Mutations of the AMH type II receptor in two extended families with persistent Müllerian duct syndrome: lack of phenotype/genotype correlation

Our goal was to compare phenotype and genotype in two extended Middle-Eastern families affected by persistent Müllerian duct syndrome due to mutations of the type II anti-Müllerian hormone receptor (AMHR-II). The first, consanguineous, family consisted of 6 boys and 2 girls, the second consisted of 4 girls and 2 boys. In family I, 4 boys and 1 girl were homozygous for a stop mutation in the 9th exon of AMHR-II, removing part of the intracellular domain of the protein. In family II, 1 girl and 1 boy were homozygous for a transversion changing conserved histidine 254 into a glutamine. Both homozygous girls were normal. In the homozygous males, the degree of development of Müllerian derivatives was variable. The uterus was well developed in 2 boys of family I and in the patient from family II; however, in 1 subject from family I, Müllerian derivatives were undetectable. Taken together, the diversity of clinical symptoms within the same sibship and the lack of correlation between the development of the Müllerian derivatives and the severity of the molecular defects suggest highly variable penetrance of the abnormal alleles and/or the existence of other genetic or epigenetic modifiers of gene expression.     

The application of quality by design in the development of the liquid chromatography method to determine empagliflozin in the presence of its organic impurities

Analysis of impurities is an important step in the quality control of pharmaceutical ingredients and final products. From drug synthesis or excipients, even in small concentrations, impurities may affect efficacy and safety. The method was developed following Quality by Design (QbD) for the analysis of the antidiabetic empagliflozin. The concept of QbD is used as a tool for the development of methods and formulations. Through predefined objectives and risk analysis, robust methodologies and reduced solvent consumption are developed. A simple HPLC method was developed and validated for the quantitative determination of empagliflozin and its organic impurities from the synthesis process. The method was carried out in a Shim-pack phenyl column with a mobile phase consisting of an acetonitrile/water mixture (72 : 28), with isocratic elution and the detector wavelength was 230 nm. The validation process, in accordance with international guidelines, shows that the method was linear, precise and accurate for empagliflozin, impurity 1 and impurity 2. Limits of detection (0.01, 0.02 and 0.01 μg mL⁻¹) and quantification (0.10, 0.10 and 0.05 μg mL⁻¹) were determined for EMPA, IMP1 and IMP2, respectively. The HPLC method for impurity determination in empagliflozin was linear, precise, accurate and robust. It can be successfully applied in the quality control of empagliflozin and the synthesis of impurities, being adequate for routine analysis.

Analysis of impurities is an important step in the quality control of pharmaceutical ingredients and final products.     

Obesity leads to higher risk of sperm DNA damage in infertile patients

There has been a growing interest over the past few years in the impact of male nutrition on fertility. Infertility has been linked to male overweight or obesity, and conventional semen parameter values seem to be altered in case of high body mass index （BMI）. A few studies assessing the impact of BMI on sperm DNA integrity have been published, but they did not lead to a strong consensus. Our objective was to explore further the relationship between sperm DNA integrity and BMI, through a 3-year multicentre study. Three hundred and thirty male partners in subfertile couples were included. Using the terminal uridine nick-end labelling （TUNEL） assay, we observed an increased rate of sDerm DNA damage in obese men （odds ratio （95% confidence interval）： 2.5 （1.2-5.1））.     

Living organ donation practices in Europe – results from an online survey

In Europe, living organ donation (LOD) is increasingly accepted as a valuable solution to overcome the organ shortage. However, considerable differences exist between European countries regarding frequency, practices and acceptance of donor–recipient relations. As a response, the Coordination Action project ‘Living Organ Donation in Europe’ ( www.eulod.eu ), funded by the Seventh Framework Programme of the European Commission, was initiated. Transplant professionals from 331 European kidney and liver transplant centres were invited to complete an online survey on living kidney donation (LKD) and living liver donation (LLD). In total, 113 kidney transplant centres from 40 countries and 39 liver transplant centres from 24 countries responded. 96.5% and 71.8% performed LKD and LLD respectively. The content of the medical screening of donors was similar, but criteria for donor acceptance varied. Few absolute contraindications for donation existed. The reimbursement policies diverged and the majority of the donors did not get reimbursed for their income loss during recovery. Large discrepancies were found between geographical European regions (the Eastern, the Mediterranean and the North‐Western). As a result of this survey we suggest several recommendations to improve quality and safety of LOD in Europe.     

