Microvascular structural entropy: A novel approach to assess angiogenesis in pituitary tumors

Entropy, a measure of the degree of disorder in a system, has recently been used in different morphologic studies to quantify regularity. Our aims were (a) to study the structural organization of the microvascular bed in prolactin (PRL)-producing adenomas and carcinomas, the most vascularized of pituitary tumors, by assessing microvascular structural entropy (MSE), and (b) to determine whether the degree of disorder of the capillary bed correlates with tumor cell proliferation as estimated by MIB-1 labeling, microvessel density (MVD), the most widely used method of quantifying blood vessel formation, and various clinicopathologic parameters (gender, age, tumor size and invasiveness). The morphometric study demonstrated statistically significant differences in MIB-1 labeling, MVD, and MSE between PRL-producing adenomas and carcinomas. Unlike MIB-1 labeling index (PRL-producing adenomas 1.5±0.27; carcinomas 15.0±4.04) and MVD (PRL-producing adenomas 2.7±0.34; carcinomas 4.2±0.72), the MSE values were significantly higher in adenomas (171.5±25.37) than in carcinomas (67.9±17.45). These results indicate that PRL-producing carcinomas have a less chaotic distribution of vessels than benign adenomas. In contrast to a lack of correlation between, microvessel density and other morphometric parameters, a strong negative correlation was found between MSE and MIB-1 labeling index (r=0.511, p=0.003). It thus appears that regular, less chaotic microvascular geometry contributes to increased proliferative activity in PRL cell tumors. Analysis of MSE may provide an independent parameter of tumor behavior, and contributes to a better understanding of the role of microvasculature in pituitary tumor progression.     

Neutralizing antibodies in gene-defective hosts

In a host with a normal immune system and a complete gene defect, the nondefective gene product will be immunogenic. Consequently, neutralizing antibodies against the respective protein can arise either 'spontaneously' or after immunization, as shown in patients and in animal models, such as knockout mice. Accordingly, patients with X-linked or homozygous autosomal gene defects are at risk of developing neutralizing antibodies, in particular after protein substitution or gene therapy. This Review compares and exemplifies the various genetic and immunological contexts that lead to 'neutralizing and generated by gene defect' or 'nagged' antibodies, and outlines implications and solutions for therapeutic strategies.     

A sensitive bioassay for chicken interleukin-18 based on the inducible release of preformed interferon-gamma

Conventional tests for measuring the biological activity of chicken interleukin (IL)-18 require primary chicken spleen cells. We now describe a sensitive bioassay that is based on interleukin-18-induced release of interferon (IFN)-gamma by a permanent chicken cell line. In B19-2D8 cells, cytoplasmically stored interferon-gamma is quickly secreted in response to interleukin-18 exposure.     

The Arabidopsis PILZ group genes encode tubulin-folding cofactor orthologs required for cell division but not cell growth

Plant microtubules are organized into specific cell cycle-dependent arrays that have been implicated in diverse cellular processes, including cell division and organized cell expansion. Mutations in four Arabidopsis genes collectively called the PILZ group result in lethal embryos that consist of one or a few grossly enlarged cells. The mutant embryos lack microtubules but not actin filaments. Whereas the cytokinesis-specific syntaxin KNOLLE is not localized properly, trafficking of the putative auxin efflux carrier PIN1 to the plasma membrane is normal. The four PILZ group genes were isolated by map-based cloning and are shown to encode orthologs of mammalian tubulin-folding cofactors (TFCs) C, D, and E, and associated small G-protein Arl2 that mediate the formation of alpha/beta-tubulin heterodimers in vitro. The TFC C ortholog, PORCINO, was detected in cytosolic protein complexes and did not colocalize with microtubules. Another gene with a related, although weaker, embryo-lethal phenotype, KIESEL, was shown to encode a TFC A ortholog. Our genetic ablation of microtubules shows their requirement in cell division and vesicle trafficking during cytokinesis, whereas cell growth is mediated by microtubule-independent vesicle trafficking to the plasma membrane during interphase.     

The Topography of Non-Linear Cortical Dynamics at Rest, in Mental Calculation and Moving Shape Perception

Differential cortical activation by cognitive processing was studied using dimensional complexity, a measure derived from nonlinear dynamics that indicates the degrees of freedom (complexity) of a dynamic system. We examined the EEG of 32 healthy subjects at rest, during a visually presented calculation task, and during a moving shape perception task. As a nonlinear measure of connectivity, the mutual dimension of selected electrode pairs was used. The first Lyapunov coefficient was also calculated. Data were tested for non-linearity using a surrogate data method and compared to spectral EEG measures (power, coherence). Surrogate data testing confirmed the presence of nonlinear structure in the data. Cognitive activation led to a highly significant rise in dimensional complexity. While both tasks activated central, parietal and temporal areas, mental arithmetic showed frontal activation and an activity maximum at T3, while the moving shape task led to occipital activation and a right parietal activity maximum. Analysis of mutual dimension showed activation of a bilateral temporal-right frontal network in calculation. The Lyapunov coefficent showed clear topographic variation, but was not significantly changed by mental tasks (p<.09). While dimensional complexity was almost unrelated to power values, nonlinear (mutual dimension) and linear (coherence) measures of connectivity shared up to 37% of variance. Data are interpreted in terms of increased cortical complexity as a result of recruitment of asynchronously active, distributed neuronal assemblies in cognition. The topography of nonlinear dynamics are related to neuropsychological and neuroimaging findings on mental calculation and moving shape perception.     

