Drug monitoring detects under- and overdosing in patients receiving 5-fluorouracil-containing chemotherapy-results of a prospective, multicenter German observational study.

INTRODUCTION: Body surface area (BSA)-based dosing of 5-fluorouracil (5-FU) results in marked inter-individual variability in drug levels, whereas determination of plasma 5-FU concentration and area under the curve (AUC) is a more precise dosing method but has not been integrated into clinical routine. We conducted a multicenter, prospective study to study 5-FU AUC distributions and assess clinical factors predicting therapeutic dosing in patients receiving BSA-dosed 5-FU. METHODS: Between June 2017 and January 2018, a total of 434 patients receiving continuous, infusional BSA-dosed 5-FU from 37 sites in Germany were included. Plasma 5-FU concentration and AUC were measured in venous blood samples at steady state. The primary objective was to determine 5-FU AUC distributions in relation to the target range, which is defined as 20-30 mg × h/l. The second objective was to explore clinical parameters that correlate with achievement of 5-FU AUC target range. RESULTS: The primary tumor was mainly located in the gastrointestinal tract (96.3%), with colorectal cancer being the most common (71.2%) tumor entity. 5-FU was administered as monotherapy (8.1%) or as part of FOLFOX (33.2%), FOLFIRI (26.3%), or other regimens (12.4%). Treatment setting was adjuvant (31.3%) or metastatic (64.5%). The median AUC was 16 mg × h/l. Only 20.3% of patients received 5-FU treatment within the target range, whereas the majority of patients (60.6%) were underdosed and 19.1% of patients were overdosed. In the univariate logistic regression, treatment setting was the only clinical parameter that significantly correlated with achievement of the target range. Patients treated in the metastatic setting had a 2.1 (95% confidence interval 1.186-3.776, P = 0.011) higher odds to reach the target range compared with patients treated in the adjuvant setting. CONCLUSIONS: The majority of patients received suboptimal doses of 5-FU using BSA dosing. Therapeutic drug monitoring of 5-FU is an option for optimized individualized cancer therapy and should be integrated into the clinical practice.

Continuous magnetic resonance perfusion imaging acquisition during systemic thrombolysis in acute stroke.

BACKGROUND: Early recanalization and increase in collateral blood supply are powerful predictors of favourable outcome in acute ischaemic stroke. The factors contributing to the heterogeneous response to intravenous thrombolysis therapy in individual patients, however, are not fully understood. The on-going single-centre 'MR perfusion imaging during thrombolysis' study uses repetitive arterial spin labelling (ASL) measurements to characterize the haemodynamic processes in acute stroke during therapy. The first milestone was to develop an appropriate infrastructure for thrombolysis in the magnetic resonance imaging (MRI) scanner without time delay and ensuring optimal patient safety and care. METHODS: Between February and December 2011, 16 patients with acute neurological symptoms suggestive of hemispheric stroke within 4.5 h after symptom onset were included. In addition to clinical data, we documented the time from onset to arrival at the hospital, start and duration of MRI examination, start of thrombolytic therapy, and complications. The decision to thrombolyse was made after a routine stroke MRI protocol. During the 60-min systemic thrombolysis, repetitive ASL perfusion imaging was acquired, providing non-invasive information on cerebral perfusion. Continuous ECG monitoring, pulse oximetry, blood pressure measurements every 5 min, and short neurological assessments every 15 min were performed in every patient. RESULTS: The median initial NIHSS score of the patients presenting with a mean of 84 min after onset was 4 (range 2-18). MRI examination was initiated within a mean of 45 min after arrival at the hospital. Five patients identified as stroke mimics were not treated with recombinant tissue plasminogen activator (rt-PA), and in 1 case with basilar artery occlusion bridging therapy was performed outside the scanner. In the remaining 10 patients, rt-PA therapy was started in the scanner directly after decision making on the basis of clinical information and baseline MRI. The mean door-to-needle time was 60 min (range 44-115) including approximately 10 min needed for acquiring informed consent. While 4 patients required antihypertensive treatment, no relevant complications were encountered. CONCLUSIONS: Fast and safe medical care in patients during systemic thrombolysis in the MRI scanner is feasible. Despite the process of obtaining informed consent, with a dedicated and experienced stroke team the door-to-needle time can be kept in a range recommended by current guidelines. Continuous real-time information about the dynamics of cerebral perfusion from ASL perfusion in acute stroke patients undergoing thrombolysis may provide additional information for the understanding of the events following acute arterial obstruction and its course.

