A systematic approach to assess locoregional differences in angiogenesis.

Skeletal muscle tissue differs with regard to the abundance of glycolytic and oxidative fiber types. In this context, capillary density has been described to be higher in muscle tissue with more oxidative metabolism as compared to that one with more glycolytic metabolism, and the highest abundance of capillaries has been found in boneward-oriented moieties of skeletal muscle tissue. Importantly, capillary formation is often analyzed as a measure for angiogenesis, a process that describes neo-vessel formation emanating from preexisting vessels, occurring, i.e., after arterial occlusion. However, a standardized way for investigation of calf muscle capillarization after surgically induced unilateral hind limb ischemia in mice, especially considering these locoregional differences, has not been provided so far. In this manuscript, a novel, methodical approach for reliable analysis of capillary density was established using anatomic-morphological reference points, and a software-assisted way of capillary density analysis is described. Thus, the systematic approach provided conscientiously considers intra-layer differences in capillary formation and therefore guarantees for a robust, standardized analysis of capillary density as a measure for angiogenesis. The significance of the methodology is further supported by the observation that capillary density in the calf muscle layers analyzed negatively correlates with distal lower limb perfusion measured in vivo.

Spatio-temporal dynamics induced by competing instabilities in two asymmetrically coupled nonlinear evolution equations.

Pattern formation often occurs in spatially extended physical, biological, and chemical systems due to an instability of the homogeneous steady state. The type of the instability usually prescribes the resulting spatio-temporal patterns and their characteristic length scales. However, patterns resulting from the simultaneous occurrence of instabilities cannot be expected to be simple superposition of the patterns associated with the considered instabilities. To address this issue, we design two simple models composed by two asymmetrically coupled equations of non-conserved (Swift-Hohenberg equations) or conserved (Cahn-Hilliard equations) order parameters with different characteristic wave lengths. The patterns arising in these systems range from coexisting static patterns of different wavelengths to traveling waves. A linear stability analysis allows to derive a two parameter phase diagram for the studied models, in particular, revealing for the Swift-Hohenberg equations, a co-dimension two bifurcation point of Turing and wave instability and a region of coexistence of stationary and traveling patterns. The nonlinear dynamics of the coupled evolution equations is investigated by performing accurate numerical simulations. These reveal more complex patterns, ranging from traveling waves with embedded Turing patterns domains to spatio-temporal chaos, and a wide hysteretic region, where waves or Turing patterns coexist. For the coupled Cahn-Hilliard equations the presence of a weak coupling is sufficient to arrest the coarsening process and to lead to the emergence of purely periodic patterns. The final states are characterized by domains with a characteristic length, which diverges logarithmically with the coupling amplitude.

Analysis of magnetosome chains in magnetotactic bacteria by magnetic measurements and automated image analysis of electron micrographs.

Magnetotactic bacteria (MTB) align along the Earth's magnetic field by the activity of intracellular magnetosomes, which are membrane-enveloped magnetite or greigite particles that are assembled into well-ordered chains. Formation of magnetosome chains was found to be controlled by a set of specific proteins in Magnetospirillum gryphiswaldense and other MTB. However, the contribution of abiotic factors on magnetosome chain assembly has not been fully explored. Here, we first analyzed the effect of growth conditions on magnetosome chain formation in M. gryphiswaldense by electron microscopy. Whereas higher temperatures (30 to 35°C) and high oxygen concentrations caused increasingly disordered chains and smaller magnetite crystals, growth at 20°C and anoxic conditions resulted in long chains with mature cuboctahedron-shaped crystals. In order to analyze the magnetosome chain in electron microscopy data sets in a more quantitative and unbiased manner, we developed a computerized image analysis algorithm. The collected data comprised the cell dimensions and particle size and number as well as the intracellular position and extension of the magnetosome chain. The chain analysis program (CHAP) was used to evaluate the effects of the genetic and growth conditions on magnetosome chain formation. This was compared and correlated to data obtained from bulk magnetic measurements of wild-type (WT) and mutant cells displaying different chain configurations. These techniques were used to differentiate mutants due to magnetosome chain defects on a bulk scale.

