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Targeted cancer therapies exploit the continued dependence of cancer cells on oncogenic mutations. Such agents can have remarkable activity against some cancers, although antitumor responses are often heterogeneous, and resistance remains a clinical problem. To gain insight into factors that influence the action of a prototypical targeted drug, we studied the action of imatinib (STI-571, Gleevec) against murine cells and leukemias expressing BCR-ABL, an imatinib target and the initiating oncogene for human chronic myelogenous leukemia (CML). We show that the tumor suppressor p53 is selectively activated by imatinib in BCR-ABL-expressing cells as a result of BCR-ABL kinase inhibition. Inactivation of p53, which can accompany disease progression in human CML, impedes the response to imatinib in vitro and in vivo without preventing BCR-ABL kinase inhibition. Concordantly, p53 mutations are associated with progression to imatinib resistance in some human CMLs. Our results identify p53 as a determinant of the response to oncogene inhibition and suggest one way in which resistance to targeted therapy can emerge during the course of tumor evolution.

The analysis of rare chromosomal translocations in myeloproliferative disorders has highlighted the importance of aberrant tyrosine kinase signaling in the pathogenesis of these diseases. Here we have investigated samples from 679 patients and controls for the nonreceptor tyrosine kinase JAK2 V617F mutation. Of the 480 myeloproliferative disorder (MPD) samples, the proportion of positive cases per disease subtype was 30 (20%) of 152 for atypical or unclassified MPD, 2 of 134 (2%) for idiopathic hypereosinophilic syndrome, 58 of 72 (81%) for polycythemia vera, 24 of 59 (41%) essential thrombocythemia (ET), and 15 of 35 (43%) for idiopathic myelofibrosis. V617F was not identified in patients with systemic mastocytosis (n = 28), chronic or acute myeloid leukemia (n = 35), secondary erythrocytosis (n = 4), or healthy controls (n = 160). Homozygosity for V617F was seen in 43% of mutant samples and was closely correlated with chromosome 9p uniparental disomy. Homozygosity was significantly less common in ET compared with other MPD subtypes. In 53 cases analyzed, the median level of PRV1 expression was significantly higher in V617F-positive cases compared with cases without the mutation. We conclude that V617F is widespread in MPDs. Detection of this acquired mutation is likely to have a major impact on the way patients with MPD are diagnosed, as well as serving as an obvious target for signal transduction therapy.

Fractalkine is the only known member of the CX(3)C-chemokine family, and so is its receptor CX(3)CR1. Fractalkine, typically is expressed by neurons where it is inserted in the plasma membrane ("chemokine on a stalk"). It can, however, be clipped off by a specific enzyme and diffuse into the extracellular space. CX(3)CR1 is primarily expressed by microglia, the phagocytes of the brain. This study was aimed at studying gene expression changes in cultured rat microglia upon fractalkine stimulation using gene chip technology. Six genes turned out to be upregulated, amongst which milk-fat globule EGF factor-8 protein (MFG-E8) was the most surprising, but also the most revealing one. We hypothesize that it serves as a bridging molecule between apoptotic cells (neurons) and microglia. Since the docking to microglia is, in part, mediated by members of the integrin family, six of these molecules have been-post hoc-included in real-time PCR confirmations of chip results. Two of them-integrin alpha(2) and integrin beta(5)-were upregulated as well. These data provide a much closer look into molecular mechanisms involved in apoptosis of neurons and their removal by microglia.

The inpatient records of 44 case patients with West Nile virus infection hospitalized in 2002 were reviewed. Sixty-five percent of the case patients had encephalitis, and 35% had aseptic meningitis. There was no significant difference in the distribution of aseptic meningitis versus encephalitis among adults aged < or =50 years compared with adults aged > or =65 years. Focal weakness, likely due to anterior horn cell involvement, was present in 10 case patients (overall rate, 23%; rate among patients with encephalitis, 34%). Case patients with focal weakness who were aged < or =50 years had monoparesis, whereas those aged > or =65 years had paraparesis or quadriparesis. The overall mortality rate was 14%, and the mortality rate in patients aged > or =65 years was 35%. Increased age was associated with an increased mortality rate. The presence of paraparesis or quadriparesis was not independently predictive of mortality.

