Invited review: Piglet survival: benefits of the immunocompetence.

Piglet mortality has a negative impact on animal welfare and public acceptance. Moreover, the number of weaned piglets per sow mainly determines the profitability of piglet production. Increased litter sizes are associated with lower birth weights and piglet survival. Decreased survival rates and performance of piglets make the control of diseases and infections within pig production even more crucial. Consequently, selection for immunocompetence becomes an important key aspect within modern breeding programmes. However, the phenotypic recording of immune traits is difficult and expensive to realize within farm routines. Even though immune traits show genetic variability, only few examples exist on their respective suitability within a breeding programme and their relationships to economically important production traits. The analysis of immune traits for an evaluation of immunocompetence to gain a generally improved immune response is promising. Generally, in-depth knowledge of the genetic background of the immune system is needed to gain helpful insights about its possible incorporation into breeding programmes. Possible physiological drawbacks for enhanced immunocompetence must be considered with regards to the allocation theory and possible trade-offs between the immune system and performance. This review aims to discuss the relationships between the immunocompetence of the pig, piglet survival as well as the potential of these traits to be included into a breeding strategy for improved robustness.

Birth of healthy calves after intra-follicular transfer (IFOT) of slaughterhouse derived immature bovine oocytes.

To circumvent the negative impacts of in vitro culture on bovine embryos, we have recently established a new method, the so called intra-follicular oocyte transfer (IFOT), enabling in vivo fertilization and in vivo development of in vitro matured oocytes up to the blastocyst stage as well as to term. In this study, we raised the question whether immature bovine oocytes could also be transferred into a pre-ovulatory follicle to support in vivo maturation prior to subsequent in vivo fertilization, in vivo development as well as to term. To unravel that question, a total of 791 immature oocytes were transferred in groups of ∼50 into pre-ovulatory follicles of 16 recipient heifers. Consequently, we were able to recollect a total of 306 structures 8 days thereafter (38.5%). All in all, 12 heifers (75%) gave embryos developed to the morula or blastocyst stage in addition to the expected native embryos. Among all recollected structures, 40.1% had developed to the morula and/or blastocyst stage, meaning a total efficiency of 17.3% based on all transferred oocytes. Of impact, IFOT-embryos reached significantly higher developmental rates to the Morula and/or blastocyst stage until day 7 compared to in vitro cultured control embryos, despite being derived from the same charge of slaughterhouse ovaries (40.1 vs. 29.3%). This implicates a beneficial effect of the follicular environment for the intrinsic quality of the fertilized embryos during maturation and for subsequent developmental rates up to the blastocyst stage. Finally, the birth of two healthy calves after transfer of frozen-thawed IFOT-derived blastocysts to final recipients established the first proof of principle that IFOT of immature bovine oocytes generates bovine blastocysts bearing developmental capacity to term. Likewise, to the best of our knowledge, these calves are the first calves derived from full in vivo development of immature slaughterhouse derived oocytes. Thus, the results of the present study clearly demonstrate that IFOT of immature slaughterhouse-derived oocytes is now a feasible technique. Since efficiencies following IFOT achieved within the present study were improved compared to previous studies, IFOT now offers an attractive option for designing new scientific experiments.

Cryosurvival of in vitro produced bovine embryos supplemented with l-Carnitine and concurrent reduction of fatty acids.