Silent gene clusters encode magnetic organelle biosynthesis in a non-magnetotactic phototrophic bacterium.

Horizontal gene transfer is a powerful source of innovations in prokaryotes that can affect almost any cellular system, including microbial organelles. The formation of magnetosomes, one of the most sophisticated microbial mineral-containing organelles synthesized by magnetotactic bacteria for magnetic navigation in the environment, was also shown to be a horizontally transferrable trait. However, the mechanisms determining the fate of such genes in new hosts are not well understood, since non-adaptive gene acquisitions are typically rapidly lost and become unavailable for observation. This likely explains why gene clusters encoding magnetosome biosynthesis have never been observed in non-magnetotactic bacteria. Here, we report the first discovery of a horizontally inherited dormant gene clusters encoding biosynthesis of magnetosomes in a non-magnetotactic phototrophic bacterium Rhodovastum atsumiense. We show that these clusters were inactivated through transcriptional silencing and antisense RNA regulation, but retain functionality, as several genes were able to complement the orthologous deletions in a remotely related magnetotactic bacterium. The laboratory transfer of foreign magnetosome genes to R. atsumiense was found to endow the strain with magnetosome biosynthesis, but strong negative selection led to rapid loss of this trait upon subcultivation, highlighting the trait instability in this organism. Our results provide insight into the horizontal dissemination of gene clusters encoding complex prokaryotic organelles and illuminate the potential mechanisms of their genomic preservation in a dormant state.

Quantification of the pathological response and fate in the lung and pleura of chrysotile in combination with fine particles compared to amosite-asbestos following short-term inhalation exposure.

The marked difference in biopersistence and pathological response between chrysotile and amphibole asbestos has been well documented. This study is unique in that it has examined a commercial chrysotile product that was used as a joint compound. The pathological response was quantified in the lung and translocation of fibers to and pathological response in the pleural cavity determined. This paper presents the final results from the study. Rats were exposed by inhalation 6 h/day for 5 days to a well-defined fiber aerosol. Subgroups were examined through 1 year. The translocation to and pathological response in the pleura was examined by scanning electron microscopy and confocal microscopy (CM) using noninvasive methods. The number and size of fibers was quantified using transmission electron microscopy and CM. This is the first study to use such techniques to characterize fiber translocation to and the response of the pleural cavity. Amosite fibers were found to remain partly or fully imbedded in the interstitial space through 1 year and quickly produced granulomas (0 days) and interstitial fibrosis (28 days). Amosite fibers were observed penetrating the visceral pleural wall and were found on the parietal pleural within 7 days postexposure with a concomitant inflammatory response seen by 14 days. Pleural fibrin deposition, fibrosis, and adhesions were observed, similar to that reported in humans in response to amphibole asbestos. No cellular or inflammatory response was observed in the lung or the pleural cavity in response to the chrysotile and sanded particles (CSP) exposure. These results provide confirmation of the important differences between CSP and amphibole asbestos.

Cultivation-independent characterization of 'Candidatus Magnetobacterium bavaricum' via ultrastructural, geochemical, ecological and metagenomic methods.

'Candidatus Magnetobacterium bavaricum' is unusual among magnetotactic bacteria (MTB) in terms of cell size (8-10 µm long, 1.5-2 µm in diameter), cell architecture, magnetotactic behaviour and its distinct phylogenetic position in the deep-branching Nitrospira phylum. In the present study, improved magnetic enrichment techniques permitted high-resolution scanning electron microscopy and energy dispersive X-ray analysis, which revealed the intracellular organization of the magnetosome chains. Sulfur globule accumulation in the cytoplasm point towards a sulfur-oxidizing metabolism of 'Candidatus M. bavaricum'. Detailed analysis of 'Candidatus M. bavaricum' microhabitats revealed more complex distribution patterns than previously reported, with cells predominantly found in low oxygen concentration. No correlation to other geochemical parameters could be observed. In addition, the analysis of a metagenomic fosmid library revealed a 34 kb genomic fragment, which contains 33 genes, among them the complete rRNA gene operon of 'Candidatus M. bavaricum' as well as a gene encoding a putative type IV RubisCO large subunit.