In 1936 Lemierre described an aggressive neck infection with a high mortality rate. In the original characterization, he describes a pharyngotonsillitis and/or peritonsillar infection followed by unilateral swelling and tenderness along the sternocleidomastoid muscle owing to septic thrombophlebitis of the internal jugular vein. Subsequent to invasion and thrombophlebitis of the internal jugular vein, Fusobacterium necrophorum septicemia occurs, with rigors, high fever, and septic thromboembolism to peripheral sites, especially the lungs and bones. This entity became known as Lemierre Syndrome. Hyperbaric Oxygen (HBO2) has been described as adjunctive treatment in two cases of postanginal septicemia. This case describes the combined approach to a case of Lemierre Syndrome in which HBO2 was added as an adjunct to the treatment, with a favorable and rapid improvement in the patient's condition.

Unlabelled: Irinotecan (CPT-11) has been shown to prolong survival and improve quality of life in comparison to best supportive care in colorectal cancer patients with pretreatment of bolus 5-fluorouracil (5-FU). After first-line 24-h high-dose (HD) 5-FU/folinic acid (FA) an objective response rate of 11% with 3-weekly CPT-11 350 mg/m was reported. In the present study we investigated weekly CPT-11 in combination with 24-h HD-5-FU/FA as second-line treatment after prior exposure to 24-h HD-5-FU. Synergy between 5-FU and CPT-11 is the rationale to combine both substances for second-line therapy in order to overcome resistance to 5-FU. Thirty-five patients were recruited in a single institution to receive 6 x weekly CPT-11 80 mg/m(2), FA 200 mg/m(2) and 24-h HD-5-FU 2000 mg/m(2). Treatment was repeated on day 57., Patient Characteristics: M/F=20/15, median WHO performance status 1, range (0-2). Thirty-four patients were evaluable for response: partial response 17% and no change 40%. Median time to progression and overall survival were 3.3 and 8.4 months, respectively. All patients were evaluable for toxicity analysis (National Cancer Institute Common Toxicity Criteria grade 3): leukocytopenia 3%, diarrhea 12% and vomiting/nausea 6%. Of the assigned doses, a median 100% of 5-FU and 92% of CPT-11 were administered during the first cycle of chemotherapy. We conclude that weekly CPT-11 and HD-5-FU/FA is an active and safe combination chemotherapy resulting in response rates in the upper range of other CPT-11-based second-line regimen. The toxicity profile in our series compared to 3-weekly CPT-11 seems favorable.

A substantial minority of patients with chronic myelogenous leukemia (CML) achieve a complete response (CR) to treatment with interferon-alpha (IFN), defined as the disappearance of Philadelphia chromosome-positive metaphases. Currently it is unclear how long IFN treatment should be continued for such patients. We used a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify levels of BCR-ABL transcripts in 297 peripheral blood specimens collected from 54 patients who had achieved CR with IFN. The median duration of observation was 1.9 years (range, 0.3-11.0 years). Total ABL transcripts were quantified as internal control and results were expressed as the ratio BCR-ABL/ABL. All 54 patients had molecular evidence of residual disease, although 3 patients were intermittently PCR negative. The median BCR-ABL/ABL ratio at the time of maximal response for each patient was 0.045% (range, 0%-3. 6%). During the period of observation 14 patients relapsed, 11 cytogenetically to chronic phase disease and 3 directly to blastic phase. The median ratio of BCR-ABL/ABL at maximal response was significantly higher in patients who relapsed than in those who remained in CR (0.49% versus 0.021%, P < 0.0001). Our findings show that the level of residual disease falls with time in complete responders to IFN, but molecular evidence of disease is rarely if ever eliminated. The actual level of minimal residual disease correlates with the probability of relapse. We suggest that for patients who reach CR, IFN should be continued at least until relatively low levels of residual leukemia are achieved. (Blood. 2000;95:62-66)

Bacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (alpha and beta). To assess the contributions of the alpha and beta subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids in Escherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different beta subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes.

