[Therapy for Ocular Graft-vs-Host Disease]. / Therapie der okulären Graft-versus-Host-Erkrankung.

Therapy for ocular graft-vs-host disease (ocular GvHD) is challenging for ophthalmologists as progress of the disease often occurs rapidly and is unforeseeable. Primary goal is the preservation or restoration of visual acuity, however, studies on ocular GvHD that have investigated therapeutic concepts are limited. In contrast, most therapeutic recommendations from consensus conferences derive from studies on dry eye diseases other than ocular GvHD. This review demonstrates the available therapies in the following categories: local, systemic, surgical and prophylactic. Primary targets are anti-inflammation, anti-fibrosis and lubrification of the ocular surface. In conclusion, studies strictly on ocular GvHD are needed to enable better evidence-based therapeutic decision-making in the future.

Meibography and meibomian gland measurements in ocular graft-versus-host disease.

Meibomian gland loss in ocular GvHD was described as a mechanism contributing to dry eye and severe damage to the ocular surface. Infrared images of upper eyelid meibomian glands from 86 ocular GvHD patients, from 10 patients after allogeneic stem cell transplantation (aSCT) without ocular GvHD, from 32 patients prior to aSCT and from 30 healthy controls were analyzed retrospectively and evaluated using two grading schemes. The upper meibomian gland area (uMGA) was calculated and set in relation to the total tarsal area of the lid. Results demonstrate that meibomian gland loss is significantly increased in patients with ocular GvHD as well as in patients prior to aSCT in comparison with controls (P between 0.05 and <0.001). Patients after aSCT without ocular GvHD had no significant difference in uMGA in comparison with controls. This study suggests that meibomian gland loss in GvHD patients is likely to be a multifactorial process that also occurs prior to aSCT, possibly due to underlying diseases and/or secondary to chemotherapy or irradiation. In addition, the question has to be addressed whether meibomian gland loss could serve as a predictor for the development of ocular GvHD. Overall, infrared meibography should be included in routine examination of patients undergoing aSCT and during follow-up.

p53-dependent and p53-independent anticancer effects of different histone deacetylase inhibitors.

BACKGROUND: Histone deacetylase inhibitors (HDACi) are promising antineoplastic agents, but their precise mechanisms of actions are not well understood. In particular, the relevance of p53 for HDACi-induced effects has not been fully elucidated. We investigated the anticancer effects of four structurally distinct HDACi, vorinostat, entinostat, apicidin and valproic acid, using isogenic HCT-116 colon cancer cell lines differing in p53 status. METHODS: Effects were assessed by MTT assay, flow-cytometric analyses of propidium iodide uptake, mitochondrial depolarisation and cell-cycle distribution, as well as by gene expression profiling. RESULTS: Vorinostat was equally effective in p53 wild-type and null cells, whereas entinostat was less effective in p53 null cells. Histone deacetylase inhibitors treatment suppressed the expression of MDM2 and increased the abundance of p53. Combination treatments showed that vorinostat enhanced the cytotoxic activity of TRAIL and bortezomib, independent of the cellular p53 status. Investigations into the effects of an inhibitor of the sirtuin class of HDAC, tenovin-1, revealed that tenovin-1-mediated cell death hinged on p53. CONCLUSION: These results demonstrate that vorinostat activates p53, but does not require p53 for inducing its anticancer action. Yet they also demonstrate that entinostat-induced cytotoxic effects partially depend on p53, indicating that different HDACi have a different requirement for p53.

[Short and simple is not always better: limitations of cognitive screening tests]. / Kurz und einfach ist nicht immer besser: Grenzen kognitiver Demenzscreenings.

The increasing prevalence of Alzheimer's dementia (AD) and limited resources in outpatient care have encouraged the distribution of cognitive screening tests, in spite of their frequently unsatisfying accuracy regarding the differentiation between incipient AD, depression and age-associated memory impairment. 8 patients with probable AD and 17 controls completed a neuropsychological follow-up two years after initial examination. Beside four screening tests a memory based testing-the-limits (TtL) paradigm as well as the German version of the California Verbal Learning Test were administered. Based on hierarchical cluster analysis we could demonstrate that only well elaborated tests, such as a plasticity based TtL paradigm, did classify AD-patients correctly. The findings confirm reservations against cognitive screening procedures in detecting dementia and suggest that dynamic test strategies offer a powerful diagnostic alternative to traditional status-oriented tests.