The pathological response and fate in the lung and pleura of chrysotile in combination with fine particles compared to amosite asbestos following short-term inhalation exposure: interim results.

The pathological response and translocation of a commercial chrysotile product similar to that which was used through the mid-1970s in a joint compound intended for sealing the interface between adjacent wall boards was evaluated in comparison to amosite asbestos. This study was unique in that it presents a combined real-world exposure and was the first study to investigate whether there were differences between chrysotile and amosite asbestos fibers in time course, size distribution, and pathological response in the pleural cavity. Rats were exposed by inhalation 6 h/day for 5 days to either sanded joint compound consisting of both chrysotile fibers and sanded joint compound particles (CSP) or amosite asbestos. Subgroups were examined through 1-year postexposure. No pathological response was observed at any time point in the CSP-exposure group. The long chrysotile fibers (L > 20 microm) cleared rapidly (T(1/2) of 4.5 days) and were not observed in the pleural cavity. In contrast, a rapid inflammatory response occurred in the lung following exposure to amosite resulting in Wagner grade 4 interstitial fibrosis within 28 days. Long amosite fibers had a T(1/2) > 1000 days and were observed in the pleural cavity within 7 days postexposure. By 90 days the long amosite fibers were associated with a marked inflammatory response on the parietal pleural. This study provides support that CSP following inhalation would not initiate an inflammatory response in the lung, and that the chrysotile fibers present do not migrate to, or cause an inflammatory response in the pleural cavity, the site of mesothelioma formation.

[Regional coordination of paediatric oncological care in northwestern Lower Saxony, Germany--network funded by health insurance companies]. / Verbund PädOnko Weser-Ems--Regionale ambulante Versorgung pädiatrisch-onkologischer Patienten aus der Weser-Ems-Region im Rahmen einer Integrierten Versorgung.

The cure rates in pediatric oncology have been substantially improved due to standardized treatment strategies and centralization of therapy. Close clinical and hematological monitoring is mandatory for patients between periods of chemotherapy for early detection and treatment of therapy-related complications such as infections. This results in frequent and time-consuming outpatient examinations for the patient and family at the oncological center in order to evaluate clinical condition and hematological findings. In widespread regions such as the Weser-Ems area in northwest Lower Saxony, Germany, the long distances between patients' home and the oncological center lead to higher risks and impairment of quality of life (QoL) for the patients and their families. Accordingly, in 2001 pediatric hospitals and practices, patient care services and patients' support groups in Weser-Ems founded a network (Verbund PädOnko Weser-Ems). The "Verbund PädOnko" aims at coordinated, high-quality regional outpatient patient treatment in order to reduce risks of long-distance transports to reach the oncological center. Since 2005 a newly established mobile care team realized 1 443 home visits covering a total of 150 300 km. Since 2007 the network has been funded by health insurance organisations. Internal and external benchmarking was performed showing that the rate of short term inpatient treatments were reduced. Treatment quality was assured and the QoL of the patients and their families was improved through the work of the network. The "Verbund PädOnko Weser-Ems" network represents a promising prototype model for the regional coordination of outpatient treatment and care of patients with rare diseases in wide spread areas.

Bacterial magnetosomes - nature's powerful contribution to MPI tracer research.

The magnetic particle spectrum (MPS) of bacterial magnetosomes, isolated from Magnetospirillum gryphiswaldense, is measured and compared to that of the current "gold standard", Resovist®. It is shown that the amplitudes of the magnetosomes' harmonics by far exceed that of Resovist®; the amplitude of the third harmonic is higher by a factor of 7, and is the highest value obtained for iron oxide nanoparticles to date.

