.g. borreliosis or tick-borne encephalitis (TBE). To investigate the validity of this assumption, we examined records of patients who had been hospitalized between 1989 and 1995, who were tested for borreliosis (n = 8003) and TBE (n = 904) and whose cerebrospinal fluid (CSF) had subsequently tested positive for intrathecal production of antibodies. The time period between the beginning of the symptoms and the time of the CSF examination ranged from one day to six weeks. Seventy-seven patients showed a production of intrathecal antibodies against Borrelia burgdorferi. Three of these patients were false positives with no history and no clinical signs of neuroborreliosis. In two cases, this was due to a non-specific cross-reaction caused by a preceding infection with syphilis. The third false positive was possibly caused by an earlier administration of immunoglobulins. Three patients showed a production of intrathecal antibodies against TBE virus. Two of these patients were false positives. In one case, we suspect that the production of intrathecal antibodies was caused by a non-specific immune reaction during an acute neuroborreliosis. One year earlier, the patient had contact with TBE virus through a vaccination against TBE. The cause of the second false positive is unclear, the clinical findings, acute encephalitis and the serological analysis suggest a cross-reaction with a virus similar to TBE. A specific intrathecal production of antibodies is not a proof for an infection of the CNS. In unclear cases, one should carry out a Western blot analysis or, if one suspects a case of TBE, a neutralization test.

Effects of multiple sclerosis and other inflammatory CNS disorders on tick-borne encephalitis serology.

The goal of the present study was to investigate whether a direct association exists between false-positive recognition of IgG antibodies and inflammatory changes in the central nervous system (CNS) and whether inflammatory diseases of the CNS affect the specificity of the enzyme-linked immunosorbent assay (ELISA) of tick-borne encephalitis (TBE) virus. A group of patients (1,815), treated in the Department of Neurology, University Hospital of the Saarland, Homburg/Saar, Germany, were tested forTBE IgG antibodies by ELISA. Several subgroups of patients with and without inflammatory changes in the CSF as well as patients with and without confirmed multiple sclerosis (MS) were investigated. Overall, 4.5% of all the 1,815 patients and 4.8% of the patients with inflammatory changes in the CSF but without MS had TBE IgG antibodies. In the subgroup with inflammatory changes in the CSF and MS, 4.4% of the patients were TBE IgG-positive. In the subgroup without inflammatory changes in the CSF, 3.8% of the patients without MS were TBE IgG-positive and 4.9% of the patients with MS were TBE IgG-positive. The rate of TBE IgG positivity was not significantly different in the subgroups with and without inflammatory changes in the CSF (P = 0.45). The comparison of the subgroups with and without MS showed no significant difference in the TBE IgG titer (P = 0.83) as well. This indicates that the specificity of the ELISA was affected neither by inflammatory changes in the CSF nor by MS.

[Ischemic or hemorrhagic stroke? Rapid diagnosis improves prognosis for the patient]. / Ischämischer oder hämorrhagischer Schlaganfall? Schnelle Diagnostik verbessert die Prognose des Patienten.

In industrialized countries, stroke is the third most common cause of death, and often leads to permanent disability. When clinical suspicion is raised, a thorough diagnostic work-up and treatment must be initiated without delay. In recent years, decisive advances have been made in this area, and the pathophysiological categorization of ischemic cerebral infarction has become accepted, with the modern imaging procedures having played a significant role. Reliable differentiation from intracerebral hemorrhage is enabled in particular by CCT and MRI, and is of great importance both for acute treatment and secondary prevention. Stroke units make available the necessary equipment and personnel. Patients who receive initial treatment on a stroke unit are already benefiting from a 31% reduction in mortality and severe disability.

[Differential diagnosis of Lyme borreliosis]. / Die wichtigsten Differenzialdiagnosen: Wanderröte oder Arzneimittelexanthem--Neuroborreliose oder MS?

In all three stages, Lyme borreliosis offers a wide range of possible differential diagnoses: even the "typical" erythema chronicum migrans may present as erysipelas, erysipeloid, erythema annulare centrifugum or a drug-induced exanthema. In the advanced stages II and III, neuroborreliosis in particular may be mimicked by various other conditions of both infectious and noninfectious etiology. Major examples are CEE (Central European Encephalitis), ehrlichiosis, chlamydial infections and multiple sclerosis. Currently, the biggest diagnostic problem is the non-standardized laboratory diagnostic work-up. For this reason, even in the presence of a positive or borderline IgG antibody result, unclear symptoms should prompt a differential diagnostic investigation.