Lipid accumulation is associated with reduced embryonic quality, causing limited survival after cryopreservation. Therefore, in the present study we aimed to reveal the effects of supplementation of a lipid reducing agent, l-carnitine and the removal of fatty acids during in vitro culture on the morphological as well as on the molecular level. To accomplish that, presumptive zygotes were cultured in 4 contrasting groups: namely SOFaa medium supplemented with BSA, (BSA), SOFaa medium supplemented with fatty acid free BSA (FAF), SOFaa medium supplemented with BSA as well as l-Carnitine (BSA + LC) and SOFaa medium concurrently supplemented with fatty acid free BSA and l-Carnitine (FAF + LC). Considering the developmental rates, no impact of different treatments was observed. Conversely, treatment groups clearly affected lipid content, with the lowest amounts detected in embryos derived from FAF and BSA + LC groups, implicating that both removal of fatty acids and supplementation of LC reduces lipid content effectively. Importantly, survival rates after cryopreservation show that LC significantly affects the kinetics of re-expansion, with the highest hatching rates detected for embryos cultured in FAF + LC (p < 0.05). Noteworthy, the highest cryotolerance did not go along with lowest lipid contents. Finally, metabolic alterations between the groups were reflected in different abundances of selected candidate genes related to lipid metabolism and oxidative stress response, like AMPKA1, ACC and PGC1 α or KEAP1 and SOD1. All in all, highly beneficial effects on survival rates after cryopreservation have been detected when embryos were cultured in absence of fatty acids and concurrent presence of l-Carnitine. Highest cryotolerance, however, did not correlate with lowest lipid contents.

Genotype-outcome correlations in pediatric AML: the impact of a monosomal karyotype in trial AML-BFM 2004.

We conducted a cytogenetic analysis of 642 children with de novo acute myeloid leukemia (AML) treated on the AML-Berlin-Frankfurt-Münster (BFM) 04 protocol to determine the prognostic value of specific chromosomal aberrations including monosomal (MK+), complex (CK+) and hypodiploid (HK+) karyotypes, individually and in combination. Multivariate regression analysis identified in particular MK+ (n=22) as a new independent risk factor for poor event-free survival (EFS 23±9% vs 53±2% for all other patients, P=0.0003), even after exclusion of four patients with monosomy 7 (EFS 28±11%, P=0.0081). CK+ patients without MK had a better prognosis (n=47, EFS 47±8%, P=0.46) than those with MK+ (n=12, EFS 25±13%, P=0.024). HK+ (n=37, EFS 44±8% for total cohort, P=0.3) influenced outcome only when t(8;21) patients were excluded (remaining n=16, EFS 9±8%, P<0.0001). An extremely poor outcome was observed for MK+/HK+ patients (n=10, EFS 10±10%, P<0.0001). Finally, isolated trisomy 8 was also associated with low EFS (n=16, EFS 25±11%, P=0.0091). In conclusion, monosomal karyotype is a strong and independent predictor for high-risk pediatric AML. In addition, isolated trisomy 8 and hypodiploidy without t(8;21) coincide with dismal outcome. These results have important implications for risk stratification and should be further validated in independent pediatric cohorts.

Fateful triad of reactive oxygen species, mitochondrial dysfunction and lipid accumulation is associated with expression outline of the AMP-activated protein kinase pathway in bovine blastocysts.

Low cryotolerance is considered as the major drawback of in vitro-produced bovine embryos and is frequently associated with a triad encompassing increased cytoplasmic lipid accumulation, enhanced levels of reactive oxygen species (ROS) and mitochondrial dysfunction. The aim of the present study was to explore the role of the AMP-activated protein kinase (AMPK) pathway in the process resulting such phenotypes. Comparative analysis under different environmental conditions revealed downregulation of AMP-activated protein kinase cytalytic subunit 1alpha (AMPKA1), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1A) and carnitine palmitoyltransferase 1 (CPT1) genes and upregulation of acetyl-CoA carboxylase α (ACC). In contrast, the presence of fatty acids within the culture medium resulted in a distinct molecular profile in the embryo associated with enhanced levels of ROS, mitochondrial dysfunction and elevated lipid accumulation in bovine embryos. Because AMPKA1 regulates PGC1A, CPT1 and ACC, the results of the present study reveal that AMPK in active its form is the key enzyme promoting lipolysis. Because AMPK1 activity is, in turn, controlled by the AMP : ATP ratio, it is possible to speculate that excessive uptake of exogenous free fatty acids could increase cellular ATP levels as a result of the disturbed ß-oxidation of these external fatty acids and could therefore bypass that molecular feedback mechanism. Subsequently, this condition would cause enhanced generation of ROS, which negatively affect mitochondrial activity. Both enhanced generation of ROS and low mitochondrial activity are suggested to enhance the accumulation of lipids in bovine embryos.

