Loss of drug-induced activation of the CD95 apoptotic pathway in a cisplatin-resistant testicular germ cell tumor cell line

Testicular germ cell tumors (TGCTs) are unusually sensitive to cisplatin. In the present study the role of the CD95 death pathway in cisplatin sensitivity of TGCT cells was studied in Tera and its in vitro acquired cisplatin-resistant subclone Tera-CP. Cisplatin induced an increase in CD95 membrane expression, which preceded the onset of apoptosis. Cisplatin-induced apoptosis was efficiently blocked by caspase-8 inhibitor zIETD-fmk in Tera cells, but only partially in Tera-CP cells. In addition, cisplatin induced FADD and caspase-8 recruitment to the CD95 receptor in Tera cells, which was not noticed in Tera-CP cells. Moreover, overexpression of vFLIP reduced apoptosis induction by cisplatin in Tera cells. CD95L-blocking experiments revealed the involvement of CD95/CD95L interactions in cisplatin-induced apoptosis of Tera cells as well as cisplatin-sensitive 833KE TGCT cells. Tera and 833KE cells, treated with low doses of cisplatin, were sensitive for an apoptosis-inducing anti-CD95 antibody. In contrast, CD95L blocking had no effect on cisplatin-induced apoptosis in Tera-CP or Scha, an intrinsic resistant TGCT cell line, nor did anti-CD95 antibody induce additional apoptosis in cisplatin-treated Tera-CP or Scha cells. Taken together, these results show that (1) cisplatin sensitivity of TGCT cells is dependent on the activation of the CD95 death pathway and (2) loss of cisplatin-induced activation of this CD95 signaling pathway may result in resistance to cisplatin.     

How Reliable Are Current Data for Assessing the Actual Prevalence of Chronic Obstructive Pulmonary Disease?

Background

Estimating COPD occurrence is perceived by the scientific community as a matter of increasing interest because of the worldwide diffusion of the disease. We aimed to estimate COPD prevalence by using administrative databases from a city in central Italy for 2002–2006, improving both the sensitivity and the reliability of the estimate.

Methods

Multiple sources were used, integrating the hospital discharge register (HDR), clinical charts, spirometry and the cause-specific mortality register (CMR) in a longitudinal algorithm, to reduce underestimation of COPD prevalence. Prevalence was also estimated on the basis of COPD cases confirmed through spirometry, to correct misclassification. Estimating such prevalence relied on using coefficients of validation, derived as the positive predictive value (PPV) for being an actual COPD case from clinical and spirometric data at the Institute of Clinical Physiology of the National Research Council.

Results

We found that sensitivity of COPD prevalence increased by 37%. The highest estimate (4.43 per 100 residents) was observed in the 5-year period, using a 3-year longitudinal approach and combined data from three sources. We found that 17% of COPD cases were misclassified. The above estimate of COPD prevalence decreased (3.66 per 100 residents) when coefficients of validation were applied. The PPV was 80% for the HDR, 82% for clinical diagnoses and 91% for the CMR.

Conclusions

Adjusting the COPD prevalence for both underestimation and misclassification of the cases makes administrative data more reliable for epidemiological purposes.     

Lymphocyte subpopulations in peripheral blood and bone marrow in patients with idiopathic myelofibrosis

In 8 patients with idiopathic myelofibrosis (IM) T and B cells were studied in view of the possibility that immunological dysfunctions are involved in initiating or contributing to the bone marrow fibrosis. In peripheral blood the absolute numbers of E-SRBC and OKT3+ lymphocytes were significantly reduced; in addition a significant decline was observed in the proportion and absolute numbers of OKT8+ cells, resulting in a reversed Leu-3a/OKT8 ratio. An impaired B cell function was observed in 4 of the 8 patients, characterized by a disturbed in vitro pokeweed mitogen stimulated immunoglobulin synthesis and low serum immunoglobulin levels. Immuno-histological studies of the bone marrow demonstrated a scarcity of T cells but normal numbers of B cells. However, no correlation was noted between the observed deviations of B and T cells and the degree of bone marrow fibrosis determined by means of bone marrow histology and serum procollagen-III levels. These data are not sufficient to support the hypothesis that immunological changes in IM are primarily involved in the process of bone marrow fibrosis.     