[Pain, fatigue, depression after borreliosis. Antibiotics used up--what next?]. / Schmerzen, Müdigkeit, Depressionen nach Borrelieninfektion. Antibiotika ausgereizt--was jetzt?

Antibiotic treatment--usually stage-dependent in terms of the active agent, duration and form of application--is the central pillar in the management of Lyme disease. In the late stages of borreliosis, symptoms may persist despite extensive and repeated antibiotic treatment. In this phase, borreliosis-typical neuropathy and neuralgia, chronic fatigue and neuropsychological deficits predominate. Irrespective of whether renewed antibiotic treatment is indicated or not, symptomatic treatment must be continued.

[Stroke therapy. What is established, what is new?]. / Schlaganfalltherapie. Was hat sich bewährt, was ist neu?

Early treatment of a stroke, for example, on a stroke unit reduces neurological deficits and decisively improves survival. Of initial importance is an optimal supply of oxygen. Fever should be rapidly reduced, and blood sugar within the normal range also improves the prognosis. Only when blood pressure is very high is--moderate--lowering recommended. On the contrary, raising the blood pressure and cardiac output may have a positive effect on cerebral bloodflow with fewer neurological deficits resulting. In the case of immobile patients or patients with higher-grade paralysis, antithrombotic measures--increasingly utilizing low molecular-weight heparins--are indicated. Last but not least, early physiotherapy and logopedic treatment have an important role to play. Special forms of therapy such as thrombolysis can be carried out only in well equipped centers with experienced personnel. The use of oxygen-binding plasma substitutes and hypervolemic hemodilution are highly promising future therapeutic options.

Indacaterol once-daily is equally effective dosed in the evening or morning in COPD.

UNLABELLED: Indacaterol is a novel, inhaled, long-acting ß(2)-agonist providing 24-h bronchodilation with once-daily (o.d.) dosing in patients with COPD. In this double-blind, incomplete block crossover study, patients with moderate-to-severe COPD were randomised to receive three treatment cycles from: indacaterol 300 µg o.d. dosed PM or AM, salmeterol 50 µg twice daily or placebo, each for 14 days. Trough FEV(1) was measured 24 h after indacaterol, and 12 h after salmeterol. Ninety-six patients (mean age: 64 years; post-bronchodilator FEV(1) 57% predicted, FEV(1)/FVC 55%) were randomised; 83 completed. After 14 days, the difference vs. placebo in trough FEV(1) for PM indacaterol was 200 mL (p < 0.001 [primary analysis]) and for AM indacaterol was 200 mL (p < 0.001). Compared with salmeterol, trough FEV(1) for PM indacaterol was 110 mL higher (p < 0.001), and for AM indacaterol was 50 mL higher (p = NS). Over 14 days, vs. placebo, both PM and AM indacaterol improved the % of nights with no awakenings (by 11.9 and 8.1 points; p < 0.01); the % of days with no daytime symptoms (by 6.7 and 5.5 points; p < 0.05); and the % of days able to perform usual activities (by 6.7 and 7.8 points; p < 0.05). Indacaterol provided 24-h bronchodilation and improvement in symptoms regardless of whether taken regularly in the morning or evening. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT00615030.

Inhibition of KSP by ARRY-520 induces cell cycle block and cell death via the mitochondrial pathway in AML cells.

Kinesin spindle protein (KSP), a microtubule-associated motor protein essential for cell cycle progression, is overexpressed in many cancers and is a potential anti-tumor target. We found that inhibition of KSP by a selective inhibitor, ARRY-520, blocked cell cycle progression, leading to apoptosis in acute myeloid leukemia cell lines that express high levels of KSP. Knockdown of p53, overexpression of XIAP and mutation in caspase-8 did not significantly affect sensitivity to ARRY-520, suggesting that the response is independent of p53, XIAP and the extrinsic apoptotic pathway. Although ARRY-520 induced mitotic arrest in both HL-60 and Bcl-2-overexpressing HL-60Bcl-2 cells, cell death was blunted in HL-60Bcl-2 cells, suggesting that the apoptotic program is executed through the mitochondrial pathway. Accordingly, inhibition of Bcl-2 by ABT-737 was synergistic with ARRY-520 in HL-60Bcl-2 cells. Furthermore, ARRY-520 increased Bim protein levels prior to caspase activation in HL-60 cells. ARRY-520 significantly inhibited tumor growth of xenografts in SCID mice and inhibited AML blast but not normal colony formation, supporting a critical role for KSP in proliferation of leukemic progenitor cells. These results demonstrate that ARRY-520 potently induces cell cycle block and subsequent death in leukemic cells via the mitochondrial pathway and has the potential to eradicate AML progenitor cells.