Measurement of the magnetic moment of single Magnetospirillum gryphiswaldense cells by magnetic tweezers.

Magnetospirillum gryphiswaldense is a helix-shaped magnetotactic bacterium that synthesizes iron-oxide nanocrystals, which allow navigation along the geomagnetic field. The bacterium has already been thoroughly investigated at the molecular and cellular levels. However, the fundamental physical property enabling it to perform magnetotaxis, its magnetic moment, remains to be elucidated at the single cell level. We present a method based on magnetic tweezers; in combination with Stokesian dynamics and Boundary Integral Method calculations, this method allows the simultaneous measurement of the magnetic moments of multiple single bacteria. The method is demonstrated by quantifying the distribution of the individual magnetic moments of several hundred cells of M. gryphiswaldense. In contrast to other techniques for measuring the average magnetic moment of bacterial populations, our method accounts for the size and the helical shape of each individual cell. In addition, we determined the distribution of the saturation magnetic moments of the bacteria from electron microscopy data. Our results are in agreement with the known relative magnetization behavior of the bacteria. Our method can be combined with single cell imaging techniques and thus can address novel questions about the functions of components of the molecular magnetosome biosynthesis machinery and their correlation with the resulting magnetic moment.

A longevity study with enhancer substances (selegiline, BPAP) detected an unknown tumor-manifestation-suppressing regulation in rat brain.

AIMS: First proof to show that (-)-deprenyl/selegiline (DEP), the first selective inhibitor of MAO-B, later identified as the first ß-phenylethylamine (PEA)-derived synthetic catecholaminergic activity enhancer (CAE) substance and (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine (BPAP), the tryptamine-derived presently known most potent, selective, synthetic enhancer substance, are specific markers of unknown enhancer-sensitive brain regulations. MAIN METHODS: Longevity study disclosing the operation of tumor-manifestation-suppressing (TMS) regulation in rat brain. Immonohistochemical identification of a fibromyxosarcoma in rats. Experiments with human medulloblastoma cell lines. Analysis of the mechanism of action of enhancer substances. KEY FINDINGS: Whereas 20/40 saline-treated rats manifested a fibromyxosarcoma, in groups of rats treated with 0.001mg/kg DEP: 15/40 rats; with 0.1mg/kg DEP: 11/40 rats (P<0.01); with 0.0001mg/kg BPAP: 8/40 rats (P<0.001); with 0.05mg/kg BPAP: 7/40 rats (P<0.01) manifested the tumor. Experiments with human medulloblastoma cell lines, HTB-186 (Daoy); UW-228-2, showed that BPAP was devoid of direct cytotoxic effect on tumor cells, and did not alter the direct cytotoxic effectiveness of temozolomide, cisplatin, etoposide, or vincristine. Interaction with distinct sites on vesicular monoamine-transporter-2 (VMAT2) is the main mechanism of action of the enhancer substances which clarifies the highly characteristic bi-modal, bell-shaped concentration-effect curves of DEP and BPAP. SIGNIFICANCE: Considering of the safeness of the enhancer substances and the finding that DEP and BPAP, specific markers of unknown enhancer sensitive brain regulations, detected the operation of an enhancer-sensitive TMS-regulation in rat brain, it seems reasonable to test in humans low dose DEP or BPAP treatment against the spreading of a malignant tumor.

The three-dimensional structure and function of Escherichia coli ribosomal RNA, as studied by cross-linking techniques.

A large number of intra-RNA and RNA-protein cross-link sites have been localized within the 23S RNA from E. coli 50 S ribosomal subunits. These sites, together with other data, are sufficient to constrain the secondary structure of the 23 S molecule into a compact three-dimensional shape. Some of the features of this structure are discussed, in particular, those relating to the orientation of tRNA on the 50 S subunit as studied by site-directed cross-linking techniques. A corresponding model for the 16S RNA within the 30 S subunit has already been described, and here a site-directed cross-linking approach is being used to determine the path followed through the subunit by messenger RNA.