Heat shock proteins and Bcl-2 expression and function in relation to the differential hyperthermic sensitivity between leukemic and normal hematopoietic cells

A major problem in autologous stem cell transplantation is the occurrence of relapse by residual neoplastic cells from the graft. The selective toxicity of hyperthermia toward malignant hematopoietic progenitors compared with normal bone marrow cells has been utilized in purging protocols. The underlying mechanism for this selective toxicity has remained unclear. By using normal and leukemic cell line models, we searched for molecular mechanisms underlying this selective toxicity. We found that the differential heat sensitivity could not be explained by differences in the expression or inducibility of Hsp and also not by the overall chaperone capacity of the cells. Despite an apparent similarity in initial heat-induced damage, the leukemic cells underwent heat-induced apoptosis more readily than normal hematopoietic cells. The differences in apoptosis initiation were found at or upstream of cytochrome c release from the mitochondria. Sensitivity to staurosporine-induced apoptosis was similar in all cell lines tested, indicating that the apoptotic pathways were equally functional. The higher sensitivity to heat-induced apoptosis correlated with the level of Bcl-2 protein expression. Moreover, stable overexpression of Bcl-2 protected the most heat sensitive leukemic cells against heat-induced apoptosis. Our data indicate that leukemic cells have a specifically lower threshold for heat damage to initiate and execute apoptosis, which is due to an imbalance in the expression of the Bcl-2 family proteins in favor of the proapoptotic family members.     

Optimal lineage principle for age-structured populations

We present a formulation of branching and aging processes that allows age distributions along lineages to be studied within populations, and provides a new interpretation of classical results in the theory of aging. We establish a variational principle for the stable age distribution along lineages. Using this optimal lineage principle, we show that the response of a population's growth rate to age-specific changes in mortality and fecundity--a key quantity that was first calculated by Hamilton--is given directly by the age distribution along lineages. We apply our method also to the Bellman-Harris process, in which both mother and progeny are rejuvenated at each reproduction event, and show that this process can be mapped to the classic aging process such that age statistics in the population and along lineages are identical. Our approach provides both a theoretical framework for understanding the statistics of aging in a population, and a new method of analytical calculations for populations with age structure. We discuss generalizations for populations with multiple phenotypes, and more complex aging processes. We also provide a first experimental test of our theory applied to bacterial populations growing in a microfluidics device.     

Most Water in the Tomato Truss Is Imported through the Xylem,Not the Phloem: A Nuclear Magnetic Resonance Flow Imaging Study