A prospective, randomized, assessor-blind, multicentre study comparing recombinant and urinary follicle stimulating hormone (Puregon versus Metrodin) in in-vitro fertilization

Urinary follicle stimulating hormone (FSH) is being used forthe treatment of human infertility. Recently, FSH manufacturedby means of recombinant DNA technology with a much higher purity(>99%) has become available. A prospective, randomized, assessor-blind,multicentre (n = 18) study was conducted in infertile womenundergoing in-vitro fertilization comparing recombinant FSH(Org 32489, Puregon®) and urinary FSH (Metrodin®). Eligiblesubjects were randomized (recombinant versus urinary FSH = 3:2)and pretreated with buserelin for pituitary suppression. FSHwas given until three or more follicles with a diameter of atleast 17 mm were seen. After oocyte retrieval, fertilizationroutines were applied according to local procedures. No morethan three embryos were replaced. In all, 585 subjects receivedrecombinant FSH and 396 urinary FSH. Significantly more oocyteswere retrieved after recombinant FSH treatment (mean adjustedfor centre 10.84 versus 8.95, P < 0.0001). Ongoing pregnancyrates per attempt and transfer in the recombinant FSH groupwere 22.17 and 25.97% respectively, and in the urinary FSH group,18.22 and 22.02% respectively (not significant). Ongoing pregnancyrates including pregnancies resulting from frozen-thawed embryocycles were 25.7% for recombinant and 20.4% for urinary FSH(P = 0.05). Compared to urinary FSH, the total dose of FSH wassignificantly lower with recombinant FSH (2138 versus 2385 IU,P < 0.0001) in a significantly shorter treatment period (10.7versus 11.3 days, P < 0.0001). No clinically relevant differencesbetween recombinant and urinary FSH were seen with respect tosafety variables. It is concluded that recombinant FSH (Puregon)is more effective than urinary FSH in inducing multifolliculardevelopment and achieving an ongoing pregnancy.     

Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells

Possible interactions of cytoskeletal elements with mechanically induced membrane currents and Ca²⁺ signals were studied in human endothelial cells by using a combined patch-clamp and Fura II technique. For mechanical stimulation, cells were exposed to hypotonic solution (HTS). The concomitant cell swelling activates a Cl^– current, releases Ca²⁺ from intracellular stores and activates Ca²⁺ influx. To interfere with the cytoskeleton, cells were loaded either with the F-actin-stabilizing agent phalloidin (10 mol/l), or the F-actin-depolymerizing substance cytochalasin B (50 mol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endothelial cells was modulated by taxol (50 mol/l), which supports polymerization of tubulin, or by the depolymerizing agent colcemid (10 mol/l) both applied to the bath. Immunofluorescence experiments show that under the chosen experimental conditions the cytoskeletal modifiers employed disintegrate the F-actin and microtubuli cytoskeleton. Neither of these cytoskeletal modifiers influenced the HTS-induced Cl^– current. Ca²⁺ release was not affected by cytochalasin B, taxol or colcemid, but was suppressed if the cells were loaded with phalloidin. Depletion of intracellular Ca²⁺ stores by thapsigargin renders the intracellular [Ca²⁺] sensitive to the extracellular [Ca²⁺], which is indicative of a Ca²⁺ entry pathway activated by store depletion. Neither cytochalasin B nor phalloidin affected this Ca²⁺ entry. We conclude that F-actin turnover or depolymerization is necessary for Ca²⁺ release by mechanical activation. The tubulin network is not involved. The Ca²⁺ release-activated Ca²⁺ entry is not modulated by the F-actin cytoskeleton.     

The nonselective cation channel in the basolateral membrane of rat exocrine pancreas

Nonselective Ca²⁺-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca²⁺ on the cytosolic side, the mean openstate probabilityP _o of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 mol/l of NPPB or DPC decreasedP _o from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 mol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reducedP _o, but in contrast to the abov mentioned substances, it induced fast flickering. Ba²⁺ (70 mmol/l) and tetraethylammonium (TEA⁺; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and 4,4-dinitro-2,2-stilbenedisulfonate (DNDS). 10 mol/l SITS applied to the cytosolic side increasedP _o from 0.5 to 0.7 and with 100 mol/l SITS the channels remained nearly permanently in its open state (P _o1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca²⁺ concentration to 0.1 mol/l, addition of 100 mol/l of SITS had no effect. Similarly, reducing bath Ca²⁺ concentration from 1.3 mmol/l in presence of 100 mol/l SITS (channels are maximally activated) to 0.1 mol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca²⁺. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca²⁺ ions.