Effect of Reynolds number, turbulence level and periodic wake flow on heat transfer on low pressure turbine blades.

The development of effective cooling methods is of major importance for the design of new gas turbines blades. The conception of optimal cooling schemes requires a detailed knowledge of the heat transfer processes on the blade's surfaces. The thermal load of turbine blades is predominantly determined by convective heat transfer which is described by the local heat transfer coefficient. Heat transfer is closely related to the boundary layer development along the blade surface and hence depends on various flow conditions and geometrical parameters. Particularly Reynolds number, pressures gradient and turbulence level have great impact on the boundary layer development and the according heat transfer. Therefore, in the present study, the influence of Reynolds number, turbulence intensity, and periodic unsteady inflow on the local heat transfer of a typical low pressure turbine airfoil is experimentally examined in a plane cascade.

Film cooling from rows of holes--effect of cooling hole shape and row arrangement on adiabatic effectiveness.

In the present study the film cooling performance in terms of the adiabatic film cooling effectiveness on the scaled suction side model of an actual guide vane was investigated. An infrared thermography measurement system was used to determine highly resolved distribution of models surface temperature. Two different film cooling hole configurations were investigated: a single row of fanshaped holes and a double row of cylindrical holes in staggered arrangement. The influence of blowing rate and mainstream turbulence level on effectiveness was investigated in a wide range for both of the injection configurations.

Effects of entrance crossflow directions to film cooling holes.

Two-dimensional distributions of local adiabatic film cooling effectiveness as well as discharge coefficients have been measured to investigate the effect of different entrance crossflow orientations and magnitudes on film-cooling performance. Operating conditions have been varied in terms of hot gas Mach number (up to 0.6), coolant crossflow Mach number (up to 0.6), coolant crossflow orientation (perpendicular or parallel with respect to the mainflow), and blowing ratio (0.5-1.5). The temperature ratio of coolant and hot gas was kept constant at 0.56 for the effectiveness tests, leading to an enginelike density ratio of 1.8. Infrared thermography was applied to perform local measurements of the surface temperatures with high resolution. The results indicate that the impact of hot gas crossflow Mach number is not very pronounced within the range of Mach numbers investigated. In contrast to this finding, the effect of internal coolant crossflow is very pronounced and strongly depends on coolant crossflow orientation and the ejected mass flow rate.

Site directed in-vitro assembly of nucleosomes.

An in-vitro system is described allowing for the assembly of nucleosomes on preselected sites of a cloned tRNA gene. The system consists of a soluble nucleoprotein fraction, a histone source, and circular DNA containing a single stranded stretch (sssDNA). Nucleosomes assemble on the sssDNA, if the three components are incubated as a highly concentrated solution in the presence of the four deoxyribonucleotidetriphosphates and of ATP. The single stranded stretch is rendered double stranded during incubation. The middle axis of one assembled nucleosome always coincides roughly with the midpoint of the original single stranded DNA stretch.

R-loop hybridization of collagen DNA: separation in Cs2SO4 gradients.

High molecular weight poly(A)RNA (26-35 S) from chicken embryo trunks which is enriched for collagen mRNA was iodinated and hybridized to DNA under conditions of R-loop formation. The R-loops were separated in Cs2SO4 gradients from the bulk of DNA yielding double stranded DNA enriched for collagen genes.

A phase relationship associates tRNA structural gene sequences with nucleosome cores.