Awareness and perception of intra-abdominal adhesions and related consequences: survey of gynaecologists in German hospitals.

OBJECTIVE: Intra-abdominal adhesion formation after abdominal surgery is the most common postsurgical complication, and the consequences are a considerable burden for patients, surgeons and health systems. Since a wide variety of factors influence adhesion formation, it is difficult to define clear guidelines on how to reduce adhesion formation in daily practice. Given this dilemma, this study assessed the awareness and perception of adhesion formation among gynaecologists in Germany in order to define a baseline for further research and education. STUDY DESIGN: The Clinical Adhesion Research and Evaluation (CARE) group of the University of Giessen designed a questionnaire that was sent to the heads of all gynaecological departments in Germany. The director or one of the surgical consultants was asked to complete the questionnaire and return it for evaluation. RESULTS: The completed questionnaire was returned by 279 of 833 gynaecological departments. Interviewed surgeons expected adhesions to form in 15% of cases after laparoscopy and 40% after laparotomy. Before surgery, 83.1% of the respondents told their patients about the risk of prior adhesion formation. More than 60% believed that postsurgical adhesion accounts for major morbidity. Infections within the abdomen, previous surgery and extensive tissue trauma were thought to have the most influence on adhesion formation. Risk of adhesion formation was thought to be highest in endometriosis and adhesiolysis surgery. The respondents agreed on performing adhesiolysis in symptomatic but not in all patients. Only 38.4% used adhesion reduction agents regularly. A total of 65.1% of a repertoire of adhesion prevention agents were familiar to the interviewed surgeons. Only 22.0% of them used anti-adhesion products in clinical practice. In general, the respondents were uncertain whether these products play an important role in adhesion reduction, represented by a range of 1.97+/-0.98% on a scale from 0 to 4. CONCLUSIONS: Even though postoperative adhesions are recognized as a major cause for morbidity, and it is widely agreed that infections, extensive tissue trauma and surgery lead to adhesion formation, there is uncertainty about the treatment and prophylactic strategies for dealing with adhesions. This dilemma reflects the awareness and perception of gynaecologists in Germany and is an initial point for further research.

[Orolingual angioedema under systemic thrombolysis with rt-PA: an underestimated side effect]. / Orolinguales Angioödem unter systemischer Thrombolyse mit rt-PA : Eine unterschätzte Nebenwirkung.

BACKGROUND: Orolingual angioedema (OA) is an unappreciated complication of acute stroke treatment with recombinant tissue plasminogen activator (rt-PA). It has been described in 2% of patients receiving thrombolysis, and it seems that patients taking angiotensin-converting enzyme inhibitors are especially at risk. Even though the presentation is generally unilateral and limited to lips and tongue, an extension of edema to the oropharynx may lead to life-threatening upper airway obstruction. MATERIAL AND METHODS: In a retrospective analysis of clinical data of 407 patients treated with systemic rt-PA thrombolysis between January 2006 and October 2008 in our department, we describe the occurrence and clinical presentation of OA. RESULTS: Nine of 407 patients (2.2%) showed clinical signs of OA. Typical presentations of OA are illustrated in case reports describing two of these patients and are completed by an overview of the current literature. DISCUSSION: Besides prophylactic inspection of the oral cavity during and after thrombolysis, therapeutic options in case of OA include early intravenous antihistaminergic therapy and protective intubation.

Cortical current density reconstruction of interictal epileptiform activity in temporal lobe epilepsy.

