RNA degradation differentially affects quantitative mRNA measurements of endogenous reference genes in human placenta

It has been highlighted that RNA quality and appropriate reference gene selection is crucial for the interpretation of RT-qPCR results in human placental samples. In this context we investigated the effect of RNA degradation on the mRNA abundance of seven frequently used reference genes in 119 human placental samples. Combining RNA integrity measurements, RT-qPCR analysis and mathematical modeling we found major differences regarding the effect of RNA degradation on the measured expression levels between the different reference genes. Furthermore, we demonstrated that a modified RNA extraction method significantly improved RNA quality and consequently increased transcript levels of all reference genes.

Effects of a single administration of prostaglandin F2alpha, or a combination of prostaglandin F2alpha and prostaglandin E2, or placebo on fertility variables in dairy cows 3-5 weeks post partum, a randomized, double-blind clinical trial

BACKGROUND: Delayed uterine involution has negative effects on the fertility of cows; use of prostaglandin F2alpha alone as a single treatment has not been shown to consistently improve fertility. Combined administration of PGF2alpha and PGE2 increased uterine pressure in healthy cows. We hypothesized, that the combination of both prostaglandins would accelerate uterine involution and have, therefore, a positive effect on fertility variables. In commercial dairy farming, the benefit of a single post partum combined prostaglandin treatment should be demonstrated. METHODS: 383 cows from commercial dairy farms were included in this study. Uterine size and secretion were evaluated at treatment 21-35 days post partum and 14 days later. Cows were randomly allocated to one of three treatment groups: PGF2alpha and PGE2, PGF2alpha or placebo. For every animal participating in the study, the following reproduction variables were recorded: Interval from calving to first insemination, days open, number of artificial inseminations (AI) to conception; subsequent treatment of uterus, subsequent treatment of ovaries. Plasma progesterone level at time of treatment was used as a covariable. For continuous measurements, analysis of variance was performed. Fisher's exact test for categorical non-ordered data and exact Kruskal-Wallis test for ordered data were used; pairwise group comparisons with Bonferroni adjustment of significance level were performed. RESULTS: There was no significant difference among treatment groups in uterine size. Furthermore, there was no significant difference among treatments concerning days open, number of AI, and subsequent treatment of uterus and ovaries. Days from calving to first insemination tended to be shorter for cows with low progesterone level given PGF2alpha and PGE2 in combination than for the placebo-group (P = 0.024). CONCLUSION: The results of this study indicate that the administration of PGF2alpha or a combination of PGF2alpha and PGE2 21 to 35 days post partum had no beneficial effect upon measured fertility variables. The exception was a tendency for a shorter interval from calving to first insemination after administration of the combination of PGF2alpha and PGE2, as compared to the placebo group. Further research should be done in herds with reduced fertility and/or an increased incidence of postpartum vaginal discharge.

The diagnostic yield of vitrectomy specimen analysis in chronic idiopathic endogenous uveitis

PURPOSE: The low diagnostic yield of vitrectomy specimen analysis in chronic idiopathic uveitis (CIU) has been related to the complex nature of the underlying disease and to methodologic and tissue immanent factors in older studies. In an attempt to evaluate the impact of recently acquired analytic methods, the authors assessed the current diagnostic yield in CIU. METHODS: Retrospective analysis of consecutive vitrectomy specimens from patients with chronic endogenous uveitis (n = 56) in whom extensive systemic workup had not revealed a specific diagnosis (idiopathic) and medical treatment had not resulted in a satisfying clinical situation. Patients with acute postoperative endophthalmitis served a basis for methodologic comparison (Group 2; n = 21). RESULTS: In CIU, a specific diagnosis provided in 17.9% and a specific diagnosis excluded in 21.4%. In 60.7% the laboratory investigations were inconclusive. In postoperative endophthalmitis, microbiological culture established the infectious agent in 47.6%. In six of eight randomly selected cases, eubacterial PCR identified bacterial DNA confirming the culture results in three, remaining negative in two with a positive culture and being positive in three no growth specimens. A double negative result never occurred, suggesting a very high detection rate, when both tests were applied. CONCLUSIONS: The diagnostic yield of vitrectomy specimen analysis has not been improved by currently routinely applied methods in recent years in contrast to the significantly improved sensitivity of combined standardized culture and PCR analysis in endophthalmitis. Consequently, the low diagnostic yield in CIU has to be attributed to insufficient understanding of the underlying pathophysiologic mechanisms.