Former studies revealed musk ambrette as a mutagen in Salmonella typhimurium TA 100 in the presence (+S9) but not in the absence (-S9) of an exogenous metabolizing system. To clarify the role of bacterial nitroreductases (NR) in the toxification of musk ambrette to mutagenic metabolites the compound was examined with the Salmonella/mammalian microsome assay using the NR deficient strain S.typhimurium TA 100 NR in the presence and absence of S9. Musk ambrette showed mutagenicity in Salmonella typhimurium TA 100 (+S9) but no mutagenicity in the NR deficient strain TA 100 NR (+S9). Additionally, no mutagenicity was detected in both TA 100 (-S9) and TA 100 NR (-S9). These results indicate the need for both mammalian microsomal enzymes and bacterial nitroreductases to cause the mutagenicity of musk ambrette.

In the present study, musk xylene (MX) and musk ketone (MK) were examined for their potency to induce toxifying enzymes in the liver of Sprague-Dawley rats, using an in vivo/in vitro model. After i.p. application of 10, 20 and 40 mg/day MX and MK over a period of 5 days, 9000 x g liver fractions (S9M) were used to study the toxification of a number of well-known pregenotoxicants in the SOS chromotest, i.e., benzo[a]pyrene (B[a]P), 2-aminoanthracene (2-AA), and aflatoxin B1 (AFB1). The genotoxic potencies of B[a]P, 2-AA and AFB1 in the presence of S9M were compared to those obtained in the presence of S9 fractions of untreated animals (S9O, negative control). S9M fractions derived from MK-treated rats showed an increased potency to toxify B[a]P, 2-AA and AFB1 in comparison to S9O fractions (for instance: TIP[toxifying induction potency] = 70 per nmol AFB1 using 10 mg MK treatment). In comparison, S9M fractions from MX-pretreated rats exhibited an increased genotoxicity only when using 2-AA (TIP = 0.04) and AFB1 (TIP = 61) as pregenotoxicants, but not when using B[a]P. To summarize the results, both MX and MK were strong inducers of toxifying liver enzymes. Therefore, these compounds seem to be cogenotoxicants for a number of well-known pregenotoxicants. Synergistic effects were found when using inducers of toxifying enzymes and pregenotoxicants in the in vivo/in vitro induction model.

In the present study a mixture containing the 11 PCB major components identified in fatty tissues of children was examined for its potency to enhance the toxification of pregenotoxicants (cogenotoxicity) in the liver. As a basis for the study GC/MS PCB analyses of 207 fatty tissue samples of children were used. The PCB mixture was produced on this basis. As a model for the identification of the cogenotoxic potency of the PCB mixtures an in vivo/in vitro enzyme induction assay was developed. The goal of the study was to clarify the question, whether the in vivo pretreatment of rats with a complex PCB pattern derived from children led to a synergism of cogenotoxicants and pregenotoxicants with regard to the enhancement of the in vitro toxification of benzo[a]pyrene (B[a]P) and 2-aminoanthracene (2-AA) to DNA reactive metabolites. Using the SOS-Chromotest as the in vitro part of the induction assay, all liver enzyme fractions of PCB pretreated rats (S9PCB) showed an increase of the toxification of the pregenotoxicants B[a]P and 2-AA in comparison to enzyme factions of untreated animals (S9(0)). With regard to the reactivity pattern it may be supposed that the PCB mixture probably induced cytochrome P450-dependent oxigenases of the subclasses CYP1A1 and CYP1A2. Additionally, it seems to be of interest that the use of S9(0) fractions did not lead to any or only to weak toxification of B[a]P and 2-AA. Thus, a synergism of cogenotoxicants and pregenotoxicants could be confirmed. PCB could be identified in fatty tissues of children in amounts up to 1 mg/kg. Additionally, pregenotoxicants like polycyclic aromates, mycotoxins and/or aminocontaining compounds, are available in almost all environmental sources. Therefore, from the present point of view, a genetic risk caused by PCB in humans (children) cannot be excluded.

