Prognostic value of O-6-methylguanine-DNA methyltransferase (MGMT) protein expression in glioblastoma excluding nontumour cells from the analysis.

AIMS: It is important to predict response to treatment with temozolomide (TMZ) in glioblastoma (GBM) patients. Both MGMT protein expression and MGMT promoter methylation status have been reported to predict the response to TMZ. We investigated the prognostic value of quantified MGMT protein levels in tumour cells and the prognostic importance of combining information of MGMT protein level and MGMT promoter methylation status. METHODS: MGMT protein expression was quantified in tumour cells in 171 GBMs from the population-based Region of Southern Denmark (RSD)-cohort using a double immunofluorescence approach. Pyrosequencing was performed in 157 patients. For validation we used GBM-patients from a Nordic Study (NS) investigating the effect of radiotherapy and different TMZ schedules. RESULTS: When divided at the median, patients with low expression of MGMT protein (AF-low) had the best prognosis (HR = 1.5, P = 0.01). Similar results were observed in the subgroup of patients receiving the Stupp regimen (HR = 2.0, P = 0.001). In the NS-cohort a trend towards superior survival (HR = 1.6, P = 0.08) was seen in patients with AF-low. Including MGMT promoter methylation status, we found for both cohorts that patients with methylated MGMT promoter and AF-low had the best outcome; median OS 23.1 and 20.0 months, respectively. CONCLUSION: Our data indicate that MGMT protein expression in tumour cells has an independent prognostic significance. Exclusion of nontumour cells contributed to a more exact analysis of tumour-specific MGMT protein expression. This should be incorporated in future studies evaluating MGMT status before potential integration into clinical practice.

Corrigendum to "Combination therapy with interferon and JAK1-2 inhibitor is feasible. Proof of concept with rapid reduction in JAK2V617F-allele burden in Polycythemia Vera" [Leuk. Res. Rep. 3 (2) (2014) 73-75].

[This corrects the article DOI: 10.1016/j.lrr.2014.05.003.].

Combination therapy with interferon and JAK1-2 inhibitor is feasible: Proof of concept with rapid reduction in JAK2V617F-allele burden in polycythemia vera.

We report a 55 year old woman with post-ET PV for 12 years, who experienced resolution of severe constitutional symptoms within 3 days, a marked reduction in splenomegaly and a rapid decline in the JAK2V617F allele burden during combination therapy with interferon-alpha2a and ruxolitinib. Within 4 weeks the patient achieved complete hematological remission with normalization of peripheral blood counts and within 10 months the JAK2V617F-allele burden was reduced from 90% to 28%. Such a rapid decline in the JAK2V617F allele burden is highly unusual in PV-patients during low-dose IFN-alpha2 monotherapy and this finding warrants a prospective study with combination therapy.

Patient satisfaction with care in the intensive care unit: can we rely on proxies?

BACKGROUND: To investigate whether next of kin can be addressed as proxy to assess patients' satisfaction with care in the intensive care unit (ICU). METHODS: Prospective observational multicentre study. Two hundred and thirty-five patients with an ICU length of stay of ≥2 days and 266 of their adult next of kin participated. Patient satisfaction was assessed by a questionnaire, distributed upon discharge from an ICU and compared with next of kin's answers. The possible range of answers was 0-100, with higher numbers indicating higher satisfaction. The main outcome measure was the extent of agreement between patients' satisfaction with care and the ratings of their next of kin. RESULTS: Patients were most satisfied concerning physicians' competence (86.7±16.3), while least satisfaction was observed for the management of agitation and restlessness (78.2±23.5). There was no significant difference between next of kin's and patients' ratings. Agreement between patients and proxies was the highest concerning overall satisfaction (Cohen's κ 0.40) and the lowest for coordination of care (0.24). Spouses/partners had a higher agreement with the patients' ratings than other proxies. CONCLUSIONS: If the patient is unable to rate his satisfaction with care in the ICU, next of kin may be taken as an appropriate surrogate. TRIAL REGISTRATION: The study has been registered at ClinicalTrials.gov, Reg No: NTC 00890513.

Clinical trial: comparison of the gastrointestinal safety of lumiracoxib with traditional nonselective nonsteroidal anti-inflammatory drugs early after the initiation of treatment--findings from the Therapeutic Arthritis Research and Gastrointestinal Event Trial.