A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit.

A large body of intra-RNA and RNA-protein crosslinking data, obtained in this laboratory, was used to fold the phylogenetically and experimentally established secondary structure of Escherichia coli 16 S RNA into a three-dimensional model. All the crosslinks were induced in intact 30 S subunits (or in some cases in growing E. coli cells), and the sites of crosslinking were precisely localized on the RNA by oligonucleotide analysis. The RNA-protein crosslinking data (including 28 sites, and involving 13 of the 21 30S ribosomal were used to relate the RNA structure to the distribution of the proteins as determined by neutron scattering. The three-dimensional model of the 16 S RNA has overall dimensions of 220 A x 140 A x 90 A, in good agreement with electron microscopic estimates for the 30 S subunit. The shape of the model is also recognizably the same as that seen in electron micrographs, and the positions in the model of bases localized on the 30 S subunit by immunoelectron microscopy (the 5' and 3' termini, the m7G and m6(2)A residues, and C-1400) correspond closely to their experimentally observed positions. The distances between the RNA-protein crosslink sites in the model correlate well with the distances between protein centres of mass obtained by neutron scattering, only two out of 66 distances falling outside the expected tolerance limits. These two distances both involve protein S13, a protein noted for its anomalous behaviour. A comparison with other experimental information not specifically used in deriving the model shows that it fits well with published data on RNA-protein binding sites, mutation sites on the RNA causing resistance to antibiotics, tertiary interactions in the RNA, and a potential secondary structural "switch". Of the sites on 16 S RNA that have been found to be accessible to chemical modification in the 30 S subunit, 87% are at obviously exposed positions in the model. In contrast, 70% of the sites corresponding to positions that have ribose 2'-O-methylations in the eukaryotic 18 S RNA from Xenopus laevis are at non-exposed (i.e. internal) positions in the model. All nine of the modified bases in the E. coli 16 S RNA itself show a remarkable distribution, in that they form a "necklace" in one plane around the "throat" of the subunit. Insertions in eukaryotic 18 S RNA, and corresponding deletions in chloroplast or mammalian mitochondrial ribosomal RNA relative to E. coli 16 S RNA represent distinct sub-domains in the structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneous spectrum of mutations in the Fanconi anaemia group A gene.

Fanconi anaemia (FA) is a genetically heterogeneous autosomal recessive disorder associated with chromosomal fragility, bone-marrow failure, congenital abnormalities and cancer. The gene for complementation group A (FAA), which accounts for 60-65% of all cases, has been cloned, and is composed of an open reading frame of 4.3 kb, which is distributed among 43 exons. We have investigated the molecular pathology of FA by screening the FAA gene for mutations in a panel of 90 patients identified by the European FA research group, EUFAR. A highly heterogeneous spectrum of mutations was identified, with 31 different mutations being detected in 34 patients. The mutations were scattered throughout the gene, and most are likely to result in the absence of the FAA protein. A surprisingly high frequency of intragenic deletions was detected, which removed between 1 and 30 exons from the gene. Most microdeletions and insertions occurred at homopolymeric tracts or direct repeats within the coding sequence. These features have not been observed in the other FA gene which has been cloned to date (FAC) and may be indicative of a higher mutation rate in FAA. This would explain why FA group A is much more common than the other complementation groups. The heterogeneity of the mutation spectrum and the frequency of intragenic deletions present a considerable challenge for the molecular diagnosis of FA. A scan of the entire coding sequence of the FAA gene may be required to detect the causative mutations, and scanning protocols will have to include methods which will detect the deletions in compound heterozygotes.

Recombinant human erythropoietin in the prevention of chemotherapy-induced anaemia in children with malignant solid tumours.