Randomised Introduction of 2-CDA as Intensification during Consolidation for Children with High-risk AML--results from Study AML-BFM 2004.

BACKGROUND: The outcome in children and adolescents with high-risk (HR) acute myeloid leukemia (AML) is still unsatisfactory. Therefore, in study AML-BFM 2004 we aimed to improve outcome of HR-patients by adding moderately dosed 2-Chloro-2-Deoxyadenosine (2-CDA) to the respective consolidation treatment backbone without increasing toxicity. The aim was to improve prognosis especially in FAB M4/M5/MLL patients, who represent the largest subgroup of HR patients. PATIENTS AND METHODS: In total, 343 children and adolescents with HR-AML were randomized to receive or not 2-CDA (6 mg/m²/d, days 1, 3) in combination with cytarabine/idarubicine (AI=500 mg/m² cytarabine 5 days continuous infusion plus 7 mg/m²/d idarubicin, days 3 and 5). RESULTS: RESULTS for patients of the AI/2-CDA arm (n=168) vs. the AI-arm (n=175) were similar: 5-year overall survival 68±4 vs. 72±4%, plogrank=0.38, event-free survival 53±4 vs. 49±4%, plogrank=0.77; cumulative incidence of relapse at 5 years: 35±4 vs. 37±4%, p(Gray)=0.89. RESULTS in patients with MLL rearrangement or FAB M4/M5 were also similar in the treatment groups. In addition, toxicities did not differ between the two arms. CONCLUSION: We conclude that additional, moderate dose 2-CDA does not improve prognosis in HR-patients when given during consolidation treatment. Its effect might be too low in this multidrug regimen, where the strongest effects are achieved during induction, or the chosen dose of 2-CDA might have been too low.

[Genetic prognostic factors in childhood acute myeloid leukemia]. / Prognostische Relevanz genetischer Aberrationen der akuten myeloischen Leukämie bei Kindern und Jugendlichen.

The survival rate of children and adolescents suffering acute myeloid leukemia (AML) has been significantly improved within the last decades. This has been achieved by a continuously intensified therapy and progress in supportive care to prevent and treat complications. In Germany, the AML-BFM trials 98 (n=413) and 2004 (n=499) enrolled 912 children and adolescents as protocol patients (1998-2010). The 5-year-overall survival was 71±2%. In the previous studies prognosis and subsequent treatment stratification based on morphology, cytochemistry and white blood cell count. Today, the identification of new genetic aberrations in AML enables a genetically determined estimation of prognosis, although treatment response must be considered for treatment stratification. The group with a favorable prognosis summarized AML with t(8;21), inv(16), t(15;17), t(1;11), and AML with normal karyotype and NPM1-mutation (n=253; EFS 74±3%, OS 88±2%). A poor prognosis (HR-group) must be expected in AML with t(4;11), t(5;11), t(6;11), t(6;9), t(7;12), t(9;22), Monosomy 7, combined FLT3/WT1-mutation, and AML with der(12p)-aberration (n=101; EFS 30±5%; OS 56±5%). The intermediate group summarizes all other subgroups especially AML with normal karyotyp, AML with FLT3-ITD or t(9;11) (n=558; EFS 43±2%; OS 64±2%). The validation of the internationally identified, genetically determined prognostic factors within the AML-BFM (Germany) study population will support treatment recommendations.

[GATA1-mutation associated leukemia in children with trisomy 21 mosaic]. / GATA1-Mutations-assoziierte Leukämien bei Kindern mit Trisomie 21-Mosaik.