DNA (760 bp) isolated from nucleosome tetramers of staphylococcal nuclease-digested chicken embryo chromatin was highly enriched for tRNA genes and subsequently cloned in E. coli chi 1776. The location of genes coding for chicken embryo tRNALys, tRNAPhe and tRNAiMet within the cloned nucleosome tetramer DNA was determined using restriction endonucleases for which single cleavage sites could be predicted from the respective tRNA base sequence. All our tRNA genes reside nonrandomly at four locations on nucleosome tetramer DNA. The spacing between the tRNA gene locations is approximately 190 bp, similar to the DNA repeat length of chicken embryo chromatin. The four tRNA gene locations were also defined in noncloned nucleosome tetramer DNA highly enriched for tRNA genes. The majority of genes coding for tRNALys, tRNAPhe and tRNAiMet, respectively, are located in equal proportion 40-45, 230, 420 and 610 bp distant from the 5' end of the tRNA-identical strand. Thus the tRNA structural gene sequences all appear to begin about 20 bp "inside" the nucleosome core. As observed with nucleosomal DNA not enriched for tRNA genes, the phase relationship between tRNA genes and nucleosome location is maintained over a distance of 4-6 subsequent nucleosomes. A cloned molecule of nucleosomal DNA containing both a tRNALys gene and a tRNAiMet gene in the same polarity reveals that a phase adjustment might be necessary for the nucleosomes between these two tRNA genes in chicken embryo chromatin.

Cloning of chicken embryo tRNA genes using single stranded nucleosomal DNA highly enriched for tRNA complementary sequences.

DNA from chicken embryo nucleosome tetramers (about 760 base pairs in size) was enriched for tRNA genes by RPC-5 chromatography. The enriched DNA was hybridized with chicken embryo total tRNA and the hybridized DNA isolated utilizing a) avidinbiotin interaction, b) diazobenzyloxymethyl paper, and c) high temperature RPC-5 chromatography. The obtained single stranded DNA highly enriched for tRNA complementary sequences was hybridized with total DNA from nucleosome monomers (140--190 base pairs in size) and the excess of non hybridized monomer nucleosome DNA removed by Sepharose 4B chromatography. The hybrid molecules obtained were made fully double stranded by incubation with E. coli DNA polymerase I, DNA ligase, and exonuclease III. DNA was inserted into plasmid pBR322 by G-C joining procedure and the recombinant DNA used to transform the E. coli strain chi 1776. More than 70% of the transformants obtained hybridize to chicken embryo total tRNA.

Purification of class A, B, and C DNA-dependent RNA polymerases from chicken embryos.

Crude nuclei were isolated from trunks of 13-day-old chicken embryos under conditions which prevent leakage of RNA polymerases from nuclei. RNA polymerases were solubilized by subsequent incubation in alkaline buffer and sonication at high salt concentration. Purification of RNA polymerases A, B, and C was achieved by conventional column chromatographic procedures. RNA polymerase B was freed from an UTP:polynucleotidyl exotransferase by chromatography on a tRNA-Sepharose column. Purified RNA polymerase A contained six putative subunits with molecular weights 190 000 (A1), 117 000 (A2), 57 000 (A3), 50 000 (A4), 25 000 (A5), 19 000 (A6); RNA polymerase B contained eight putative subunits with molecular weights 98 000 (B2'), 86 000 (B2''), 155 000 (B3), 44 000 (B4), 31 000 (B5), 28 000 (B6), 26 000 (B7), 19 000 (B8); RNA polymerase C contained nine putative subunits with molecular weights 170 000 (C1), 117 000 (C2), 84 000 (C3), 60 000 (C4), 49 000 (C5), 36 000 (C6), 33 000 (C7), 22 000 (C8), 19 000 (C9).

Reverse transcription of tRNA.

The 3' terminus of tRNA was enzymatically elongated by an oligo(A) tail. A fragment of DNA polymerase I (E. coli) was used in the presence of manganese to phase and synthesize a cleavable primer at the oligo(A)-tRNA template. When the threedimensional structure of oligo(A)-tRNA is being unfolded under conditions where the primer is still hybridized at the oligo(A) tail, the DNA polymerase I fragment transcribes oligo(A)-tRNA into DNA. Reverse transcription is slowed down and its fidelity suspended by the 1-methyladenine in oligo(A)-tRNAPhe(yeast). The reaction is stopped by the highly modified Y-base present in this template. Approximately full length transcripts can be obtained from oligo(A)-tRNA3Gly(E.coli). The transcription products were characterized by sequence analysis.