Role of coupling delay in oscillatory activity in autonomous networks of excitable neurons with dissipation.

We study numerically effects of time delay in networks of delay-coupled excitable FitzHugh-Nagumo systems with dissipation. Generation of periodic self-sustained oscillations and its threshold are analyzed depending on the dissipation of a single neuron, the delay time, and random initial conditions. The peculiarities of spatiotemporal dynamics of time-delayed bidirectional ring-structured FitzHugh-Nagumo neuronal systems are investigated in cases of local and nonlocal coupling topology between the nodes, and a first-order nonequilibrium phase transition to synchrony is established. It is shown that the emergence of an oscillatory activity in delay-coupled FitzHugh-Nagumo neurons is observed for smaller values of the coupling strength as the dissipation parameter decreases. This can provide the possibility of controlling the spatiotemporal behavior of the considered neuronal networks. The observed effects are quantified by plotting distributions of the maximal Lyapunov exponent and the global order parameter in terms of delay and coupling strength.

Spiral and target wave chimeras in a 2D lattice of map-based neuron models.

We study the dynamics of a two-dimensional lattice of nonlocally coupled-map-based neuron models represented by Rulkov maps. It is firstly shown that this discrete-time neural network can exhibit spiral and target waves and corresponding chimera states when the control parameters (the coupling strength and the coupling radius) are varied. It is demonstrated that one-core, multicore, and ring-shaped core spiral chimeras can be realized in the network. We also reveal a novel type of chimera structure-a target wave chimera. We explore the transition from spiral wave chimeras to target wave structures when varying the coupling parameters. We report for the first time that the spiral wave regime can be suppressed by applying noise excitations, and the subsequent transition to the target wave mode occurs.

Synchronization of spiral wave patterns in two-layer 2D lattices of nonlocally coupled discrete oscillators.

The paper describes the effects of mutual and external synchronization of spiral wave structures in two coupled two-dimensional lattices of coupled discrete-time oscillators. Each lattice is given by a 2D N×N network of nonlocally coupled Nekorkin maps which model neuronal activity. We show numerically that spiral wave structures, including spiral wave chimeras, can be synchronized and establish the mechanism of the synchronization scenario. Our numerical studies indicate that when the coupling strength between the lattices is sufficiently weak, only a certain part of oscillators of the interacting networks is imperfectly synchronized, while the other part demonstrates a partially synchronous behavior. If the spatiotemporal patterns in the lattices do not include incoherent cores, imperfect synchronization is realized for most oscillators above a certain value of the coupling strength. In the regime of spiral wave chimeras, the imperfect synchronization of all oscillators cannot be achieved even for sufficiently large values of the coupling strength.

R-CHOEP-14 improves overall survival in young high-risk patients with diffuse large B-cell lymphoma compared with R-CHOP-14. A population-based investigation from the Danish Lymphoma Group.

BACKGROUND: Optimal treatment of young patients with high-risk diffuse large B-cell lymphoma (DLBCL) remains a matter of debate and requires improvement. The combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) with addition of etoposide (CHOEP) has in other patient groups been shown to be effective. Further improvement has been accomplished with the use of rituximab in combination with the regimens every 2 weeks (R-CHOP-14, R-CHOEP-14). The aim of the present retrospective population-based study was to compare R-CHOP-14 with R-CHOEP-14 in a cohort of high-risk patients aged 18-60 years with two or more risk factors (stage III-IV, elevated lactate dehydrogenase levels, performance status 2-4). To our knowledge, this is the first study comparing these two regimens in this patient group. METHODS: We obtained data for the period 2004-2009 from the Danish Lymphoma Database. One hundred and fifty-nine patients were eligible to enter the study. Primary end point was overall survival (OS) and secondary end points were response to treatment, progression-free survival (PFS) and safety. RESULTS: Four-year OS was superior in the R-CHOEP-14 group: 75% compared with 62% for R-CHOP-14 (P=0.04). This superiority was also seen for PFS: 4-year PFS was 70% for the R-CHOEP-14 group compared with 58% for the R-CHOP-14 group (P=0.02). CONCLUSION: R-CHOEP-14 is a promising regimen for young patients with high-risk DLBCL with improved OS and PFS compared with R-CHOP-14.