Mutations of the hematopoietic transcription factor GATA1 (GATA1s) are pathognomonic in newborn with transient leukemia and children with Down syndrome and myeloid leukemia (ML-DS). Both TL and ML-DS can also occur in children with trisomy 21 mosaic.Between 2002 and 2011, 15 newborns and infants were diagnosed with DS mosaic. 9 of them presented with TL and 8 children suffered from ML-DS; 2 of them with a history of TL. In children without stigmata the special morphology and immunophenotype of blasts triggered the screening for GATA1 mutation and trisomy 21 mosaic.All newborns with TL achieved complete remission (CR). Due to clinical symptoms caused by the leukemic blasts, in 3 children low-dose cytarabine was applied. 1 patient died due to cardiac defect. In all patients GATA 1 s was confirmed. 6 children with ML-DS were initially treated according the AML-BFM protocol. After ML-DS was confirmed, therapy was continued with the intensity reduced schedule according to the ML-DS 2006 protocol. All children are still in CR (follow-up 1.8-7 years, median 2.7 yrs). 2 children with unknown trisomy 21 mosaic were diagnosed as acute megakaryoblastic leukemia (AMKL) and treated according the high risk arm of the AML-BFM 2004 including allogeneic stem cell transplantation in one child). GATA1 mutation was identified retrospectively. Both children are alive in CR.GATA1s associated leukemia has to be excluded in all young children with AMKL (<5 years old) to prevent overtreatment. Treatment with reduced intensity seems sufficient in children trisomy 21 mosaic and ML-DS.

Favorable outcome in infants with AML after intensive first- and second-line treatment: an AML-BFM study group report.

Infants <1 year of age have a high prevalence of prognostically unfavorable leukemias and a presumed susceptibility to treatment-related toxicities. A total of 125 infants with acute myeloid leukemia (AML) were treated in studies AML-BFM-98 (n = 59) and -2004 (n = 66). Treatment regimens of both studies were comparable, consisting of intensive induction followed by four courses (mainly high-dose cytarabine and anthracyclines). Allogeneic-hematopoietic stem-cell-transplantation (allo-HSCT) in 1st remission was optional for high-risk (HR) patients. Most infants (120/125=96%) were HR patients according to morphological, cytogenetic/molecular genetic and response criteria. Five-year overall survival was 66 ± 4%, and improved from 61 ± 6% in study-98 to 75 ± 6% in study-2004 (P(logrank) 0.14) and event-free survival rates were 44 ± 6% and 51 ± 6% (P(logrank) 0.66), respectively. Results in HR infants were similar to those of older HR children (1-<2- or 2-<10-year olds, P(logrank) 0.90 for survival). Survival rates of HSCT in 1st remission, initial partial response and after relapse were high (13/14, 2/8 and 20/30 patients, respectively). The latter contributes to excellent 5-year survival after relapse (50±8%). Despite more severe infections and pulmonary toxicities in infants, treatment-related death rate was identical to that of older children (3%). Our data indicate that intensive frontline and relapse AML treatment is feasible in infants, toxicities are manageable, and outcome is favorable.

Consequent and intensified relapse therapy improved survival in pediatric AML: results of relapse treatment in 379 patients of three consecutive AML-BFM trials.

Relapse remains the major cause of treatment failure in pediatric acute myeloid leukemia (AML). We analyzed the clinical characteristics, treatment response to relapse treatment and overall survival (OS) of 379 children with AML relapse treated according to three consecutive frontline protocols of the AML-Berlin/Frankfurt/Muenster study group (AML-BFM-87/-93/-98). Of 313 treated patients with data on remission status, 198 children (63%) achieved a second complete remission (CR2). There were no significant differences in remission rates and OS for the intensive reinduction treatment schedules used. The 5-year OS rate was 23% for the total group and 29% for patients treated with curative intent. OS rates increased with study periods from 18 to 34% (P(log rank)=0.012), whereas the proportion of patients receiving only palliative treatment decreased from 23 to 11% (P(CMH)=0.005). Late relapse, no allogeneic stem cell transplantation (SCT) in CR1, age <10 years and favorable cytogenetics were independent favorable prognostic factors for survival. Achievement of CR2 was the most important prognostic factor (OS 44 vs 3%; P(log rank)<0.0001). Overall, one-third of children with relapsed AML can be cured today. SCT in CR2 is recommended for most patients, although its impact on CR2 is discussed.