OBJECTIVE: To investigate the value of cortical current density (CCD) reconstruction in localizing intracranial generators of interictal epileptiform activity in mesial and lateral temporal lobe epilepsy (TLE). METHODS: Non-linear minimum L(1)-norm CCD reconstruction (with current sources restricted to the individual cortical surface and a realistic boundary element method (BEM) head model) was used to localize and to study the propagation of interictal epileptiform EEG activity in 13 pre-surgical patients with TLE. RESULTS: In all but one patient with mesial temporal lesions, an initial activation maximum corresponding to the ascending part of averaged sharp waves was found in the ipsilateral anterior basolateral temporal lobe, mostly extending up to the affected mesial structures whose resection rendered the patients seizure-free. In all 3 patients with lateral temporal lesions, the activation was initially confined to temporal neocortex immediately adjacent to the epileptogenic lesion. Towards the peak of sharp waves, two patients showed a propagation of interictal activity to anterior and posterior and partly contralateral temporal regions. A conventional EEG analysis based on amplitude maxima or phase reversal would have missed the initial onset zone. CONCLUSIONS: The findings demonstrate that CCD reconstruction can be a valuable additional non-invasive component in the multimodal pre-surgical evaluation of epilepsy patients.

Predominantly BCR-ABL negative myeloid precursors in interferon-alpha treated chronic myelogenous leukemia: a follow-up study of peripheral blood colony-forming cells with fluorescence in situ hybridization.

The mechanism and target cell of the life-prolonging effect of interferon-alpha (IFN-alpha) in chronic myelogenous leukemia (CML) are controversial. We studied the influence of IFN-alpha treatment on the frequency of malignant hematopoietic precursor cells in the peripheral blood (PB) of CML patients during the course of the disease. PB 10-day colony-forming cells (PB-CFCs) were assessed with regard to their quantity, lineage distribution, and BCR-ABL status, as determined by fluorescence in situ hybridization (FISH). PB-CFC numbers were determined in 39 patients (29 in the chronic phase, 6 in an advanced stage, and 4 with progression to an advanced stage during follow-up). Thirty-one patients were evaluated either once or several times to determine the BCR-ABL status of the colonies. BCR-ABL negative PB-CFCs were detectable at diagnosis in 5 of 11 patients. A major reduction of BCR-ABL positive colonies to <25% of PB-CFCs was observed in 10/13 determinable IFN-alpha treated patients in early and late chronic phases, indicating a high proportion of BCR-ABL negativity at the clonogenic cell level. In contrast, only 3 of these patients had a cytogenetic response of <25% Philadelphia chromosome (Ph1)-positive metaphases in bone marrow cytogenetics. Treatment with IFN-alpha and/or hydroxyurea (HU) during chronic phase was accompanied by a reduction of PB-CFCs to subnormal levels (median 24 CFCs/ml) compared to controls (median 207 CFCs/ml), untreated patients in chronic phase (median 25,979 CFCs/ml), and patients with advanced disease (median 6,047 CFCs/ml). In blast crisis (6 patients), all colonies tested were BCR-ABL positive. Our results show that IFN-alpha treatment leads to a marked reduction of malignant myeloid precursor cells in the PB of CML patients, which exceeds the degree of cytogenetic remission. This offers an explanation for the good therapeutic efficacy and even life-prolonging effect of IFN-alpha, which is also observed in cytogenetic non-responders.

Novel chicken CXC and CC chemokines.

Upon stimulation with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes factors with cytokine activity. To characterize these molecules, representational difference analysis with RNA of LPS-induced and uninduced HD-11 cells was performed. Two cDNA clones were isolated that code for polypeptides with structural features of chemokines. cDNA K60 codes for a novel CXC chemokine of 104 residues including a putative signal peptide of 20 amino acids at the N-terminus. It is 67% identical to the previously cloned chicken chemokine 9E3/CEF4. K60 exhibits a similar degree of sequence identity to human interleukin 8 and other related CXC chemokines (about 50%), rendering straight-forward predictions of its biological properties difficult. cDNA K203 codes for a novel CC chemokine of 89 amino acids including a putative N-terminal signal peptide of 21 residues. It is 43% identical to a previously characterized chicken protein with homology to mammalian macrophage inflammatory protein 1beta (MIP-1beta). K203 exhibits about 50% sequence identity to human MIP-1beta and other related CC chemokines.

Fatal liver failure associated with valproate therapy in a patient with Friedreich's disease: review of valproate hepatotoxicity in adults.