Characterization and validation of a chemiluminescent assay based on a 1,2-dioxetane for rapid detection of enterococci in contaminated water and comparison with standard methods and qPCR.

AIMS: To evaluate the potential for using a novel chemiluminescence-based enzyme assay for rapid detection of enterococci in water contaminated with faecal waste. METHODS AND RESULTS: The novel assay (EntLight) was based on the enzymatic hydrolysis of the chemiluminescent 1,2-dioxetane [(4-methoxy-4(3-ß-d-glucoside-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7·3·1·0(2,7) ]tridec-2,7-ene] specific for ß-d-glucosidase. The specificity of the proposed EntLight assay was characterized using 26 different Enterococcus strains and 10 bacterial genera other than Enterococcus. With an analysis time of ≤8 h, the assay was found to be sensitive and specific. Validation experiments were carried out using water samples contaminated with raw municipal wastewater in comparison with qPCR and ISO standard methods. EntLight was successfully applied to detect enterococci in contaminated water within ≤8 h, and the proposed assay correlated well with both qPCR and ISO standard methods (R(2) > 0·776). CONCLUSIONS: EntLight can be applied to rapid and simple detection of viable enterococci in water contaminated with faecal matter. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel EntLight assay and qPCR have the potential to be used as methods for early warning (1-7 h) of faecal pollutions in different water types.

Application of mussels as biosamplers for characterization of faecal pollution in coastal recreational waters.

Sources of faecal pollution in coastal recreational waters may be identified by analysing different host associated microorganisms or molecular markers. However, the microbial targets are often present at low numbers in moderately impacted waters, and often exhibit significant temporal and spatial variability in waters with fluctuating faecal loads. This patchy occurrence can limit successful detection of relevant targets in microbial source tracking studies. In this study, we explored the possibility for using the blue mussel (Mytilus edulis) as a biosampler for accumulation of faecal bacteria relevant for microbial source tracking. Non-contaminated blue mussels were transferred to three coastal recreational waters affected by faecal pollution of unknown origin. Molecular markers associated with animal and human waste were targeted by PCR and compared in seawater and mussel samples. The results demonstrated that transplanted mussels in simple enclosures accumulated and retained elevated levels of molecular markers associated with different types of faecal pollution. The targets included a novel putative human associated E. coli subgroup B2 VIII clone, and animal and human associated markers in enterococci (esp, M19, M66, M90, and M91). Human (sewage) associated markers including esp and M66 were sometimes not detectable in seawater samples despite known wastewater contamination, whereas the markers were detectable in mussels. We suggest that transplanted mussels should be considered as potential biosamplers in studies focusing on identifying source of faecal pollution in low or moderately impacted recreational waters. Bioaccumulation of molecular markers in mussels for several days may represent the water quality better than traditional grab samples from the water column.

Validation and clinical implication of a quantitative real-time PCR determination of MDR1 gene expression: comparison with semi-quantitative PCR in 101 patients with acute myeloid leukemia.

INTRODUCTION: The multidrug resistance protein 1 (MDR1) has the capacity to extrude chemotherapeutics and has been implicated in treatment failure in acute myeloid leukemia (AML). Previous methods for determination of MDR1 expression have included dye exclusion, demonstration of P-glycoprotein by flow cytometry and/or immunohistochemistry, and molecular polymerase chain reaction (PCR)-based assays for RNA expression. However, these assays have either proven difficult to standardize or tedious to perform. We have therefore designed a real-time quantitative (RQ)-PCR based assay measuring MDR1 gene expression and validated it in AML patients by direct comparison with a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay. PATIENTS AND METHODS: Bone marrow or peripheral blood from 101 AML patients diagnosed (1987-96) at our department were assessed for quantitative expression of MDR1 employing TaqMan RQ-PCR. These data were compared with results obtained by a semi-quantitative competitive PCR assay employing an artificial internal RNA construct. RESULTS: While the RQ-PCR method was able to determine MDR1 gene expression in a continuous fashion over five logs, the semi-quantitative PCR only yielded data in a discontinuous fashion and over four logs at best. Compared with the MDR1 positive and negative cell lines 8226 DOX40 and REH AML cells exhibited variation of 10 PCR cycles, equivalent to a 1000-fold difference. A significant correlation was observed between the two methods, Spearman's correlation coefficient = -0.502, P-value = 10-5. CONCLUSION: We conclude that, RQ-PCR is a novel methodology, which enables sensitive and quantitative measurement of MDR1 gene expression. This assay is moreover suitable because of its high throughput for longitudinal follow-up and large number of patients.