In this study, we demonstrate nuclear magnetic resonance flow imaging of xylem and phloem transport toward a developing tomato (Solanum lycopersicum) truss. During an 8-week period of growth, we measured phloem and xylem fluxes in the truss stalk, aiming to distinguish the contributions of the two transport tissues and draw up a balance between influx and efflux. It is commonly estimated that about 90% of the water reaches the fruit by the phloem and the remaining 10% by the xylem. The xylem is thought to become dysfunctional at an early stage of fruit development. However, our results do not corroborate these findings. On the contrary, we found that xylem transport into the truss remained functional throughout the 8 weeks of growth. During that time, at least 75% of the net influx into the fruit occurred through the external xylem and about 25% via the perimedullary region, which contains both phloem and xylem. About one-half of the net influx was lost due to evaporation. Halfway through truss development, a xylem backflow appeared. As the truss matured, the percentage of xylem water that circulated into the truss and out again increased in comparison with the net uptake, but no net loss of water from the truss was observed. The circulation of xylem water continued even after the fruits and pedicels were removed. This indicates that neither of them was involved in generating or conducting the circulation of sap. Only when the main axis of the peduncle was cut back did the circulation stop.Fruits are terminal organs that depend completely on long-distance transport to supply them with sugars and water for growth. Water is imported by means of both the xylem and the phloem, whereas sugars are only imported by means of the phloem. Fruits have to compete for water with the rest of the plant, and for that reason, xylem influx is expected to be sensitive to changes in plant water potential. Xylem influx into fruits may thus be lower during the day and higher during the night. When in the apoplast the water potential is especially low, for instance, when the plant is transpiring a lot of water during a hot day, fruits may even experience a xylem efflux and lose water to the vegetative parts of the plant (Johnson et al., 1992; Guichard et al., 2005). It has been suggested that in several species, in order to reduce the sensitivity of fruits to changes in plant water status, during fruit development the xylem connection between fruit and plant is reduced or even severed (Findlay et al., 1987; Lang, 1990; Creasy et al., 1993; Lang and Ryan, 1994; van Ieperen et al., 2003; Drazeta et al., 2004). In contrast to the xylem, the phloem is expected to be relatively insensitive to diurnal changes in water potential (Ehret and Ho, 1986; Ho et al., 1987). For instance, in the main stem of a number of plants, the phloem was found not to respond to diurnal differences in plant water status, whereas the xylem did (Peuke et al., 2001; Windt et al., 2006).The tomato (Solanum lycopersicum) plant has been the subject of many studies dealing with long-distance transport to fruits and has been chosen as a model system in this study as well. It has been estimated that in tomato fruits, about 80% to 90% of the influx of sap takes place by means of the phloem (Ho et al., 1987; Plaut et al., 2004; Guichard et al., 2005). It has been proposed that the low xylem contribution is due to the presence of some form of restriction in the xylem connection between plant and fruit, possibly in the knuckle (Lee, 1989; van Ieperen et al., 2003). Despite the expected low xylem contribution and limited conductivity of the xylem connection between plant and fruit, fruits have been shown to exhibit a diurnal pattern of growth. In most cases, fruits have been observed to grow fastest at night (Lee, 1989; Grange, 1995; van de Sanden and Uittien, 1995; Guichard et al., 2005). The opposite has been found to occur as well (Ehret and Ho, 1986; Pearce et al., 1993), but in these cases, the faster daytime growth was probably caused by a low diurnal stress environment. In a number of studies, even an efflux of xylem sap and fruit shrinkage during the day was reported (Johnson et al., 1992; Leonardi et al., 1999, 2000). It has been proposed that, if the phloem and xylem operate under different diurnal cycles or if their relative contributions can be modified in any way by adjusting the environmental conditions in a greenhouse, it might become possible to control and regulate fruit yield as well as fruit quality and taste.Considering the importance of fruit for the world''s food production, surprisingly little is known about the dynamics of sap flow to fruits. Since the conception of the cohesion tension theory (Dixon and Joly, 1894) and the Munch pressure flow hypothesis (Münch, 1930), there has been a decent theoretical understanding of the basic forces that govern phloem and xylem flow. It has already been attempted to apply this understanding to model fruit growth for a variety of fruits and applications (e.g. Daudet et al., 2002). However, many of the parameters that are needed to model long-distance transport to fruits are currently outside of experimental reach. First, little is known about the pressure and water potential gradients that drive flow to fruits. The xylem and the phloem are extremely sensitive to invasive experimentation and are easily disturbed, and the water potentials in the fruits’ symplast and apoplast are difficult to assess. Second, it is not clear whether xylem and phloem sap only enters the fruit (unidirectional flow), or if return flow is possible as well, and if it is, under which conditions it may occur. As the results of this study show, NMR flow imaging can provide answers to these important questions.