[Antirheumatic therapy and reproduction. The influence on fertility, pregnancy and breast feeding]

Antirheumatic drugs can have a negative effect on reproduction in both men and women. Possible negative effects are impairment of fertility, harmful effects on the fetus and adverse effects on the breastfed child. In women non-steroidal antiinflammatory drugs (NSAID) and cyclophosphamide can impair fertility. In men infertility can result from the use of salazopyrine and cyclophosphamide. A desire for children should be taken into account before the start of disease modifying drugs (DMARD). Treatment with NSAID is possible at some stages of pregnancy as well as during lactation. A limited number of DMARD is compatible with pregnancy and is presented. Cytostatic drugs and leflunomide must be prophylactically withdrawn before a planned pregnancy. TNF alpha antagonists should be discontinued at the start of pregnancy. Safe birth control must be practised during therapy with drugs that are gonadotoxic or teratogenic. Treatment with immunosuppressive drugs during lactation is limited because of insufficient documentation of safety for the breastfed child.

Role of endogenous Fas (CD95/Apo-1) ligand in balloon-induced apoptosis, inflammation, and neointima formation

BACKGROUND: Fas (CD95/Apo-1) ligand (FasL)-induced apoptosis in Fas-bearing cells is critically involved in modulating immune reactions and tissue repair. Apoptosis has also been described after mechanical vascular injury such as percutaneous coronary intervention. However, the relevance of cell death in this context of vascular repair remains unknown. METHODS AND RESULTS: To determine whether FasL-induced apoptosis is causally related to neointimal lesion formation, we subjected FasL-deficient (generalized lymphoproliferative disorder [gld], C57BL/6J) and corresponding wild-type (WT) mice to carotid balloon distension injury, which induces marked endothelial denudation and medial cell death. FasL expression in WT mice was induced in injured vessels compared with untreated arteries (P<0.05; n=5). Conversely, absence of functional FasL in gld mice decreased medial and intimal apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling [TUNEL] index) at 1 hour and 7 days after balloon injury (P<0.05; n=6). In addition, peritoneal macrophages isolated from gld mice showed no apoptosis and enhanced migration (P<0.05; n=4). In parallel, we observed increased balloon-induced macrophage infiltrations (anti-CD68) in injured arteries of FasL-deficient animals (P<0.05; n=6). Together with enhanced proliferation (bromodeoxyuridine index; P<0.05), these events resulted in a further increase in medial and neointimal cells (P<0.01; n=8) with thickened neointima in gld mice (intima/media ratio, x3.8 of WT; P<0.01). CONCLUSIONS: Our data identify proapoptotic and antiinflammatory effects of endogenous FasL as important factors in the process of neointimal lesion formation after balloon injury. Moreover, they suggest that activation of FasL may decrease neointimal thickening after percutaneous coronary intervention.

A polymorphism within the equine CRISP3 gene is associated with stallion fertility in Hanoverian warmblood horses

Fertility of stallions is of high economic importance, especially for large breeding organisations and studs. Breeding schemes with respect to fertility traits and selection of stallions at an early stage may be improved by including molecular genetic markers associated with traits. The genes coding for equine cysteine-rich secretory proteins (CRISPs) are promising candidate genes because previous studies have shown that CRISPs play a role in the fertilising ability of male animals. We have previously characterised the three equine CRISP genes and identified a non-synonymous polymorphism in the CRISP1 gene. In this study, we report one non-synonymous polymorphism in the CRISP2 gene and four non-synonymous polymorphisms in the CRISP3 gene. All six CRISP polymorphisms were genotyped in 107 Hanoverian breeding stallions. Insemination records of stallions were used to analyse the association between CRISP polymorphisms and fertility traits. Three statistical models were used to evaluate the influence of single mutations, genotypes and haplotypes of the polymorphisms. The CRISP3 AJ459965:c.+622G>A SNP leading to the amino acid substitution E208K was significantly associated with the fertility of stallions. Stallions heterozygous for the CRISP3 c.+622G>A SNP had lower fertility than homozygous stallions (P = 0.0234). The pregnancy rate per cycle in these stallions was estimated to be approximately 7% lower than in stallions homozygous at this position.