MDR1 gene expression and drug resistance of AML cells.

We investigated the cellular drug resistance to aclarubicin (Acla), cytosine arabinoside (Ara-C), daunorubicin (Dau), doxorubicin (Dox), etoposide (Etop) and mitoxantrone (Mitox) using the MTT assay at time of disease presentation in 93 cases of acute myeloid leukaemia (AML). In 31 cases we concomitantly investigated MDR1 (multiple drug resistance 1 gene) expression (semi-quantitative competitive RT-PCR) of the leukaemic cells. Drug resistance towards Dau, Dox and Etop was correlated to the MDR1 expression of the AML cells (P<0.05) with high MDR1 expression being associated with high drug resistance towards these drugs. Although the data did not allow firm conclusions to be drawn on the correlation between MDR1 expression and drug resistance towards Ara-C and Mitox, the drug resistance towards Acla clearly was not correlated to, or dependent on, the MDR1 expression level of the AML blast cells. In addition, when examining the cross-activities among the six drugs distinct patterns emerged. Thus, high to very high degrees of cross-activity were found to exist between Dau, Dox, Etop and Mitox, whereas Ara-C had moderate cross-activity with the other drugs except Acla, which showed absent to moderate cross-activity with the other drugs. We conclude that MDR1 gene expression is of significance for cellular drug resistance towards specific (MDR1-related) drugs in AML, whereas it is not of significance regarding drug resistance towards other drugs, which is the case with the anthracycline Acla. We suggest that in the place of other more or less complicated ways to circumvent MDR1-mediated drug resistance, Acla may be used to replace Dau, Dox and other MDR1-related drugs if proven as potent as the drug it is to substitute.

[Diagnosis of minimal residual disease in autologous bone marrow transplantation]. / Diagnostik af minimal residual sygdom ved autolog knoglemarvstransplantation.

ABMT (autologous bone marrow transplantation) is being increasingly used in the treatment of malignant diseases, including the acute leukaemias. Although ABMT seems to be superior to conventional chemotherapy in terms of disease-free survival, an unacceptably high frequency of relapse after ABMT remains a major problem. As such relapse may be due to malignant cells in the graft or residual malignant cells surviving in the patient after preconditioning therapy, it is essential to be able to detect and eradicate residual malignant cells. The article presents a review of available methods for the detection of minimal residual disease in conjunction with ABMT, especially regarding their relative sensitivity and specificity.

Pregnancy zone protein: a re-evaluation of serum levels in healthy women and in women suffering from breast cancer or trophoblastic disease.

Pregnancy zone protein (PZP) and alpha 2-macroglobulin (alpha 2M) serum concentrations were studied in healthy female donors, in women suffering from benign and malignant breast tumours, and in relation to normal and abnormal pregnancies. PZP was found to be useless as a tumour marker. Thus, PZP levels in breast cancer patients did not differ from those of fibroadenoma patients or healthy women. There was no correlation between PZP (or alpha 2M) concentrations and the pTNM-classification or metastatic burden of the breast cancer patients. Moreover, PZP levels were unaffected by cancer treatment and the course of disease. Neither patients nor control donors showed any age-dependent increase in circulating PZP and the mean serum value (8.38 +/- 4.83 mg/l, mean +/- SD) determined in a population of 154 non-pregnant women was considerably lower than that of most previous reports. Serum concentrations were unchanged during the normal menstrual cycle, but increased during pregnancy. However, late pregnancy sera (35th gestational week) contained significantly less PZP than previously reported by others, and non-pregnancy levels were observed in one out of 22 cases. Results obtained in hydatidiform mole patients were similar to findings in normal pregnancy. Neither serum 17 beta-oestradiol nor morphological differentiation between complete and partial mole showed any correlation with circulting PZP levels. Apart from a moderate increase during gestation, alpha 2M concentrations showed little variation between the populations examined.

