Prospective evaluation of clinical scoring systems in infants with bronchiolitis admitted to the intensive care unit.

The objective of this investigation was to compare different scoring systems to assess the severity of illness in infants with bronchiolitis admitted to a tertiary paediatric intensive care unit (PICU). Over an 18-year period (1990-2007), infants with bronchiolitis aged up to 12 months and admitted to the PICU were prospectively scored using the Pediatric Risk of Mortality III (PRISM III) score, the Organ System Failure (OSF) score and the Acute Physiologic Score for Children (APSC) within 24 h. Infants were compared as to whether or not bronchiolitis was associated with respiratory syncytial virus (RSV). There was no difference between 113 RSV-positive and 80 RSV-negative infants regarding gestational age, birth weight, rate of premature delivery or bronchopulmonary dysplasia (BPD). The PRISM III score differed significantly between RSV-positive and RSV-negative cases (3.27 ± 0.39 vs. 1.96 ± 0.44, p = 0.006), as did the OSF score (0.56 ± 0.05 vs. 0.35 ± 0.06, p = 0.049) and the APSC (5.16 ± 0.46 vs. 4.1 ± 0.53, p = 0.048). All scores were significantly higher in the subgroup with mechanical ventilation (p < 0.0001). The mean time of ventilation was significantly higher in the RSV-positive group compared to the RSV-negative group (6.39 ± 1.74 days vs. 2.4 ± 0.47 days, p < 0.001). Infants suffering from RSV-positive bronchiolitis had higher clinical scores corresponding with the severity of bronchiolitis.

Quality of life measurement in oncology--a matter of the assessment instrument?

Two widely used quality of life questionnaires, European Organization for Research and Treatment of Cancer Core (EORTC QLQ-C30) & Functional Assessment of Cancer Therapy-General (FACT-G), were examined for their comparability using four different groups of cancer patients. During a follow-up investigation, 418 cancer patients (Hodgkin's disease, breast cancer, bone marrow transplantation (BMT), chronic lymphatic leukaemia (CLL)) completed both the EORTC QLC-C30 and the FACT-G during the same session. For an illustration of the differences between the two Quality of Life (QoL) instruments, pairs of diagnostic groups were formed and their QoL scores using the EORTC QLQ-C30 and FACT-G compared. The corresponding subscales of the EORTC-QLC-C30 and the FACT-G show only low to moderate intercorrelations across all four groups of cancer patients studied. In particular, a comparison of pairs, namely Hodgkin's disease versus breast cancer patients and BMT versus CLL patients, highlights substantial differences in the corresponding subscales of the EORTC QLQ-C30 and the FACT-G. The results of the QoL investigations should not be interpreted independently of the instrument used and an interpretation of results must be based on the contents of items of the respective questionnaires.

Analysis of Bcl-2 protein expression in chronic lymphocytic leukemia. A comparison of three semiquantitation techniques.

A number of studies revealed that high expression of the proto-oncogene bcl-2 correlated with poor prognosis or resistance to chemotherapy in some tumors but predicted a favorable clinical course in other neoplasias. In these studies, however, different immunologic techniques for Bcl-2 detection were used, raising the question of whether the methods applied were comparable. Using chronic lymphocytic leukemia (CLL) cells, the aims of our study were as follows: (1) to determine the reproducibility of Bcl-2 semiquantitation by immunocytochemistry, flow cytometry, or immunoblotting; (2) to study the agreement between results obtained by these methods; and (3) to examine the association between Bcl-2 expression in tumor cells of 99 patients with CLL and clinical parameters. We found that determination of Bcl-2 expression by immunocytochemistry was reproducible and the results were comparable with those of flow cytometry and immunoblotting. In the patient collective examined, Bcl-2 expression did not reflect the extent of tumor mass, but higher levels were found more often in patients with progressive disease.

Expression of functional interleukin-15 receptor and autocrine production of interleukin-15 as mechanisms of tumor propagation in multiple myeloma.