PURPOSE: Valproate (VPA)-associated hepatotoxicity is usually considered a problem of young children with polytherapy, mental retardation, and underlying metabolic defects. METHODS: An adult patient with fatal liver failure during treatment with VPA is presented, and a review of the literature on other adult patients is given. RESULTS: A 29-year-old female patient with Friedreich's ataxia and partial seizures with acute liver failure during VPA treatment is reported. The first symptoms of liver failure (i.e., apathy during febrile upper airway infection) occurred 2 months after starting VPA therapy. VPA was discontinued 10 days later on hospital admission, when she had hepatic encephalopathy and severe bleeding diathesis. The patient died of severe liver failure and bronchopneumonia after 4 weeks of supportive treatment. CONCLUSIONS: Twenty-six adult patients (>17 years) with VPA-associated fatal hepatotoxicity have been reported in the literature. Of the 26 adult patients, three were receiving VPA monotherapy. The age ranged between 17 and 62 years. The duration of VPA treatment before the first symptom varied between 7 days and 6 years. Twelve of the 26 affected adults had no underlying disease or a clearly nonmetabolic and non-hepatic disease. Therefore VPA-associated severe side effects also must be considered in adult patients without any evidence of a metabolic defect or underlying neurologic disease.

Does long-term culture favor normal clonogenic cells from interferon-treated patients with chronic myelogenous leukemia?

We have tested whether peripheral blood mononuclear cells (PBMNCs) from interferon (IFN)-treated patients may lose residual BCR-ABL sequence-positive progenitor cells when long-term cultured for 35 days on allogeneic stromal cells. IFN-treated patients have low white blood cell counts and a fair number of BCR-ABL-negative colony-forming cells in the peripheral blood. Particularly, IFN responders show increased numbers of normal hematopoietic cells. We have quantitatively analyzed progenitor cells in PBMNCs of IFN-treated patients by combining the clonogenic assay in semisolid medium with interphase fluorescent in situ hybridization (FISH). Thus, the identification is possible of the BCR-ABL status of colony-forming progenitor cells. In IFN-treated patients, the number of BCR-ABL-positive CFCs is considerably decreased and BCR-ABL-negative CFCs appear in the peripheral blood. We could show that after LTC for 35 days of the same PBMNCs on irradiated allogeneic normal stromal cells residual BCR-ABL sequence-positive CFCs were still present. In some cases the relative number of BCR-ABL sequence-positive CFCs was found to be increased after LTC. A minor proportion of blood samples from IFN-treated patients did not give rise to CFCs after LTC on allogeneic stromal cells (three of 10 patients). Inter- and intraindividual variations can be found with regard to loss or gain of BCR-ABL sequence-positive colonies after LTC. We conclude that early CML progenitor cells persist in the peripheral blood of IFN-treated patients and that a certain proportion may survive long-term culture.

Sex chromosome linkage of chicken and duck type I interferon genes: further evidence of evolutionary conservation of the Z chromosome in birds.

Type I interferons (IFNs) are a family of proteins that are predominantly expressed in response to viral infection. Two serologically distinct forms of type I IFN, designated ChIFN1 and ChIFN2, have recently been recognized in the chicken. ChIFN1 is encoded by a cluster of ten or more intronless genes, whereas ChIFN2, whose primary sequence is 57% identical, is encoded by a single intronless gene. By fluorescence in situ hybridization we now demonstrate that the genes for ChIFN1 and ChIFN2 are all located on the short arm of the chicken Z chromosome. This assignment was confirmed by results that showed that DNA from male (ZZ) chickens yielded approximately twofold stronger Southern blot signals with ChIFN1 and ChIFN2 hybridization probes than DNA from females (ZW). Attempts to determine differences in IFN production between male and female chickens failed owing to a high degree of variation in virus-induced IFN expression between individuals of both sexes. Sex linkage of IFN genes was also observed in domestic ducks: fluorescence in situ hybridization of duck metaphase chromosomes with a duck type I IFN probe was confined to the terminal region of the long arm of the Z chromosome. Thus, in contrast to mammals, which have their IFN genes on autosomes, birds have the type I IFN genes on the sex chromosome.

Promoter structures and differential responses to viral and nonviral inducers of chicken type I interferon genes.