Long-term antibiotic treatment with roxithromycin in patients with multiple sclerosis.

BACKGROUND: There are conflicting results concerning an association between Chlamydia pneumoniae and MS (multiple sclerosis). In the present study, we investigated a possible therapeutic option with antibiotics. PATIENTS AND METHODS: In our randomized, placebo-controlled double-blind study, 28 patients with the confirmed diagnosis of MS [61% relapsing-remitting MS (RR-MS), 32% secondary chronic-progressive MS (SP-MS) and 7% primary chronic progressive MS (PP-MS)] were treated over a time period of 12 months with three cycles of a 6-week oral antibiotic therapy with roxithromycin (300 mg per day) or placebo. RESULTS: No significant differences were observed in patients with RR-MS regarding the expanded disability status scale (EDSS) and the relapse rate when comparing treatment with roxithromycin and placebo. CONCLUSION: Our study shows that the patients with MS do not profit from a long-term antibiotic treatment with roxithromycin compared to placebo treatment. A causative connection between bacterial infections with C. pneumonia and MS therefore does seem very unlikely.

Fractioning the Hooper: a multiple-choice response format.

The Hooper Visual Organization Test (HVOT) provides an excellent illustration of the multifactorial nature of most neuropsychological tests. Although the HVOT clearly requires certain visual perceptual skills, the test also demands that the subject produce an overt verbal response - i.e., the name of the object that has been cut up and rearranged. Thus, individuals with disorders of confrontation naming may obtain low scores on the HVOT by virtue of their anomia, even if the primary perceptual skills that the HVOT purports to assess are intact. The present study was designed to minimize the demands of object naming on HVOT performance, by using a multiple choice format of the HVOT. Fourteen individuals with lateralized injury resulting from either cerebral vascular accident or cerebral contusion were administered the Boston Naming Test (BNT) and the standard version of the HVOT. Approximately 24 hours later, subjects were administered the Multiple-Choice Hooper Visual Organization Test (MC-HVOT). The MC-HVOT consisted of the 30 original HVOT stimuli presented with four response choices, including the correct response and three foils. A paired sample t test revealed that anomic subjects achieved a significantly greater number of correct responses on the MC-HVOT then under the standard HVOT administration. Subjects with both right and left hemisphere involvement benefited from diminished naming demands. Overall HVOT performance significantly improved when the object naming demand was reduced, resulting in a clearer assessment of visual integration skills. These findings may have significant implications for both interpretation of impairment and formulation of treatment recommendations.

Tick-borne encephalitis diagnosis in patients with inflammatory changes in the cerebrospinal fluid in a region with very low prevalence.

Tick-borne encephalitis (TBE)-IgG antibodies are used for the serologic detection of antigen contact caused by TBE infection or immunization. In the present study, enzyme-linked immune sorbent assay (ELISA) results from a group of patients with inflammatory changes in the cerebrospinal fluid (CSF) were re-examined using Western blot technology. The result of the TBE-IgG-ELISA was positive in 47 of the 904 sera samples tested. Retesting the sera with a Western blot confirmed this result in only 31.8% of the positive cases. In 134 of the 904 sera, the ELISA result was borderline. In 5.5% of these sera, the Western blot reacted specifically. The remaining 723 sera samples tested negative with the ELISA. Of these sera, 15 were selected randomly and retested with the Western blot; none of them tested positive. The high number of false positive ELISA results can be explained by the highly selected group of patients and the low prevalence of TBE in the region studied. In patients with meningitis or encephalitis with positive ELISA results and uncharacteristic clinical symptoms, the treating physician should consider the possibility of nonspecific reactions involving inflammatory mediators or cross-reactivity with other flaviviruses. The ELISA-mediated diagnosis of TBE should therefore be verified by means of the patient's history and clinical symptoms, as well as further serologic tests including the Western blot, the hemagglutination test and the neutralization test.