Reliable detection of macrolide-resistant Helicobacter pylori via fluorescence in situ hybridization in formalin-fixed tissue.

Macrolide-resistant Helicobacter (H.) pylori represent an increasing therapeutic problem. Macrolide resistance is usually determined phenotypically in vitro with methods such as E-test or agar dilution test. A prerequisite for those tests, however, is bacterial culture that is not routinely set up in the course of gastroscopy. In contrast, formalin-fixed, paraffin-embedded biopsies are regularly available from patients who have undergone gastroscopy. In such biopsies macrolide-resistant H. pylori can be detected by the genotype-based technique of fluorescence in situ hybridization (FISH). Experience gained by this new method, however, is still extremely limited, especially in formalin-fixed tissue. Therefore, we retrospectively investigated formalin-fixed, paraffin-embedded biopsy specimens by FISH in 104 patients suffering from therapy-resistant H. pylori gastritis. To test the accuracy of FISH, we initially examined specimens from 53 patients for whom results of the E-test were available. Next we analyzed biopsies from another 51 patients that had been selected since phenotypical resistance testing had failed despite documented culturing attempts. In all 104 patients, H. pylori was detected by FISH and could thus be investigated for macrolide resistance. Overall, macrolide-resistant bacteria were found in 71 patients (68.3%). In 49 of 53 patients (92.4%), FISH and E-test returned identical results (no significant discordance according to McNemar's chi(2)-test). Taken together, our study demonstrates that FISH is a highly sensitive and reliable method for detecting macrolide-resistant H. pylori in formalin-fixed, paraffin-embedded biopsy specimens, which represents the routine method of processing tissue obtained upon gastroscopy.     

Evolution of resource competition between mutually dependent digital organisms

We study the emergence and dynamics of competing strains of digital organisms in a world with two depletable resources. Consumption of one resource produces the other resource as a by-product, and vice versa. As a consequence, two types of mutually dependent organisms emerge that each prey on the waste product of the other. In the absence of mutations, that is, in a purely ecological setting, the abundances of the two types of organisms display a wide range of different types of oscillations, from regular oscillations with large amplitude to irregular oscillations with amplitudes ranging from small to large. In this regime, time-averaged abundance levels seem to be controlled by the relative fitness of the organisms in the absence of resources. Under mutational pressure, on the other hand, populations evolve that seem to avoid the oscillations of intermediate to large amplitudes. In this case, the relative fitness of the organisms in the presence of resources plays an important role in the time-averaged abundance levels as well.     

Infantile hemangioma is a proliferation of beta 4-negative endothelial cells adjacent to HLA-DR-positive cells with dendritic cell morphology

Although hemangioma is referred as to the most common tumor in infancy, the underlying pathogenetic events and the biologic origin of this benign vascular neoplasm have remained obscure. By using immunohistochemistry on frozen sections of infantile hemangiomas, we show here that proliferating endothelial cells abundantly expressed alpha(v)beta(3) but lacked beta(4) integrins. Instead, regressing and involuting infantile hemangiomas due to treatment with IFN-alpha showed positive staining of beta(4) integrin, which might point to the angiogenic significance of beta(4) integrin in infantile hemangiomas. Moreover, immunofluorescence analysis revealed the existence of HLA-DR(+), mostly CD68(+) and partly DC-SIGN/CD209(+) cells with dendritic cell morphology in the intimate vicinity of hemangiomatous vessels. Such cells were also detected in the dermal microvascular unit in normal skin. The coupled occurrence of vascular structures and perivascular cells that were stained positive with markers of monocyte or macrophage or dendritic cells might suggest that the development of infantile hemangioma is a result of vasculogenesis, that is, the formation of primitive blood vessels from angioblasts, rather than of angiogenesis, that is, the sprouting of capillaries from preexisting vessels.     

Effect of polyphenolic compounds on the renal Na+,K(+)-ATPase during development and persistence of hypertension in rats

It has been suggested that polyphenolic substances provide protection against the risk factors of cardiovascular diseases. The present study was designed to investigate whether application of red wine polyphenols influences the kinetic properties of the renal Na+,K(+)-ATPase in rats with hypertension (164 +/- 8 mmHg) that was experimentally induced by the NO synthase inhibitor N(G.) -nitro-L- arginine methyl ester (L-NAME). Polyphenols in a dose of 40 mg kg(-1) day(-1) in drinking fluid induced different effects on the properties of the renal Na+,K(+)-ATPase depending on the mode of their administration. Preventive application of polyphenols during the development of hypertension (144 +/- 5 mmHg) partially protected the Na+,K(+)-ATPase molecule against hypertension-induced deterioration via increased capability of the enzyme to bind ATP and/or Na+ as suggested by decrease of Km and KNa, respectively, even to values lower than in controls. However, polyphenols did not prevent the hypertension-induced reduction of the number of active Na+,K(+)-ATPase molecules as shown by similar V(max) values as compared to the hypertensive L-NAME group. The above protection is probably secured by a NO-dependent mechanism as suggested by 150% increase of the NO synthesis. Additional treatment of already hypertensive animals with polyphenols (153 +/- 8 mmHg) resulted in partial restoration of the Na+,K(+)-ATPase affinities especially for sodium as indicated by significant diminution of KNa. However, polyphenols in this mode of application did not slow down the L-NAME-induced decrease in the number of Na+,K(+)-ATPase molecules in the kidney as suggested by additional significant decrease in V(max) values when comparing this group with the control group and also the hypertensive L-NAME group. In this case the polyphenols affected the Na,K-ATPase molecule in a NO-independent way as indicated by the fact that polyphenols failed to restore normal NO synthesis.     