Estimating Long-Distance Transport to Fruits

So far, the most important methods to estimate xylem and phloem influx in fruits have been the subtractive method (Lang and Thorpe, 1989) and the mineral accumulation method (Ho et al., 1987). In the subtractive method, the contribution of xylem and phloem are estimated by heat girdling the pedicel (fruit stalk) of a fruit. Heat girdling destroys the sieve tubes, stopping phloem influx, while the xylem is assumed to remain intact and functional. By comparing the growth of nongirdled fruits to that of girdled fruits, the phloem contribution can be estimated. The most critical assumption in this method is that the xylem sap flow is not affected by heat girdling. However, the validity of this assumption is not evident. First, because xylem and phloem flow to fruits are coupled. Xylem influx is driven by a water potential difference between the xylem and the fruit symplast, which is maintained by osmotically active compounds (sugars), which in turn are imported by means of the phloem. Fishman et al. (2001) showed that the coupling between phloem and xylem influx could give rise to significant errors when using the pedicel girdling technique. A second reason is that heat girdling may profoundly affect xylem function. The xylem tissue may apparently escape heat girdling unscathed, as demonstrated by Guichard et al. (2005), but if the surrounding cells are damaged, it is not unlikely that functional damage will occur. For instance, it has been proposed that the cells that surround the xylem protect it against embolisms by preventing the entry of air (Hacke and Sperry, 2001). Van Ieperen et al. (2003) found that in the tomato pedicel, the abscission zone is the site of highest xylem resistance and that only a few xylem conduits traverse it. If an obstruction would occur in these conduits, either by embolisms or by particles of debris, it could significantly affect xylem resistance and have large implications for xylem transport to the fruit.In the second method, the mineral composition of the fruit is used to estimate the relative xylem and phloem contribution. Ho et al. (1987) measured calcium accumulation, net water import, and fruit respiration in tomato fruits. The xylem contribution to fruit growth was then estimated based on a number of assumptions: (1) the calcium content of phloem sap can be neglected compared to that of xylem sap; (2) the calcium content of xylem sap is similar to that measured in root stump exudate; and (3) xylem backflow from fruits does not occur. However, in view of current knowledge, the first and third assumptions are questionable. Calcium is used in signal transduction and as such is known to be present in the phloem. The question is, in what concentration. In phloem sap exudate of castor bean (Ricinus communis) and eucalyptus (Eucalyptus globulus), calcium concentrations have been found that were about 66% and 25% of the concentration in the root stump exudate, respectively (Pate et al., 1998; Peuke et al., 2006). In Banksia prionotes, the calcium concentration in phloem exudate was even found to be 10 times higher than that of the xylem sap (Pate and Jeschke, 1995). It should be noted that in these studies phloem sap was harvested by cutting. This may have elicited a wounding response, causing elevated calcium levels in the phloem (Knoblauch et al., 2001). Still, we argue that these findings illustrate that the calcium concentration in the phloem cannot be assumed to be negligible, especially when the majority of influx of sap is thought to take place via the phloem. The assumption that backflow does not take place also may not hold. In a number of studies, backflow from tomato fruits has already been observed, especially under summer conditions or high vapor deficit (Johnson et al., 1992; Leonardi et al., 1999, 2000; Guichard et al., 2005). The subtractive and the mineral accumulation method thus are likely to be subject to large systematic errors. Better methods to estimate or measure long-distance transport to fruits are needed.