Endogenous bone marrow derived cells express retinal pigment epithelium cell markers and migrate to focal areas of RPE damage

PURPOSE: The aim of the present study was to investigate whether bone marrow-derived cells (BMCs) can be induced to express retinal pigment epithelial (RPE) cell markers in vitro and can home to the site of RPE damage after mobilization and express markers of RPE lineage in vivo. METHODS: Adult RPE cells were cocultured with green fluorescence protein (GFP)-labeled stem cell antigen-1 positive (Sca-1(+)) BMCs for 1, 2, and 3 weeks. Cell morphology and expression of RPE-specific markers and markers for other retinal cell types were studied. Using an animal model of sodium iodate (NaIO(3))-induced RPE degeneration, BMCs were mobilized into the peripheral circulation by granulocyte-colony stimulating factor, flt3 ligand, or both. Immunocytochemistry was used to identify and characterize BMCs in the subretinal space in C57BL/6 wild-type (wt) mice and GFP chimeric mice. RESULTS: In vitro, BMCs changed from round to flattened, polygonal cells and expressed cytokeratin, RPE65, and microphthalmia transcription factor (MITF) when cocultured in direct cell-cell contact with RPE. In vivo, BMCs were identified in the subretinal space as Sca-1(+) or c-kit(+) cells. They were also double labeled for GFP and RPE65 or MITF. These cells formed a monolayer on the Bruch membrane in focal areas of RPE damage. CONCLUSIONS: Thus, it appears that BMCs, when mobilized into the peripheral circulation, can home to focal areas of RPE damage and express cell markers of RPE lineage. The use of endogenous BMCs to replace damaged retinal tissue opens new possibilities for cell replacement therapy in ophthalmology.

[Endogenous hypercortisolism and immunologically-mediated disease: three cases]

We report three women with hypercortisolism presenting with symptoms and signs of Cushing's syndrome. In two of the patients, initial symptoms of hypercortisolism were associated with spontaneous amelioration of previously known atopic dermatitis and psoriasis, respectively. DIAGNOSTIC PROCEDURES: Diagnosis was established by demonstrating both lack of responsiveness to dexamethasone (1mg) suppression test and increased 24-hour urine cortisol secretion. One patient had a low serum ACTH level indicating Cushing's syndrome of adrenal origin. In the other two patients hypercortisolism proved to be ACTH-dependent, the source being the pituitary, as demonstrated by CRH stimulation test (elevation of ACTH and cortisol by 35 % and 20 %, respectively) and sampling of the petrosus sinus. In both patients imaging confirmed the presence of a pituitary adenoma.

Spatial Assessment of Erosion and its Impact on Soil Fertility in the Tajik Foothills

Efficient planning of soil conservation measures requires, first, to understand the impact of soil erosion on soil fertility with regard to local land cover classes; and second, to identify hot spots of soil erosion and bright spots of soil conservation in a spatially explicit manner. Soil organic carbon (SOC) is an important indicator of soil fertility. The aim of this study was to conduct a spatial assessment of erosion and its impact on SOC for specific land cover classes. Input data consisted of extensive ground truth, a digital elevation model and Landsat 7 imagery from two different seasons. Soil spectral reflectance readings were taken from soil samples in the laboratory and calibrated with results of SOC chemical analysis using regression tree modelling. The resulting model statistics for soil degradation assessments are promising (R2=0.71, RMSEV=0.32). Since the area includes rugged terrain and small agricultural plots, the decision tree models allowed mapping of land cover classes, soil erosion incidence and SOC content classes at an acceptable level of accuracy for preliminary studies. The various datasets were linked in the hot-bright spot matrix, which was developed to combine soil erosion incidence information and SOC content levels (for uniform land cover classes) in a scatter plot. The quarters of the plot show different stages of degradation, from well conserved land to hot spots of soil degradation. The approach helps to gain a better understanding of the impact of soil erosion on soil fertility and to identify hot and bright spots in a spatially explicit manner. The results show distinctly lower SOC content levels on large parts of the test areas, where annual crop cultivation was dominant in the 1990s and where cultivation has now been abandoned. On the other hand, there are strong indications that afforestations and fruit orchards established in the 1980s have been successful in conserving soil resources.