The expression of c-fos, c-jun, and c-myc genes is regulated by heat shock in human lymphoid cells.

The effect of heat shock on the expression of the nuclear protooncogenes c-fos, c-jun, and c-myc was studied in human lymphoid cells. Heat shock caused an increase in c-fos and c-jun mRNA levels and a decrease in c-myc mRNA levels in pre-B (Hyon) and T (DND-41) cell lines as well as in freshly isolated normal human thymocytes. The changes in the mRNA levels of these protooncogenes in Hyon cells were most pronounced at 42 and 43 degrees C; kinetic analysis demonstrated that the changes could be detected within 30 min of heat shock. Altered transcription of c-fos and c-myc genes was the primary effect of heat shock. Secondarily, heat shock of Hyon cells stabilized the c-myc mRNA level by increasing its half-life from 24 to 45 min. The overall effect of heat shock on c-myc mRNA level, however, was a marked inhibition of its transcription. These results demonstrate that the transcription of nuclear protooncogenes is regulated by heat shock indicating a role for nuclear protooncogenes in the stress response of lymphoid cells.

Analysis of the decreased NK (natural killer) activity in lung cancer patients, using whole blood versus separated mononuclear cells.

The aim of this study was to analyze whether a whole blood assay would give a more correct measure of NK activity than assays using separated mononuclear cells (SMNC). We found that the NK activity of whole blood was higher than the NK activity of SMNC in the 28 lung cancer patients investigated (p = 0.01), whereas this difference between the assays could not be demonstrated in the 29 healthy controls. Since no differences were found between the NK activity of washed blood, SMNC, and monocyte-depleted lymphoid cells, there was no indication that the lower NK activity of SMNC in comparison with whole blood was due to cell loss or to a systematic disturbing effect due to monocytes. The possible effect of plasma factors on the whole blood NK activity was analyzed by comparing whole blood and washed blood. The NK activity of whole blood was increased in comparison with washed blood in the lung cancer patients (p less than 0.0001) indicating a stimulatory effect of plasma. Further, the finding that the reactive capability of lymphocytes from cancer patients was higher than in controls could indicate preactivation of the lymphocytes from the cancer patients due to the presence of stimulatory plasma factors. The NK activity of lung cancer patients was lower than the NK activity of healthy controls. The difference was found to be smaller with whole blood than with SMNC as effector cells, although both differences were significant. The decreased NK activity of cancer patients could be due to blocking immune complexes (IC), but we found no evidence for circulating or cell-bound IC in the lung cancer patients.

Human lung cancer--a comparative study of the levels of circulating immune complexes in pulmonary blood draining the tumor area and peripheral venous blood.

Our objective was to investigate whether low levels of circulating immune complexes (cICs) in peripheral venous blood of cancer patients could be due to removal of cIC released at the tumor site during passage to the peripheral veins. In 54 patients with primary lung cancer, we therefore compared the cIC levels as detected by 3 different assays in paired samples from the pulmonary vein draining the tumor area and from a peripheral vein. Only 6 of the 54 patients had significantly increased pulmonary vein cIC levels as compared to the corresponding peripheral vein levels. The peripheral vein levels of these 6 patients were all within the normal range, and in none of these patients was the difference between the 2 sites of analysis--although significant--of such a magnitude that the pulmonary vein cIC level appeared higher than the normal range, i.e., "positive" for cIC. Positive cIC levels were only found in 11% of lung cancer patients (irrespective of the site of measurement). Thus, our present data, together with our previous findings indicating no significant difference between peripheral venous blood cIC levels in cancer patients and normal controls, contradict the theory of tumor cells expressing new antigens resulting in the formation of tumor-associated cICs.

Female haemophilia A in a family with seeming extreme bidirectional lyonization tendency: abnormal premature X-chromosome inactivation?