Multiplex polymerase chain reaction.

The polymerase chain reaction (PCR) is a widely utilized assay for specifically amplifying small fragments of DNA. Multiplex PCR is the amplification of more than one DNA fragment per reaction and has many potential uses. When more than one primer set per reaction tube is utilized, the total number of tubes in any one experiment may be reduced, conserving expensive reagents and decreasing possible contamination. Multiplex PCR allows for an assay of the gene of interest and assures that the amplification process proceeds as expected with the use of a companion control genome primer set. Multiplex PCR is useful in assaying DNA extracted from samples of immunocompromised patients in which more than one infectious agent may be suspected such as simultaneous EBV and CMV detection. Multiplex PCR offers many advantages over single reaction PCR and has been found to be an useful adjunct in our laboratory.     

Comparison of viable cell yield from excised versus aspirated adipose tissue

The correction of soft-tissue defects by adipose tissue transplantation often produces poor and unpredictable results. The implantation of isolated and cultured preadipocytes offers a solution to this problem since these cells differentiate into adipocytes when implanted in vivo. A field of major interest is to maximize the yield of preadipocytes isolated from adipose tissue showing only low contamination with other cell types. Aspiration and excision are two concurrent clinical ways of harvesting adipose tissue for the isolation of preadipocytes. This tissue is usually discarded after surgery. In this study, the yield of preadipocytes obtained from liposuction material was compared to that of excised adipose tissue. Furthermore, we determined the loss of precursor cells if isolation of preadipocytes was delayed for 24 h. Preadipocytes were isolated from the stromal cell fraction of human subcutaneous adipose tissue samples. Harvesting of adipose tissue by suction was performed according to the Coleman procedure (manually applied negative pressure using a 10-ml syringe with a blunt tip cannula). Isolation was either carried out within 60 min after extraction or after storage for 24 h in culture medium at 4 degrees C. Isolated preadipocytes were cultured for 24 h, trypsinized and counted in a Neubauer chamber. Our results show clearly that the yield of preadipocytes isolated from liposuction material (within 60 min after extraction and after 24 h of storage) is higher than the cell yield from excised adipose tissue. Overnight storage for 24 h leads to a significant loss of preadipocytes in excised tissue but not in liposuction material. The high yield of cells isolated from liposuction material proves that extraction by suction does not damage the stromal cell fraction in the adipose tissue. If cell isolation is not performed immediately after the operation, liposuction material is clearly the better alternative for storage.     

The “Mozart Effect”: An Electroencephalographic Analysis Employing the Methods of Induced Event-Related Desynchronization/Synchronization and Event-Related Coherence

The event-related responses of 18 individuals were recorded while they were listening to 3 music clips of 6 s duration which were repeated 30 times each. The music clips differed in the level of their complex structure, induced mood, musical tempo and prominent frequency. They were taken from Mozart's sonata (K. 448), and Brahms' Hungarian dance (no. 5). The third clip was a simplified version of the theme taken from Haydn's symphony (no. 94) played by a computer synthesizer. Significant differences in induced event-related desynchronization between the 3 music clips were only observed in the lower-1 alpha band which is related to attentional processes. A similar pattern was observed for the coherence measures. While respondents listened to the Mozart clip, coherence in the lower alpha bands increased more, whereas in the gamma band a less pronounced increase was observed as compared with the Brahms and Haydn clips. The clustering of the three clips based on EEG measures distinguished between the Mozart clip on the one hand, and the Haydn and Brahms clips on the other, even though the Haydn and Brahms clips were at the opposite extremes with regard to the mood they induced in listeners, musical tempo, and complexity of structure. This would suggest that Mozart's music--with no regard to the level of induced mood, musical tempo and complexity--influences the level of arousal. It seems that modulations in the frequency domain of Mozart's sonata have the greatest influence on the reported neurophysiological activity.     

Modulation of the responses of human osteoblast-like cells to physiologic mechanical strains by biomaterial surfaces

In a previous study we demonstrated that MG-63 cells cultured on Ti-6Al-4V discs covered by alumina ceramic and submitted to intermittent mechanical strain (IMS) presented morphological alteration associated with enhanced differentiation. Here we examine how the mechanical response of osteoblasts can be modulated by the nature of the substrate. MG-63 cells were cultured on four materials: polystyrene and Ti-6Al-4V (average roughness = 0.48 microm) as smooth substrates; Ti-6Al-4V (average roughness = 5.76 microm) and Ti-6Al-4V covered with alumina (average roughness = 5.21 microm) as rough substrates. Mechanical strains were applied for 15 min, three times a day for 1-5 days with a 600 microstrains magnitude and a 0.25 Hz frequency. IMS stimulated alkaline phosphatase activity by 25-35% on all substrates and had no effect on cell growth on either substrate. Fibronectin (FN) was chosen as representative of cell-matrix interaction. FN production was increased by 60% after 1 day of stretching only on alumina-coated discs. FN organization examined on smooth substrates was affected by 5 days of IMS, showing a thickening of the fibres. The same modifications induced by IMS were previously observed on alumina-covered discs. Vinculin expression was not affected by IMS whatever the substrate. Cell-cell interactions were determined by N-cadherin immunoblotting. N-cadherin expression was increased by IMS specifically on rough substrates. Our results suggest that the nature of the surface did not influence the up-regulation of alkaline phosphatase activity induced by IMS, but modulates specifically cell-substrate as well as cell-cell interactions in response to IMS.