Interleukin-15 (IL-15) induces proliferation and promotes cell survival of human T and B lymphocytes, natural killer cells, and neutrophils. Here we report the constitutive expression of a functional IL-15 receptor (IL-15R) in 6 of 6 myeloma cell lines and in CD38(high)/CD45(low )plasma cells belonging to 14 of 14 patients with multiple myeloma. Furthermore, we detected IL-15 transcripts in all 6 myeloma cell lines, and IL-15 protein in 4/6 cell lines and also in the primary plasma cells of 8/14 multiple myeloma patients. Our observations confirm the existence of an autocrine IL-15 loop and point to the potential paracrine stimulation of myeloma cells by IL-15 released from the cellular microenvironment. Blocking autocrine IL-15 in cell lines increased the rate of spontaneous apoptosis, and the degree of this effect was comparable to the pro-apoptotic effect of depleting autocrine IL-6 by antibody targeting. IL-15 was also capable of substituting for autocrine IL-6 in order to promote cell survival and vice versa. In short-term cultures of primary myeloma cells, the addition of IL-15 reduced the percentage of tumor cells spontaneously undergoing apoptosis. Furthermore, IL-15 lowered the responsiveness to Fas-induced apoptosis and to cytotoxic treatment with vincristine and doxorubicin but not with dexamethasone. These data add IL-15 to the list of important factors promoting survival of multiple myeloma cells and demonstrate that it can be produced and be functionally active in an autocrine manner. (Blood. 2000;95:610-618)

Differential expression of cell-cell adhesion proteins and cyclin D in MEK1-transdifferentiated MDCK cells.

Overexpression of a constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 (caMEK1) in epithelial Madin-Darby canine kidney (MDCK)-C7 cells disrupts morphogenesis, induces an invasive phenotype, and is associated with a reduced rate of cell proliferation. The role of cell-cell adhesion molecules and cell cycle proteins in these processes, however, has not been investigated. We now report loss of E-cadherin expression as well as a marked reduction of beta- and alpha-catenin expression in transdifferentiated MDCK-C7 cells stably expressing caMEK1 (C7caMEK1) compared with epithelial mock-transfected MDCK-C7 (C7Mock1) cells. At least part of the remaining alpha-catenin was coimmunoprecipitated with beta-catenin, whereas no E-cadherin was detected in beta-catenin immunoprecipitates. In both cell types, the proteasome-specific protease inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN) and lactacystin led to a time-dependent accumulation of beta-catenin, including the appearance of high-molecular-weight beta-catenin species. Quiescent as well as serum-stimulated C7caMEK1 cells showed a higher cyclin D expression than epithelial C7Mock1 cells. The MEK inhibitor U-0126 inhibited extracellular signal-regulated kinase phosphorylation and cyclin D expression in C7caMEK1 cells and almost abolished their already reduced cell proliferation rate. We conclude that the transdifferentiated and invasive phenotype of C7caMEK1 cells is associated with a diminished expression of proteins involved in cell-cell adhesion. Although beta-catenin expression is reduced, C7caMEK1 cells show a higher expression of U-0126-sensitive cyclin D protein.

Differential sensitivity of CD4+ and CD8+ T lymphocytes to the killing efficacy of Fas (Apo-1/CD95) ligand+ tumor cells in B chronic lymphocytic leukemia.

B-chronic lymphocytic leukemia (B-CLL) is characterized by cellular and humoral immune defects resulting in increased rates of infection and disturbed immune surveillance against cancer cells as well as by the expansion of slowly proliferating tumor cells. We found increased Fas receptor (FasR) expression in peripheral blood CD4+ and CD8+ cells of B-CLL patients compared with the equivalent cells of healthy donors. Although increased Fas receptor expression was significant in both T-lymphocytic subsets, only CD4+ cells from B-CLL patients underwent apoptosis after treatment with the agonistic Fas antibody CH11. In CD4+ cells of B-CLL patients, the Fas-sensitivity also correlated with a CD4+/CD8+ ratio below the lower threshold of healthy individuals (<1.0). By contrast, FasR expression in the CD19(+) fraction of B-CLL patients was downregulated compared with normal controls, and this was associated with an insensitivity to CH11-induced apoptosis. The B-CLL cell line EHEB as well as CD19(+) cells from B-CLL patients constitutively expressed Fas ligand (FasL). The FasL was functionally active, as the B-CLL cell line as well as T-cell-depleted CD19+ B-CLL fractions were able to kill target T-acute lymphatic leukemia (T-ALL) cells in vitro. This effect was inhibited by the antagonistic FasR-antibody ZB4, the neutralizing anti-FasL monoclonal antibody (MoAb) NOK-2 or by transfection of the caspase inhibitor crmA. These data point to the fact that expression of FasL on CD19(+) B-CLL cells, together with enhanced susceptibility of CD4+ T cells toward FasL-bearing effector cells, are causally linked to the relative reduction of CD4+ cells occurring during B-CLL progression. These findings could explain the inversion of the ratio of CD4+/CD8+ cell numbers, which may be causally linked to the immune deficiency observed in these patients and to the expansion of the neoplastic clone in B-CLL.

Rapid activation of Na+/H+-exchange in MDCK cells by aldosterone involves MAP-kinase ERK1/2.