Function of the mitochondrial outer membrane as a diffusion barrier in health and diseases.

The mitochondrial outer membrane separates the intermembrane space from the cytosol. The whole exchange of metabolites, cations and information between mitochondria and the cell occurs through the outer membrane. Experimental evidence is reviewed supporting the hypothesis of dynamic ADP compartmentation within the intermembrane space. The outer membrane creates a diffusion barrier for small molecules (adenine nucleotides, creatine phosphate, creatine etc.) causing rate-dependent concentration gradients as a prerequisite for the action of ADP shuttles via creatine kinases or adenylate kinases. If the outer membrane becomes leaky, cytochrome c and apoptosis-inducing factor can be released, leading to apoptosis, and as a bioenergetic consequence the cytosolic phosphorylation potential decreases. Leaky outer membranes can be detected in saponin-skinned fibres with spectrophotometric and oxygraphic methods. This is of special interest in respect to acute impairment of mitochondria during ischaemia/reperfusion.

Baker's yeast-mediated synthesis of endo-2-cineolylol, a starting compound for potential cosmetic odourants.

Baker's yeast-mediated reduction of (+)-1,3,3-trimethyl-2-oxabicyclo[2,2,2]octan-6-one yields the corresponding 1,3,3-trimethyl-2-oxabicyclo[2,2,2]octan-6-ol as endo configuration in optically pure form. To study the reaction behaviour we have developed a RP-HPLC method. The synthesis and the olfactive character of some esters obtained from the endo-2-cineolylol are reported. A comparative odour evaluation between these esters and the corresponding compounds synthesized from the endo-exo alcohol mixture was performed.

Derivatization procedure and HPLC determination of 2-ethoxyethanol in cosmetic samples.

Ethylene glycol monoethyl ether or 2-ethoxyethanol finds a wide industrial application as a solvent for lacquers, inks, dyes, household products and as a surfactant. It is also found in cosmetics such as nail products, face cleansers, liquid soaps, oral care products, hair colours and fixatives. The potential hazard to human health of 2-ethoxyethanol following inhalation and dermal exposure has been recently reviewed and the European Cosmetic, Toiletry and Perfumery Association (COLIPA) has issued recommendations suggesting its non-use as a cosmetic ingredient. Therefore a simple and fast monitoring method is necessary for routine control to identify and quantify 2-ethoxyethanol in raw materials and finished cosmetics. We have developed a sensitive and selective method to determine 2-ethoxyethanol in complex matrices by precolumn derivatization with 1-naphthyl isocyanate and RP-HPLC analysis. Four laboratory-made cosmetic formulations (a nail lacquer remover, a baby oil, a skin lotion and an emollient O/W emulsion) containing three known amounts of 2-ethoxyethanol (0.1%, 2.0%, 5.0%) have been studied. The obtained results show that this chromatographic procedure provides a good estimate of the true concentration of 2-ethoxyethanol in complex matrices and it is reliable for routine analyses in quality control.

Application of high-performance liquid chromatography in the analysis of direct dyes in semipermanent hair colouring cosmetics.