BACKGROUND: The large (n = 18 325) Therapeutic Arthritis Research and Gastrointestinal Event Trial (TARGET) study demonstrated a significant gastrointestinal benefit with lumiracoxib 400 mg o.d. (4x the recommended dose in osteoarthritis) vs. naproxen 500 mg b.d. or ibuprofen 800 mg t.d.s. AIM: To investigate how early a reduction in ulcer complications could be detected with lumiracoxib vs. nonselective nonsteroidal anti-inflammatory drugs in TARGET. METHODS: Pointwise 95% confidence intervals were generated for the between-treatment differences in Kaplan-Meier estimates for definite or probable upper gastrointestinal ulcer complications (ulcer complications) and for all ulcers. RESULTS: In patients not on aspirin, there was a significant reduction in all ulcers by day 8 and in ulcer complications by day 16 with lumiracoxib compared with both nonselective nonsteroidal anti-inflammatory drugs combined, by day 6 (all ulcers) and day 14 (ulcer complications) vs. naproxen and by day 32 (all ulcers) and day 33 (ulcer complications) vs. ibuprofen. CONCLUSION: Even with short-term use, there are gastrointestinal safety benefits for lumiracoxib vs. nonselective nonsteroidal anti-inflammatory drugs.

Does ICU length of stay influence quality of life?

BACKGROUND: Patients with prolonged stay in the intensive care unit (ICU) use a disproportionate share of resources. However, it is not known if such treatment results in impaired quality of life (QOL) as compared to patients with a short length of stay (LOS) when taking into account the initial severity of illness. METHODS: Prospective, observational case-control study in a university hospital surgical and trauma adult ICU. All patients admitted to the ICU during a 1-year period were included. Patients with a cumulative LOS in the ICU > 7 days, surviving up to 1 year after ICU admission and consenting were identified (group L, n = 75) and matched to individuals with a shorter stay (group S). Matching criteria were diagnostic group and severity of illness. Health-related quality of life (HRQOL) was assessed 1 year after admission using the short-form 36 (SF-36) and was compared between groups and to the general population. Further, overall QOL was estimated using a visual analogue scale (VAS) and willingness to consent to future intensive care, and was compared between groups L and S. RESULTS: Based on ANCOVA, a significant difference between groups L and S was noted for two out of eight scales: role physical (P = 0.033) and vitality (P = 0.041). No differences were found for the physical component summary (P = 0.065), the mental component summary (P = 0.267) or the VAS (P = 0.316). Further, there was no difference in expectation to consent to future intensive care (P = 0.149). As compared to the general population, we found similar scores for the mental component summary and for three of eight scales in group L and five of eight scales in group S. CONCLUSIONS: When taking into account severity of illness, HRQOL 1 year after intensive care is comparable between patients with a short and a long LOS in the ICU. Thus, prolonged stay in the ICU per se must not be taken as an indicator of future poorer HRQOL. However, as compared to the general population, significant differences, mostly in physical aspects of QOL, were found for both groups of patients.

Differentiation of foot-and-mouth disease virus infected animals from vaccinated animals using a blocking ELISA based on baculovirus expressed FMDV 3ABC antigen and a 3ABC monoclonal antibody.

A blocking ELISA that differentiated foot-and-mouth disease virus (FMDV) infected animals from vaccinated animals was developed which uses baculovirus expressed FMDV 3ABC non-structural protein as antigen and monoclonal antibody against FMDV 3ABC non-structural protein as capture and detector antibody. Sera from naive, vaccinated and infected cattle, sheep and pigs were examined. The specificity of the test was high. Non-specific reactions observed in particular in sera of cattle and sheep could be removed by filtration and inactivation. Positive reactions were obtained for sera from cattle infected with all seven serotypes of FMDV. The test detected antibodies from days 7 or 9 following experimental infection of non-vaccinated cattle and sheep, and in cattle strong positive reactions persisted for up to 395 days after infection. In vaccinated cattle that became carriers after challenge with homologous FMDV, positive reactions were obtained in all but one case. In some of these cattle the antibody response was detected late in comparison to the non-vaccinated infected cattle. The test gave results that compared favourably with two commercial ELISA's when used to test sera from cattle, pigs and sheep collected after experimental or natural infection. The blocking ELISA based on recombinant FMDV 3ABC antigen and a monoclonal antibody to 3ABC is a promising tool for FMD control and eradication campaigns, where vaccination has been carried out.

Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting the 3D gene of FMDV. The assay was validated for the efficacy to detect all known FMDV serotypes. The test method was linear over a range of at least 7 orders of magnitude and the detection limit was below the equivalent of 10 genomic copies. Analysing recent African probang samples the method was able to detect FMDV in materials from both cattle and buffalo. When compared to traditional virus cultivation the virus detection sensitivity was similar but the RT-PCR method can provide a laboratory result much faster than virus cultivation. The real-time PCR method confirms the identity of the amplicon by melting point analysis for added specificity and at the same time allows the detection of mutations in the probe region. As such, the described new method is suitable for the robust real-time detection of index cases caused by any serotype of FMDV.

Resource use in the ICU: short- vs. long-term patients.

BACKGROUND: Intensive care medicine uses a disproportionate share of medical resources, and little is known about the distribution of resources between different patient groups. METHODS: In this prospective observational study, all patients admitted between 1 January 1998 and 31 December 1999 to our medical-surgical university's ICU were assigned to one of two groups according to length of stay (LOS): patients staying more than 7 days in the unit (group L) and those staying a maximum of 7 days (group S). Resource use was estimated using TISS-28, number of nursing shifts, use of mechanical ventilation, and use of renal replacement therapy. Further, SAPS II and ICU and hospital mortalities were recorded. RESULTS: Of 5481 patients, 583 (10.6%) were in group L and 4898 in group S (89.4%). Patients in group L were more severely sick upon admission than those in group S. Patients in group L stayed a total of 9726 days in the ICU (52.5% of the total LOS). In group L, 69.2% of all shifts with respiratory support and 80.1% of all shifts with renal replacement were used. Further, group L patients consumed 53.4% (909225) of all TISS points provided. The ICU-mortality rates were 14.4% in group L and 7.2% in group S, and the hospital mortality rates were 19.9% and 9.8%, respectively. A mean of 1898 TISS points was used per patient surviving the hospital stay in group L compared with 190 points in group S. CONCLUSIONS: In this university-based, medical-surgical adult ICU, 11% of all patients stayed more than 7 days in the unit and consumed more than 50% of all resources. Thus, a highly disproportionate amount of resources were used per survivor in group L compared with those in group S.

Conservation and heterogeneity of the glutamate-rich protein (GLURP) among field isolates and laboratory lines of Plasmodium falciparum.

Genetic variation of the glutamate-rich protein (GLURP) of Plasmodium falciparum was analysed in 29 field isolates and 15 laboratory lines of diverse geographical origin, by DNA sequencing of the non-repetitive 5'-region (R0) of the glurp gene. Polymorphism with respect to the length of the GLURP R2 repeat region was also analysed by a polymerase chain reaction method. As reference, the nucleotide sequence of the highly polymorphic 3'-region of the circumsporozoite protein gene was determined in the same isolates. It was found that even in the presence of variations in the GLURP R2 repeat region, immunodominant parts of the GLURP R0 region are surprisingly well conserved and the conservation is most pronounced in isolates from locations with very high malaria transmission. This might indicate that the R0 structure plays an important role in the parasite.

Anti-CD95 (APO-1/Fas) autoantibodies and T cell depletion in human immunodeficiency virus type 1 (HIV-1)-infected children.

Advanced stages of HIV-1-infection are characterized by progressive CD4+ T cell depletion. Peripheral T cells from HIV-1+ donors show accelerated apoptosis in vitro. The CD95 (APO-1/Fas) receptor/ligand system is involved in this process. To further study deregulation of the CD95 system in peripheral T cells during HIV-1-infection, we measured CD95-expression on CD4+ and CD8+ T cells together with serum levels of soluble CD95 (sCD95) and anti-CD95 autoantibodies in HIV-1+ children and healthy controls. Anti-CD95 levels in HIV-1+ children were significantly elevated when compared to uninfected controls, whereas serum levels of sCD95 were not different. In HIV-1+ children, CD95-expression on CD4+ and CD8+ T cells increased with age. A strong correlation between depletion of CD4+ cells in vivo and increase in CD95-expression on CD4+ T cells was observed. In contrast, such a correlation was not found for CD8+ T cells. A negative correlation between anti-CD95 autoantibody levels and CD4+ T cell counts, that was predicted by multiple linear regression analysis of pooled data, was found in individual patients observed longitudinally by repeated measurements. Since anti-CD95 autoantibodies isolated from HIV-infected adults have previously been shown to induce apoptosis of sensitive target cells in vitro, we speculate that the interaction of these antibodies with CD95-positive and CD95-sensitive T cells in vivo might be involved in progressive T cell loss during HIV-1-infection.