NMR Flow Imaging

Over the last 10 years, it has been demonstrated that NMR flow imaging can provide an excellent tool to measure xylem and phloem transport (Van As, 2007). NMR flow imaging does not only give information about the average flow velocity, such as heat pulse based methods do, but gives access to all properties of the flowing water, such as the flow conducting area, the distribution of flow velocities, and the volume flow, all on a per pixel basis (Scheenen et al., 2000b). So far, studies have been conducted measuring flow in the stem of a variety of plants, ranging from castor bean seedlings (Köckenberger et al., 1997) to fully developed tomato, castor bean, and tobacco (Nicotiana tabacum) plants, and a small poplar tree (Populus spp.; Windt et al., 2006). The technique has been used to study the diurnal variation in long-distance transport (Peuke et al., 2001; Windt et al., 2006), the effects of cold girdling (Peuke et al., 2006), and xylem embolism repair (Scheenen et al., 2007) and has been used as a reference technique to provide detailed velocity maps for comparison with different heat pulse methods (e.g. Helfter et al., 2007; D. Chavarro, C.W. Windt, M.W. Lubczynski, J. Roy, and H. Van As, unpublished data). These studies have in common that flow was only measured in the main stem of the plant. This is a convenient place to do flow imaging for a variety of reasons. In comparison with other flow-conducting structures in the plant, the stem is large, sturdy, and stable. It conducts the largest fluxes, and the xylem and phloem can be easily distinguished on the basis of their direction of flow. These properties make imaging xylem and phloem transport relatively easy.

Aims and Research Questions

In this study, we used NMR flow imaging to measure long-distance transport to fruits. As a model plant, tomato was chosen. The anatomy of the tomato truss, as well as the dimensions of the magnetic resonance imaging (MRI) device and its components, made it impossible to image the pedicel of a single fruit. The pedicels were too short and too close together to fit them with the radio frequency (RF) coil that is needed for MRI. For this reason, we chose to perform flow imaging on the peduncle of tomato, measuring the transport toward the entire developing truss. After fitting the plant in the imager, it was impossible to remove the plant without damaging it. The plant was therefore left in the imager and allowed to grow there for 8 weeks. In this period, we continuously monitored long-distance transport into the truss, aiming to answer the following questions: (1) can xylem and phloem flow into the truss be visualized and distinguished; (2) what transport tissues conduct sap into the truss during truss development; (3) is phloem and xylem transport into the truss unidirectional, or does backflow occur; and (4) can NMR flow imaging be used to draw up a quantitative balance of xylem and phloem influx into the truss?     

Coronary stent implantation without balloon predilatation: a single-center experience

OBJECTIVE: Assessment of safety and efficacy of coronary stent deployment, without balloon predilatation. BACKGROUND: With newer high-performance balloon-premounted stents it has become more common to attempt coronary stent deployment without balloon pre- or postdilatation. METHODS: During 1998 524 coronary angioplasties were performed in the authors' institute, of which 279 resulted in coronary stenting. Of these 101 (36.2%) were stents without balloon predilatation (SWBP). PTCA was performed according to standard technique using mostly 7 F. guiding catheters, and 'rapid exchange' balloons and pre-mounted stents. RESULTS: Seventy-two patients had acute coronary syndromes (41 acute MI or post-MI angina, 28 unstable angina, 10 rescue PTCA after failed thrombolysis). Mean age was 56.4 3 11.1 years, 84.5% were males. Sixty per cent of the lesions were ACC-AHA type B2 or C. Target arteries were LAD 57.6%, LCX 21.2%, RCA 14.1% and SVG 7.1%. Procedure time was 18.2 3 17.3 minutes. Mean heparin dose was 3850 3 1570 units. Twenty-two patients received abciximab prior to stent deployment. Seven stents were not deployed without previous balloon dilatation and were retrieved safely via the guiding catheters and deployed after balloon dilatation. There was no stent embolization, ectopic suboptimal or partial stent deployment. Immediate angiographic success was obtained in 95 patients (94.1%). Minimal lumin diameter (MLD) increased from 0.27 3 0.15 to 3.23 3 2.1 mm. There were two in-hospital deaths (1.9%) due to cardiogenic shock. An intra-aortic balloon pump was required in eight patients. Two patients (1.9%) experienced subacute stent thrombosis. CONCLUSION: SWBP in selective groups of patients and lesions is feasible and safe. Larger randomized comparative trials are needed to assess the benefits and cost saving of this approach.     

Intraspecies genomic groups in Enterococcus faecium and their correlation with origin and pathogenicity

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.