A method for the identification and the quantification of direct dyes in semipermanent hair colouring cosmetics by using a reversed phase high-performance liquid chromatography (HPLC) with gradient elution and diode array detector (DAD) detection is presented. A standard mixture of 18 commonly marketed dyestuffs (11 nitroderivatives, two anthraquinones and five Arianor dyes) was studied. The major problems in the analysis of the mixture of direct dyes is the different chemical structure and especially the wide range of polarities. However we succeeded in the complete separation of the standard dyes. A qualitative analysis of eight colouring commercial products selected from different shades and a quantitative analysis of three of them was carried out. Materials, methods and results are reported.

Synthesis of ethylammonium iodides of omega-dialkylaminoethyl ethers of 5-(arylmethylene)-1,3,3-trimethyl-2-oxabicyclo [2.2.2.] octan-6-hydroxyimines as potential cosmetic ingredients.

The synthesis of nine quaternary ammonium iodides derived from omega-dialkylaminoethyl ethers of 5-(arylmethylene)-1,3, 3-trimethyl-2-oxabicyclo[2.2.2]octan-6-hydroxyimines, as potential cosmetic ingredients, is described. They are routinely prepared starting from cineole aminoethers by reaction with iodoethane and their physics-chemical data are reported. These substances were studied for their UV absorption and three of these compounds were also submitted to microbiological assays on five test organisms. The substances have their UV absorption maxima at 284-321 nm, and one compound is active on Staphylococcus aureus, Streptococcus faecalis and Candida albicans. These preliminary findings seem to indicate that some of these compounds could be considered as potential UV sunscreens; one compound, having a moderate antimicrobial activity, could be considered a potential active compound in cosmetics as deodorants, toothpastes, mouthwashes and other oral care products.

Esters of 1,3,3,-trimethyl-2-oxabicyclo [2.2.2]octan-6-ols: synthesis, odour evaluation and in vitro preliminary toxicity assays.

Synopsis The synthesis of a series of alkyl and arylesters of 1,3,3,-trimethyl-2-oxabicyclo[2.2.2]octan-6-ols (2-cineolylols) is described. All cineole esters obtained were tested for their olfactive character; the esters derived from aryl acyl chlorides were odourless, while aliphatic esters showed interesting multipurpose aromas. Some of these compounds exhibited fruity, woody, green, pine oil and violet-like notes and some showed aromas interesting for foodstuffs. In vitro toxicity tests were carried out on the cyclopropyl ester of 2-cineolylols, the most promising of these compounds as a potential perfume ingredient. In this study, cultured mouse fibroblast L-929 and human keratinocyte NCTC 2544 cell lines were used. The results obtained with the evaluation of three different physiological end points showed that the tested compound possess much lower cytotoxicity than sodium dodecylsulphate (SDS) used as positive control.

Appearance of gastrin and somatostatin in the human fetal stomach, duodenum and pancreas.

The gestational time of appearance of gastrin and somatostatin in the human fetal stomach, duodenum and pancreas was examined. Immunoreactive gastrin (IRG) is detected in antral, duodenal and pancreatic extracts of a 7.0-cm (crown-heel length) fetus. More IRG is extracted from the duodenum than the antrum. Duodenal IRG concentration from fetuses of 16.0--26.0 cm are higher than younger fetal and adult concentrations. Antral IRG concentrations are one tenth of the adult contents. Very small IRG concentrations are present in the human fetal pancreas. Gastrin immunohistochemical staining is positive first in duodenal (6.5-cm fetus) and later in antral (12.5-cm fetus) mucosa; pancreatic tissue is negative for gastrin immunohistochemistry. Type IV cells are encountered in antral and duodenal mucosa of 4.0-cm fetuses; other endocrine cells appear with fetal growth. Not until much later in gestation (21.0 cm) do typical G cells appear. These results suggest that early in fetal life gastrin is produced by the type IV cell. Somatostatin immunohistochemical staining is positive in stomach, duodenum and pancreas in 6.5-cm fetuses. Immature D cells are found in antral and duodenal mucosa of 5.0-cm fetuses and mature D cells in 11.0-cm fetuses.