Two serologically distinct type I interferons (IFNs), designated ChIFN1 and ChIFN2, are known in the chicken. ChIFN1 is encoded by a family of 10 or more genes, whereas ChIFN2 is encoded by a single gene. We show here that ChIFN1 and ChIFN2 transcripts are both strongly induced by Newcastle disease virus in primary chicken macrophages. By contrast, oral administration of the imidazoquinoline S-28463, which selectively induces IFN-alpha in mammals, led to a rapid accumulation of ChIFN1 (but not ChIFN2) transcripts in adult chicken spleen and thymus. The 5'-upstream region of the ChIFN2 gene contains a NF-kappaB consensus motif flanked by a sequence element that could serve as a binding site for transcription factor IRF-1, reminiscent of mammalian IFN-beta promoters, and it mediated powerful virus inducibility in a duck fibroblast cell line when cloned in front of a promoterless luciferase reporter gene. The 5'-upstream region of the cloned ChIFN1 gene contains two putative binding sites for IRF-1, but lacks NF-kappaB-binding sites, and it did not respond well to virus in transfected cells. Thus, the promoters of ChIFN1 and ChIFN2 genes not only exhibited differential responses to nonviral inducers in vivo, but also differed in structure and response to virus in transfected cells. These findings indicate that ChIFN2 represents the avian homolog of mammalian IFN-beta, whereas ChIFN1 seems to correspond to mammalian IFN-alpha.

A chicken homolog of mammalian interleukin-1 beta: cDNA cloning and purification of active recombinant protein.

Upon induction with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes an activity that stimulates the synthesis of a CXC chemokine in the chicken fibroblast cell line CEC-32. We used a cDNA expression cloning strategy in COS cells to characterize this activity. The isolated cDNA clone codes for a polypeptide of 267 amino acids which lacks a hydrophobic N-terminal domain that could serve as secretory signal. Sequence homology and structural features indicate that this protein is the chicken homolog of mammalian interleukin-1 beta (ChIL-1 beta). Northern blot analysis showed that ChIL-1 beta RNA is quickly induced in blood monocyte-derived macrophages reaching maximal levels within one hour after onset of LPS treatment. To test for biological activity of putative mature ChIL-1 beta, a cDNA fragment comprising amino acids 106 to 267 of the open reading frame was expressed in Escherichia coli so that the resulting polypeptide carried a histidine tag at its N-terminus for easy purification by nickel chelate affinity chromatography. Purified His-ChIL-1 beta potently induced CXC chemokine RNA synthesis in CEC-32 cells. When injected intravenously into adult chickens, it quickly induced a transient increase in serum corticosterone levels.

Dominant-negative mutants of human MxA protein: domains in the carboxy-terminal moiety are important for oligomerization and antiviral activity.

Human MxA protein is an interferon-induced 76-kDa GTPase that exhibits antiviral activity against several RNA viruses. Wild-type MxA accumulates in the cytoplasm of cells. TMxA, a modified form of wild-type MxA carrying a foreign nuclear localization signal, accumulates in the cell nucleus. Here we show that MxA protein is translocated into the nucleus together with TMxA when both proteins are expressed simultaneously in the same cell, demonstrating that MxA molecules form tight complexes in living cells. To define domains important for MxA-MxA interaction and antiviral function in vivo, we expressed mutant forms of MxA together with wild-type MxA or TMxA in appropriate cells and analyzed subcellular localization and interfering effects. An MxA deletion mutant, MxA(359-572), formed heterooligomers with TMxA and was translocated to the nucleus, indicating that the region between amino acid positions 359 and 572 contains an interaction domain which is critical for oligomerization of MxA proteins. Mutant T103A with threonine at position 103 replaced by alanine had lost both GTPase and antiviral activities. T103A exhibited a dominant-interfering effect on the antiviral activity of wild-type MxA rendering MxA-expressing cells susceptible to infection with influenza A virus, Thogoto virus, and vesicular stomatitis virus. To determine which sequences are critical for the dominant-negative effect of T103A, we expressed truncated forms of T103A together with wild-type protein. A C-terminal deletion mutant lacking the last 90 amino acids had lost interfering capacity, indicating that an intact C terminus was required. Surprisingly, a truncated version of MxA representing only the C-terminal half of the molecule exerted also a dominant-negative effect on wild-type function, demonstrating that sequences in the C-terminal moiety of MxA are necessary and sufficient for interference. However, all MxA mutants formed hetero-oligomers with TMxA and were translocated to the nucleus, indicating that physical interaction alone is not sufficient for disturbing wild-type function. We propose that dominant-negative mutants directly influence wild-type activity within hetero-oligomers or else compete with wild-type MxA for a cellular or viral target.