Family interaction and the course of schizophrenic illness. Results of a multivariate prospective study.

The aim of the present study was to assess the prognostic relevance of relatives' interactive behaviour towards the patient, as covered by the Münster Family Interview (MFI), to the further course of the schizophrenic illness. The MFI is a family interview (of the whole family, including the patient) designed to record the emotional family atmosphere based on the concept of expressed emotion (EE). The ratings take place directly after the interview on five scales (criticism, hostility, overinvolvement, resignation and warmth), the resignation scale being added to the 'classic' EE scales. Ninety-nine families of outpatients diagnosed with schizophrenia according to the DSM-III were examined with the MFI during a home visit. The patients were seen 1 and 2 years after the first examination. The target criteria selected for the prognostic significance of the interaction measurements were: rehospitalisation within 2 years; extent of symptoms after 1 year, and psychosocial skills after 1 year. The significance of the interaction dimensions was verified in regression models. The control variable used in the regression models was the Strauss-Carpenter scale. Regression models were produced for the total group and for a subgroup of moderately ill patients. All target criteria yielded serviceable prediction models. The most important variable for prediction was the control variable, the Strauss-Carpenter scale. However the interaction variables made additional contributions to the prognosis, especially in the subgroup of moderately ill patients. The best MFI scale for all the outcome criteria was resignation; criticism predicted only the symptomatology, and emotional overinvolvement the level of social functioning after 1 year. In conclusion, practical work with families of schizophrenic patients should emphasise the protective function of relatives towards patients more strongly.

Expressed-emotion development and course of schizophrenic illness: considerations based on results of a CFI replication.

This study examines the correlation between development of expressed emotion (EE) in relatives and course of illness of 99 DSM-III schizophrenic patients. Patients whose relatives were high EE at baseline and at the 2nd CFI approximately 20 months later had a poor prognosis at the very outset of the study and an unfavourable course of illness. They had a higher rehospitalisation rate, more symptoms, lower psychosocial assessment, and a poorer 2-year and even 8-year outcome. Patients from families with a fluctuating EE or a consistently low EE had better courses. Expressed emotion is therefore a valid predictor not only of symptomatic relapses, but also of other important aspects of schizophrenia. The connection between EE index and course of illness seems not to be simply reactive or causal, but complex and non-uniform.

Expressed emotion in an 8 year follow-up.

This study addresses the questions if and for which patients expressed emotion (EE) has a predictive validity in an 8 year follow-up study and if this is dependent on the residential form, especially on patients and relatives living together. Eight years after a baseline examination (Camberwell Family Interviews [CFI], global assessment scale [GAS], symptom score) 69 of 99 patients were reexamined. The number and duration of rehospitalizations as well as the symptom and GAS scores were recorded. These was no difference between outcome of high and low EE patients with a duration of illness of less than 4.5 years and for those living with partners. Patients from high EE parents with a longer duration of illness (> 4.5 years) at the outset of the study were significantly more often and longer in hospital. They had significantly higher symptom and GAS scores at the outset of the study and after 8 years. The EE effect was equal in the first and second 4 years of the follow-up and was independent from patients and parents living together. The results emphasize the significance of the EE index as a long-term predictor for the course of schizophrenic illness. The independence of the prediction from living together is an argument against a simple causal interpretation of the connection between EE and the course of schizophrenia.

Molecular mechanisms of APO-1/Fas(CD95)-mediated apoptosis in tolerance and AIDS.

The cell surface molecule APO-1/Fas(CD95), a member of the Tumor Necrosis Factor (TNF) receptor/Nerve Growth Factor (NGF) receptor superfamily mediates apoptosis upon cross-linking by agonistic antibodies or its ligand. Recent findings suggest that APO-1/Fas(CD95) and its ligand are the key molecules for antigen receptor-induced apoptosis in activated mature T cells. Here we propose a mechanism for antigen receptor-induced apoptosis of activated T cells via APO-1 ligand mediated autocrine suicide. This mechanism may also contribute to the depletion of CD4+ T cells in AIDS.

Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120.