We studied a female child with mild classical haemophilia A, presenting with a F VIII deficiency similar to that detected in her maternal grandfather. Investigations on several occasions showed that the obligate carrier mother of the proposita had normal VIII:C activity, whereas her likewise obligate carrier sister had a typical carrier VIII:C/vWf:Ag pattern. The child was a phenotypically normal female with normal karyotype. Her father had no clinical or biochemical signs of haemophilia A. RFLP-analysis using DX13 and St14 probes each elicited one allele (5.8 and 3.4 kb, respectively) segregating along with the affected F VIII gene from the hemizygous grandfather to both his daughters and further to the haemophilic female child. The paternity of the child was analyzed using various red cell and HLA antigens and RFLP by p29C, a probe detecting polymorphic hypervariable TaqI and PstI fragments in the pseudoautosomal areas of the X- and Y-chromosomes. All results obtained were concordant with the declared paternity. RFLP-analysis, using single (Pst I) and double digestion (Pst I/Hha I) of DNA and a PGK probe, revealed a remarkable difference in hybridization fragments, strongly suggesting hypermethylation, and in consequence, preferential X-chromosome inactivation in the proposita. This points to extreme lyonization as the most plausible explanation for haemophilia A in this female child. A familial tendency to abnormal premature X-chromosome inactivation is speculated.

A polyclonal IgM-RF enzyme-linked immunosorbent assay for the detection of circulating immune complexes.

A microplate-adapted polyclonal IgM-rheumatoid factor enzyme-linked immunosorbent assay (pIgM-RF ELISA) for the detection of circulating immune complexes (cIC) is presented. The assay involves the competitive binding of cIC and horseradish peroxidase conjugated aggregated human IgG (HRP-AHG) to solid-phase bound polyclonal IgM-RF (pIgM-RF). Aggregated human IgG (AHG) inhibited the binding of HRP-AHG to pIgM-RF in a dose-dependent way. The detection limit of the assay was about 125 ng AHG/ml diluted serum. The coefficients of variation for the assay varied from 5.0 to 14.7% for intra-assay runs and from 4.5 to 13.8% for inter-assay runs. The levels of cIC in sera from 29 patients with systemic lupus erythematosus (SLE), 85 untreated patients with breast cancer and 105 blood bank donors were studied by the pIgM-RF ELISA. Increased levels of cIC were demonstrated in 41.4% of the SLE group, in 8.2% of the breast cancer group, and in 1.9% of the normal control group. The difference in cIC activity between the SLE group and the normal control group was statistically significant.

Applications of immunoperoxidase techniques in specificity testing of monoclonal antibodies (Mabs) against von Willebrand factor (vWf).

The present communication describes the production of a new series of murine Mabs against von Willebrand factor (vWf) in which specificity was tested using immunoperoxidase techniques. Seven Mabs showed specific reactivity with native and disaggregated vWf, whereas no binding was found to material from patients with severe homozygous (or doubly heterozygous) von Willebrand's disease (vWd) or factor VIII coagulant antigen (VIII:Ag). These Mabs are thought to carry separate specificities as only slight or no competitive activity was detected. Four Mabs partially inhibited the ristocetin-induced platelet agglutination and three interacted with vWf-binding to type I collagen. All antibodies bound to the complete range of vWf multimers of normal plasma. Excellent binding and detection properties of Mabs were found in asymmetrical two-site enzyme linked immunosorbent assays (ELISA) for quantitation of vWf antigen (vWf:Ag). One particular antibody (Mab vWf-33) discriminated vWf material from a number of subtype II vWd plasmas tested.

Primary lung cancer. A controlled study of preoperative and postoperative levels of circulating immune complexes.

Three complement-dependent and one rheumatoid factor-dependent immune complex assays were used to analyze the sera taken before surgery from 70 unselected and previously untreated lung cancer patients, from 30 of them after surgery, and from 31 healthy controls. Plasma levels of complement split product C3d were also analyzed. The levels of circulating immune complexes (cIC) and C3d were essentially the same in samples taken from lung cancer patients before surgery and from healthy controls. By the four immune complex assays, increased levels were found in 0 to 10% of the preoperative lung cancer patients compared to 3% of the healthy controls (not significant). The postsurgical tumor, lymph node, metastasis (pTNM) stage of the lung cancer was not reflected in the levels of cIC or C3d. Paired comparisons of the cIC and C3d levels before and after surgery did not show significant differences. Thus, we found no evidence for the occurrence of cancer-related cIC in lung cancer patients.