A novel BCR-ABL fusion gene (e6a2) in a patient with Philadelphia chromosome-negative chronic myelogenous leukemia.

A novel variant of the chimeric BCR-ABL mRNA transcript was detected in a patient with Philadelphia chromosome-negative (Ph-) chronic myelogenous leukemia (CML) by multiplex reverse-transcription polymerase chain reaction (RT-PCR). Sequence analysis of the fusion region of the amplified cDNA fragment showed an in-frame joining of exon e6 of the BCR gene and exon a2 of the ABL gene, giving rise to an e6a2 BCR-ABL transcript. This finding was confirmed by Southern blot analysis using a specific probe corresponding to intron 6 of the BCR gene, whereas conventional Southern blot for rearrangement of the major breakpoint cluster region (M-bcr) was negative. Western blot studies detected a BCR-ABL protein slightly larger than p185 BCR-ABL. Metaphase fluorescence in situ hybridization showed an insertion of ABL material into the BCR region without reciprocal BCR translocation. The findings in this case show that atypical BCR-ABL transcripts are detectable even in Ph- CML patients without M-bcr-rearrangements. Multiplex PCR using primers that allow for amplification of all known BCR-ABL transcripts is an appropriate method to exclude these rare variants.

A family of genes coding for two serologically distinct chicken interferons.

Southern blot analysis and screening of a genomic lambda phage library with the previously cloned chicken interferon (IFN) cDNA indicated that the chicken genome contains at least 10 IFN genes. A particularly strongly hybridizing phage clone that we analyzed in more detail carried a head to tail arrangement of three intron-less IFN genes that differed from each other and from the cloned chicken IFN cDNA by only a few base changes. The primary translation products of these three IFN genes consist of 193 amino acids, and the mature proteins are composed of 162 amino acids. All three genes of this IFN family, designated IFN1, yielded active chicken IFN when expressed individually in transfected COS7 cells. A weakly hybridizing phage clone contained an additional intron-less chicken IFN gene, designated IFN2, whose product was 57% identical to chicken IFN1. Southern blot analysis suggested that the chicken genome contains a single IFN2 gene. The primary translation product of IFN2 consists of 203 amino acids, and the mature protein is composed of 176 amino acids. Purified recombinant chicken IFN2 from Escherichia coli had a specific antiviral activity of about 10(6) units/mg, which was about 20-fold lower than that of chicken IFN1 purified in parallel. The antiviral activity of chicken IFN2 from E. coli or from transfected COS7 cells could not be neutralized by antiserum to recombinant chicken IFN1. Thus, like mammals, the chicken has a large number of type I IFN genes that code for at least two serologically distinct antiviral activities.

Verbal memory deterioration after unilateral infarct of the internal capsule in an adolescent.

After an infarction in the territory of the anterior perforating arteries, a 15-year-old, previously healthy, left-handed patient developed considerable verbal long term memory disturbances which could be followed up and tested for more than nine months. An extensive memory test battery was used to determine spared and impaired functions. The patient had remote memory disturbances with respect to personal events for the last 5 years and problems in all verbal task tested which required remembering items over time periods exceeding an hour. The patient was indistinguishable from control subjects on short-term memory tests and on a number of nonverbal learning and recognition tests. The crucial lesion for the observed deficits in the genu of the left internal capsule was assumed to have disrupted the anterior and inferior thalamic peduncles, fornix (column), stria terminalis, anterior commissure and the medial part of the globus pallidus. This infarct, therefore, most likely damaged traversing fibres which intercommunicate within the Papez circuit and the basolateral limbic circuit and which in part provide access to cortical memory representing areas.