This prospective, randomised pilot study was designed to evaluate safety, feasibility and efficacy of recombinant human erythropoietin (rhEPO) in the prevention and treatment of chemotherapy-induced anaemia in children with solid tumours. 20 children (age 4-18 years) undergoing cyclic combination chemotherapy were randomised either to a control group or to receive rhEPO at a dose of 150 U/kg/dose subcutaneously three times/week for a minimum of 12 weeks or three chemotherapy cycles. Of 15 evaluable patients, 8 were randomised to the rhEPO group and 7 to the control group. RhEPO-treated patients showed an increase in the haematocrit over the first 8 weeks of therapy, with a significantly higher mean haematocrit at week 8 (33.2 +/- 2.1% versus 39.3 +/- 4.2% in the control and rhEPO groups, respectively, P < 0.05). Similarly, significantly higher haemoglobin concentrations could be demonstrated in the rhEPO group by week 8 (11.06 +/- 1.35 g/dl versus 13.11 +/- 1.13 g/dl in the control and rhEPO groups, respectively, P < 0.05), with higher precycle haemoglobin before chemotherapy cycles 3 and 4 and higher midcycle haemoglobin between cycles 3 and 4. There was a trend towards a reduction of transfusion requirements during the 3rd month of therapy in rhEPO patients. The results of this pilot study indicate a significant benefit of rhEPO in children treated with intensive combination chemotherapy regimens. Further studies should target issues such as appropriate dosing, timing and duration of rhEPO therapy in children with cancer.

Gene expression of neuropeptide Y and neuropeptide-Y1 receptor in relation to proto-oncogene tyrosine kinase A, nerve-growth-factor low-affinity-receptor and the transcription factor N-myc in human neuroblastomas.

The expression of neuropeptide Y (NPY) and NPY-Y1 receptor (NPY-Y1R) in relation to that of tyrosine kinase A (trkA), nerve-growth-factor low-affinity-receptor (LNGFR) and the transcription factor N-myc was studied in 26 neuroblastomas and one ganglioneuroma by quantitative Northern-blot analysis. A correlation of NPY-Y1R with LNGFR (r = 0.85, p < 0.01) and trkA (r = 0.38, p < 0.05), respectively, could be shown, while no correlation between NPY and its receptor NPY-Y1R was observed. Comparison of a high and a low level NPY expressing group revealed that the high NPY expressing group also had high LNGFR and high trkA levels while the low NPY expressing group had low LNGFR and low trkA levels which were significantly different (NPY: p = 0.035, trkA: p = 0.008, LNGFR p = 0.004). Dividing the tumors in a high and a low N-myc expressing group showed that the low N-myc expressing group contained both high and low trkA expressing tumors while the high N-myc expressing group exclusively were low level trkA expressing tumors. The frequency distribution in both groups concerning trkA expression showed a significant difference (p < 0.01). In conclusion the coexpression of NPY-Y1R and LNGFR or trkA may indicate a similar gene regulation during ontogenesis of the peripheral nervous system.

Apoptosis and acute lymphocytic leukemia in children.

We examined in vivo spontaneous and prednisolone-induced apoptosis in peripheral blood samples of 23 children with ALL by flow cytometric and morphologic methods. There was no significant spontaneous apoptosis before the therapy. Six hours after prednisolone therapy, increased apoptosis was found in 19 of 23 cases. In one case, the apoptosis of blast cells could not be compared with the clinical data, because the patient died in sepsis during the induction therapy. In 18 of 22 evaluable cases, the in vivo apoptosis correlated with the decrease of leukemic blasts during the first 8 days of prednisolone monotherapy. In 20 of 22 children, a correlation was found between in vivo prednisolone-induced apoptosis and clinical outcome. The p53 gene expression was elevated in 2 of 10 patients. No elevation in the expression of bcl-2 gene was observed. In 6 of 23 cases, the glucocorticoid receptors were measured. The correlation of clinical responsiveness and gcR mRNA shows less parallelism than does the apoptosis correlation.