A prospective study of circulating immune complexes in patients with breast cancer.

Levels of circulating immune complex (cIC) and complement split product C3d were studied in 86 patients with breast cancer (BC), 22 patients with benign breast disease (BD), and 72 age- and sex-matched blood-bank donors (NC), using solid-phase Clq-protein A RIA, Clq-anti-IgG RIA, anti-C3d anti-IgG RIA, and polyclonal IgM-rheumatoid factor ELISA for clC detection. No significant differences in cIC and C3d levels were found between the groups. The incidence of raised cIC levels varied from 4.9 to 8.2% in the BC group and from 4.5 to 22.7% in the BD group in comparison with 2.9 to 3.0% in the NC group. Using the solid-phase polyclonal IgM-rheumatoid factor ELISA we found that the cIC levels of patients with stage-III cancer were significantly higher than those of patients with stage-I or stage-II cancer. However, the other tests showed no relationship to tumor burden. Likewise, an effect of mastectomy on the cIC levels was also only detectable by one of the assays, i.e., the post-mastectomy levels of cIC as measured by the solid-phase anti-C3d anti-IgG RIA were significantly lower than the pre-mastectomy levels. Serial analyses of cIC and C3d levels were performed pre-operatively, one month post-operatively and every 3 months during the first year after mastectomy in 46 of the patients. During a I-year observation period, 7 patients developed metastatic disease. The occurrence of metastatic disease was not, however, preceded by characteristic changes in serially determined cIC and C3d levels.

Prognostic value of serial determinations of circulating immune complex levels in malignant melanoma.

A total of 50 melanoma patients free of distant metastatic disease and 54 healthy controls were analyzed for circulating immune complexes (cIC) and complement split product (C3d), using solid-phase Clq-anti-IgG radioimmunoassay (RIA), Clq-protein A RIA, and anti-C3d anti-IgG RIA for cIC detection. No significant differences in cIC and C3d levels could be demonstrated between the controls and the 31 patients with primary malignant melanoma analyzed before surgery. To evaluate the prognostic value of serial measurements, samples from the 50 patients were taken at regular intervals for 4 to 27 months (median, 20 months). Surgery was the only treatment given. Significant changes in the cIC and C3d levels were defined by reference to the changes that occurred in 23 of the 54 healthy controls observed for a period of 6 to 55 months (median, 23 months). During the period of serial sampling, recurrent disease developed in 8 of the patients. In only 3 of these 8 patients (versus 10 of 42 patients without recurrence) did significant changes occur, and the changes occurred either at the same time or after the clinical diagnosis of recurrence. During the entire clinical observation period of 6 years, a total of 11 patients developed recurrences. Significant changes were only observed in 4 of these 11 patients versus 8 of 37 patients without recurrence. In conclusion, changes in cIC and/or C3d levels were not found to be indicative of early or long-term recurrence of malignant melanoma.

Characteristics of the von Willebrand factor in virus inactivated F VIII concentrates: the impact of heat treatment.

The known transmission of viral diseases, particularly AIDS (HIV, LAV, HTLV-III), has led to the mandatory use of virus-inactivated coagulation factor concentrates for treatment of bleeding disorders due to deficient or abnormal synthesis of the factor VIII/von Willebrand factor complex. The present investigation was undertaken to study the influence of heat-treatment on the von Willebrand factor (vWf). Using normal plasma as reference material, we studied the influence of low-purification steps in a simple cryo-plasma and a unrefined freeze-dried cryoprecipitate. For comparison, non-heated and heat-inactivated concentrates of different manufacture representing varying heat-treatment protocols were studied using quantitation of von Willebrand factor antigen (vWf:Ag) by electroimmunoassay and ELISA, and investigation of vWf multimeric composition. A locally produced factor VIII concentrate was studied before and after exposure to 68 degrees C for 72 hours (dry state). Whenever possible, commercial preparations manufactured prior to the heat-treatment era were compared with the present product. The locally produced high purity concentrate elicited only minor changes in oligomeric satellite pattern, which did not change after dry heat exposure. In principle, no major differences were found between non-heated and pasteurized commercial concentrates of same manufactural origin.