The mineralocorticoid aldosterone is essential for the adequate regulation of electrolyte homeostasis, extracellular volume and blood pressure. As a steroid hormone it influences cellular functions by genomic actions. Previously it has been shown that aldosterone can activate Na+/H+-exchange (NHE) by a rapid, nongenomic mechanism. Because (1) NHE can be regulated by ERK1/2 (extracellular signal-regulated kinase) and (2) steroids have been reported to rapidly activate ERK1/2, we tested the hypothesis that activation of NHE by aldosterone involves ERK1/2, using MDCK-C11 cells. We show that nanomolar concentrations of aldosterone induce a rapid, non-genomic activation of NHE, which is characterized by an increased affinity for H+ with minor changes in the maximum transport rate. Accordingly, aldosterone led to an increase of cytosolic steady-state pH. The aldosterone-induced activation of NHE was prevented by the two specific inhibitors of ERK1/2 activation, PD 98059 (2.5 x 10(-5) mol/l) and U0126 (10(-5) mol/l). Furthermore, in the presence of U0126 there was no aldosterone-induced increase of steady-state pH. Finally, aldosterone induced a rapid phosphorylation of ERK1/2, indicating its ability to activate ERK1/2. The data presented here support the hypothesis that the rapid activation of NHE by aldosterone at nanomolar concentrations involves ERK1/2. Thus, in certain cell types, the MAPK cascade may represent an additional pathway mediating rapid aldosterone effects.

Expression of Apo-1/Fas (CD95), Bcl-2, Bax and Bcl-x in myeloma cell lines: relationship between responsiveness to anti-Fas mab and p53 functional status.

The down-regulation of apoptosis may be an essential mechanism for tumour cell expansion in slowly proliferating tumours such as multiple myeloma. We studied eight myeloma cell lines for the presence of Bcl-2, which inhibits apoptosis, of Bax, which counteracts Bcl-2, of Bcl-x(L) and Bcl-x(S), which act in an anti- and pro-apoptotic fashion, respectively, and of Apo-1/Fas, which induces programmed cell death, when activated by the Apo-1/Fas ligand or the relevant monoclonal antibody (mab). All cell lines constitutively expressed homogenous amounts of Bcl-2, but displayed different amounts of Bax and Bcl-x proteins. The Apo-1/Fas antigen could be detected in seven out of eight myeloma lines, but expression levels varied considerably. The relative expression levels of Apo-1/Fas correlated with that of Bax, but not with that of Bcl-2 or Bcl-x subtypes. Furthermore, the effectiveness of the Apo-1/Fas mab was associated with the relative expression levels of the Apo-1/Fas and with that of the Bax antigen, but not with that of the Bcl-2 and Bcl-x antigens. We further showed that wild-type p53 function is not required for Apo-1/Fas-induced apoptosis, nor is it necessary for the expression of Bax or Apo-1/Fas antigens in myeloma. In conclusion, our results suggest a p53-independent co-regulation of Apo-1/Fas and Bax, as well as a role for Bax in Apo-1/Fas-induced apoptosis in myeloma.

Constitutive expression of Fas (Apo-1/CD95) ligand on multiple myeloma cells: a potential mechanism of tumor-induced suppression of immune surveillance.

The Fas (Apo-1/CD95) ligand (FasL) plays a central role in the elimination of target cells by effector T lymphocytes and in the suppression of cellular immune responses against nonmalignant and malignant cells. We show the expression of FasL on the surface of neoplastic plasma cells. We provide evidence that the FasL is functionally active because five of five neoplastic plasma cell lines tested killed CEM-C7H2 T-acute lymphoblastic leukemia (T-ALL) cells. The effect was mediated via the Fas (Apo-1/CD95) receptor molecule because blocking of Fas on the target cells or the FasL on the tumor cells by receptor- and ligand-specific monoclonal antibodies (MoAbs), respectively, protected T cells from being killed by myeloma cells. In addition, overexpression of the cowpox virus protein CrmA, a molecule with inhibitory potential on caspase-1 and caspase-8, specifically involved in Fas-induced signaling, protected T cells from being destroyed by the neoplastic cells or the agonistic anti-Fas MoAb. The potential of the malignant plasma cells to extinguish target T cells was independent of their own sensitivity to the agonistic anti-Fas MoAb, and FasL-positive (FasL+) CEM-C7H2 T cells were incapable of killing myeloma cells. Our results suggest that tumor cell-induced suppression of the immune system may be exerted via the FasL active on malignant plasma cells. Furthermore, loss of Fas expression or insensitivity to the agonistic anti-Fas MoAb do not seem to be prerequisites for myeloma cells to defeat T cells via Fas/FasL interaction.