Methotrexate administered by 6-h and 24-h infusion: a pharmacokinetic comparison.

The pharmacokinetics of 8 g/m2 methotrexate (MTX) was compared following short (6 h) and long (24 h) infusions of the drug to 11 children with osteogenic sarcoma (OS; 42 infusion) and 28 children with acute lymphoblastic leukemia (ALL: 118 infusions), respectively. No difference was observed in the first-phase half-life, in systemic clearance or in the volume of distribution of the drug (P greater than 0.05). The concentration of MTX at the end of the infusion was approximately 4-fold higher when the drug was given over only 6 h. However, patients receiving 24-h infusions had approximately 9-fold higher levels by 24 h after the beginning of the infusion. The area under the data curve from start of the MTX infusion until the beginning of folinic acid rescue administration was significantly higher in patients with osteogenic sarcoma (6-h infusions), while the area under the log-data curve was significantly longer in the ALL group (24-h infusions) for the same period. The latter parameter is considered to be characteristic for the concentration-time-effect relationship. The longer duration of MTX administration (with delayed rescue) is thought to be more beneficial from the pharmacokinetic aspect. Patients with osteogenic sarcoma had significantly lower concentrations of MTX at the end of their last treatment with MTX than at the end of the first infusion. Patients developing MTX toxicity had shorter half-lives of MTX in the beta phase. It is suggested that cisplatin induced tubular loss of MTX and folinic acid is responsible for these observations. A wider application of clinical pharmacologic findings in the practice of the administration of cytostatics is indicated.

Prognostic importance of systemic clearance of methotrexate in childhood acute lymphoblastic leukemia.

Pharmacokinetic studies of methotrexate have been carried out in 21 children with acute lymphoblastic leukemia diagnosed in 1981. Children were treated with intermediate dose (500 mg/m2) methotrexate in keeping with the 1981 ALL treatment Protocol of the Hungarian Childhood Leukemia Working Group. Of the 21 children, 8 relapsed, and 13 are in continuous complete remission. In the relapsed patients significantly increased systemic clearance of methotrexate was observed at the time of the second methotrexate treatment cycle compared with the calculated value after the first administration of the drug. No such change in the clearance was found in patients who are still in remission. There was no difference between children who relapsed or who are in remission in the elimination half-time of the drug. Age, sex, WBC at diagnosis, and systemic clearance of methotrexate were found to be connected with the probability of relapse in the patients studied. The possible reasons for the prognostic role of systemic methotrexate clearance are discussed.

Proteinuria due to suboptimal hydration with high-dose methotrexate therapy.

One of the major complications after high-dose methotrexate (HDMTX) infusions is renal damage. We investigated the occurrence of proteinuria after HDMTX administration in children with pediatric malignancies (acute lymphoid leukaemia, osteosarcoma Burkitt's lymphoma). In the period 1989-1990 we gave 52 HDMTX courses to 24 children. During this period, prehydration and extra urinary alkalisation were performed only if the urinary specific gravity was over 1010 or if the urinary pH fell below 7. Using this schedule the mean values obtained for protein extraction were: before the therapy, 0.12 +/- 0.03 g/m2; on day 1 after MTX treatment, 0.38 +/- 0.06 g/m2; and on day 2 after the MTX infusion, 0.39 +/- 0.11 g/m2 (P < 0.01). A significant increase in proteinuria (> 0.2 g/m2 post- vs pretreatment) was detectable in 54% of the patients. In the period 1991-1992 we modified the hydration-alkalisation schedule to include i.v. prehydration for 18-24 h at 3 l/m2/day with a 0.45% NaCl-5% glucose solution along with sodium bicarbonate and posthydration for 72 h with the same solution. On this protocol the mean values determined for the urinary protein content were all in the normal range (pretreatment, 0.03 g/m2/day; day 1, 0.05 g/m2/day; and day 2, 0.08 g/m2/day). These findings were significantly different from the previous results (P < 0.05).