In the present study a new in vivo/in vitro animal model was used to study the ability and potency of musk ketone and musk xylene to induce liver specific oxygenases (in vivo) which are necessary of toxify different premutagens, pregenotoxicants and/or precarcinogens to the ultimate DNA damaging agents. Therefore, rats were pretreated with 10, 20 and 40 mg/d nitro musk (NMV) for 5 days by intraperitoneal (i.p.) injection. Then the postmitochondrial fractions of the hepatocytes (S9M) were used to examine the metabolic potency for toxification of the pregenotoxicants benzo[a]pyrene (B[a]P) and 2-aminoanthracene (2-AA) using the SOS chromotest (in vitro). Furthermore, musk xylene, musk ketone, musk ambrette, musk moskene and musk tibetene were examined for their mutagenicity in the Salmonella/microsome assay using S. typhimurium TA97, TA98, TA100 and TA102 and for their genotoxicity in the SOS chromotest using Escherichia coli PQ37 (sfiA::lacZ) in the presence and absence of an exogenous metabolizing system (S9 of PCB induced rats = S9A). Both musk ketone and musk xylene were identified als inducers of toxifying enzymes (oxygenases) in rat liver. Using the in vivo/in vitro model these isoenzyme inductions led to a metabolisation (toxification) of the pregenotoxicants benzo[a]pyrene (B[a]P) and/or 2-aminoanthracene (2-AA) (cogenotoxicity). Using S9M fractions of rats which were i.p.-pretreated with 5 x 40 mg musk ketone the induction factor in the SOS chromotest was IFmax = 4.0 by using 1 nmole B[a]P and IFmax > 4.0 by using 20 nmole 2-AA. Thus, musk ketone seems to be a Cytochrome P450 1A1 and 1A2 isoenzyme inducer in mammals. On the other hand the S9M fractions of musk xylene pretreated rats showed only a toxification of 2-AA (IFmax = 3.0). Therefore, a synergistic effect of enzyme inducers, i.e. musk xylene and musk ketone, and pregenotoxicants, i.e. B[a]P and 2-AA, regarding DNS damaging effects was identified. Musk ambrette showed high mutagenicity in S. typhimurium TA100 (500 His(-)-revertants per mumole, +S9A). Unexpectedly, these DNA damaging effects were not caused by bacterial nitroreductases but by rat S9A metabolisation (!). SOS inducing DNA damages in E. coli PQ37 were not produced (IFmax < 1.5). On the basis of the results presented and under consideration of the concentrations of NMV, other cogenotoxicants and pregenotoxicants such as B[a]P and 2-AA in environmental samples and human tissues, a genotoxic risk for humans has to be assumed.

Enucleated human eyes were banded with metal and silicone bands to produce reductions in their diameter of 2 mm and 4 mm. The ocular rigidity produced by each banding material at each diameter was measured in the pressure range of 10 mmHg to 40 mmHg. Metal bands produced mild reductions in ocular rigidity that were significantly (P less than 0.05 to 0.01) lower than the control ocular rigidities in some pressure ranges. Silicone bands produced large reductions in ocular rigidity that were significantly (P less than 0.01) lower than ocular rigidities observed in metal-banded or control conditions in all pressure ranges. The influence of the elastic silicone banding material on ocular rigidity was greater than the influence of altered shape and wall stress that occurred with metal banding.

We studied the intraocular pressure changes produced in five eye bank eyes by Perkins, Tono-Pen, and Schiötz tonometry performed by experienced and inexperienced personnel. When all users were considered together, Perkins tonometry produced a mean intraocular pressure increase of 0.7 mm Hg, significantly less than the mean increase of 12.1 mm Hg produced by Tono-Pen tonometry (P less than .05) or the mean increase of 16.5 mm Hg produced by Schiötz tonometry (P less than .01). There was no statistically significant difference between the intraocular pressure increase produced by Tono-Pen or Schiötz tonometry. Tonometry performed by inexperienced Tono-Pen users and experienced or inexperienced Schiötz users produced a significantly greater increase in intraocular pressure than that performed by experienced Tono-Pen users (P less than .05), and an extremely significant increase compared to tonometry performed by experienced or inexperienced Perkins users (P less than .01). The marked increase in intraocular pressure produced by Tono-Pen tonometry suggests that hand-held electronic applanation tonometers should be used with caution in eyes with a weakened cornea or sclera.