The depletion of CD4+ T cells in AIDS is correlated with high turnover of the human immunodeficiency virus HIV-1 and associated with apoptosis. The molecular mechanism of apoptosis in HIV infection, however, is largely unknown. T-cell apoptosis might be affected by viral proteins such as HIV-1 Tat and gp120 (refs 10, 11). T-cell-receptor (TCR)-induced apoptosis was recently shown to involve the CD95 (APO-1/Fas) receptor. We show here that HIV-1 Tat strongly sensitizes TCR- and CD4(gp120)-induced apoptosis by upregulation of CD95 ligand expression. Concentrations of Tat found to be effective in cultures of HIV-1-infected cells were also observed in sera from HIV-1-infected individuals. Taken together, our results indicate that HIV-1 Tat and gp120 accelerate CD95-mediated, activation-induced T-cell apoptosis, a mechanism that may contribute to CD4+ T-cell depletion in AIDS.

Identification of soluble APO-1 in supernatants of human B- and T-cell lines and increased serum levels in B- and T-cell leukemias.

The cell-surface protein APO-1 is a member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor superfamily. APO-1 mediates apoptosis in susceptible cells upon stimulation with the monoclonal antibody anti-APO-1 or upon binding of its natural ligand. Soluble receptors had previously been identified for most members of the NGF/TNF receptor superfamily. Recently, a soluble form of APO-1 (sAPO-1) was described. We established a sandwich enzyme-linked immunosorbent assay to detect sAPO-1 in culture supernatants of human cell lines and in human sera. sAPO-1 was found in culture supernatants of different human B- and T-cell lines. Molecular weights of sAPO-1 and membrane APO-1 were similar. In addition, in comparison to healthy donors, sera from patients with different high- and low-grade malignant B- and T-cell leukemias and lymphomas contained increased levels of sAPO-1. These findings may have implications for the growth of leukemias and the diagnostic monitoring of individual patients.

Hydrogen peroxide as a potent activator of T lymphocyte functions.

During inflammatory processes infiltrating cells produce large amounts of reactive oxygen intermediates (ROI). Increasing evidence suggests that ROI besides being cytotoxic may act as important mediators influencing various cellular and immunological processes. In this study, we have investigated the effects of hydrogen peroxide on several aspects of lymphocyte activation. In ESb-L T lymphoma cells, micromolar concentrations of hydrogen peroxide rapidly induced activation of the transcription factor NF-kappa B, whereas DNA-binding activity of the transcription factor AP-1 was virtually not affected. In addition, hydrogen peroxide induced early gene expression of interleukin-2 (IL-2) and the IL-2 receptor alpha chain. The stimulation of IL-2 expression was found to be conferred by a kappa B-like cis-regulatory region within the IL-2 gene promoter. In contrast to these activating effects, addition of hydrogen peroxide was largely inhibitory on cell proliferation which is consistent with a general requirement of thiol compounds for lymphocyte proliferation. However, hydrogen peroxide significantly increased T cell proliferation when applied for a short period under reducing conditions. These data indicate that ROI may act as an important competence signal in T lymphocytes inducing early gene expression as well as cell proliferation.

Activation of the mouse IL-2 gene by okadaic acid: synergy with interleukin-1.

Interleukin-1 (IL-1) has potent immunoregulatory and inflammatory functions. Its activity is mediated by an 80-kDa receptor on the cell surface and leads to activation of other genes. The underlying molecular events are largely unknown. We investigated the role of phosphatases in activation of the IL-2 gene in EL4 thymoma cells. We found that the protein phosphatase PP1 and PP2A inhibitor okadaic acid (OA) alone was able to significantly stimulate IL-2 production by the IL-1-responsive EL4 subline EL4 5D3 and also by the IL-1-nonresponsive EL4 subline EL4D6/76. In the IL-1-responsive cell line OA strongly synergized with phorbol myristate acetate (PMA) and IL-1. In the IL-1-nonresponsive cell line OA synergized with PMA but not with IL-1. Under suboptimal conditions of PMA/OA synergy an additional synergistic effect of IL-1 was shown. This was true for IL-2 and IL-6 production. Sphingomyelinase or sphingosine had no detectable effect. The kinetics of OA- and PMA-induced expression of IL-2 mRNA and IL-2 protein was different. PMA induced maximal expression between 6 and 12 h and was almost undetectable at 24 h. OA-induced expression was first obvious at 12 h and continued longer than 36 h. In both cases IL-1 caused no shift in kinetics, but potentiated the effects of the different tumor promoters. Utilizing IL-2 promoter-CAT constructs we showed in transfection experiments that the synergistic effect was also evident on the transcriptional level. We conclude from the data that phosphatases play an important role for IL-2 expression and that IL-1 can use additional pathways of activation that are different from events induced by PMA or OA.