Functional late outgrowth endothelial progenitors isolated from peripheral blood of burned patients

Background

Bioengineered skin substitutes are increasingly considered as a useful option for the treatment of full thickness burn injury. Their viability following grafting can be enhanced by seeding the skin substitute with late outgrowth endothelial progenitor cells (EPCs). However, it is not known whether autologous EPCs can be obtained from burned patients shortly after injury.

Methods

Late outgrowth EPCs were isolated from peripheral blood sampled obtained from 10 burned patients (extent 19.6 ± 10.3% TBSA) within the first 24 h of hospital admission, and from 7 healthy subjects. Late outgrowth EPCs were phenotyped in vitro.

Results

In comparison with similar cells obtained from healthy subjects, growing colonies from burned patients yielded a higher percentage of EPC clones (46 versus 17%, p = 0.013). Furthermore, EPCs from burned patients secreted more vascular endothelial growth factor (VEGF) into the culture medium than did their counterparts from healthy subjects (85.8 ± 56.2 versus 17.6 ± 14 pg/mg protein, p = 0.018). When injected to athymic nude mice 6 h after unilateral ligation of the femoral artery, EPCs from both groups of subjects greatly accelerated the reperfusion of the ischaemic hindlimb and increased the number of vascular smooth muscle cells.

Conclusions

The present study supports that, in patients with burns of moderate extension, it is feasible to obtain functional autologous late outgrowth EPCs from peripheral blood. These results constitute a strong incentive to pursue approaches based on using autotransplantation of these cells to improve the therapy of full thickness burns.     

Benefits of small volume and small syringe for bone marrow aspirations of mesenchymal stem cells

Purpose

Aspirating bone marrow from the iliac crest using small volumes of 1–4 ml with a 10-ml syringe has been historically proposed for harvesting adult mesenchymal stem cells and described as a standard technique to avoid blood dilution. The disadvantage of repeated small aspirations is that there is a significantly increased time to harvest the bone marrow. However, it is not known if a large volume syringe can improve the rate of bone marrow aspiration without increasing blood dilution, thus reducing the quality of the aspirate. We compared the concentrations of mesenchymal stem cells obtained under normal conditions with two different size syringes.

Methods

Thirty adults (16 men and 14 women with a mean age of 49?±?14 years) underwent surgery with aspiration of bone marrow from their iliac crest. Bilateral aspirates were obtained from the iliac crest of the same patients with a 10-ml syringe and a 50-ml syringe. Cell analysis determined the frequencies of mesenchymal stem cells (as determined by the number of colonies) from each size of syringe. The cell count, progenitor cell concentration (colonies/ml marrow) and progenitor cell frequency (per million nucleated cells) were calculated. All bone marrow aspirates were harvested by the same surgeon.

Results

Aspirates of bone marrow demonstrated greater concentrations of mesenchymal stem cells with a 10-ml syringe compared with matched controls using a 50-ml syringe. Progenitor cell concentrations were on average 300 % higher using a 10-ml syringe than matched controls using a 50-ml syringe (p?<?0.01).

Conclusions

In normal human donors, bone marrow aspiration from 30 patients demonstrated a reduced mesenchymal stem cell number in aspirates obtained using a larger volume syringe (50 ml) as compared with a smaller volume syringe (10 ml).     

The role of mast cells in neuroinflammation

Mast cells (MCs) are densely granulated perivascular resident cells of hematopoietic origin and well known for their pathogenetic role in allergic and anaphylactic reactions. In addition, they are also involved in processes of innate and adaptive immunity. MCs can be activated in response to a wide range of stimuli, resulting in the release of not only pro-inflammatory, but also anti-inflammatory mediators. The patterns of secreted mediators depend upon the given stimuli and microenvironmental conditions, accordingly MCs have the ability to promote or attenuate inflammatory processes. Their presence in the central nervous system (CNS) has been recognized for more than a century. Since then a participation of MCs in various pathological processes in the CNS has been well documented. They can aggravate CNS damage in models of brain ischemia and hemorrhage, namely through increased blood–brain barrier damage, brain edema and hemorrhage formation and promotion of inflammatory responses to such events. In contrast, recent evidence suggests that MCs may have a protective role following traumatic brain injury by degrading pro-inflammatory cytokines via specific proteases. In neuroinflammatory diseases such as multiple sclerosis, the role of MCs seems to be ambiguous. MCs have been shown to be damaging, neuroprotective, or even dispensable, depending on the experimental protocols used. The role of MCs in the formation and progression of CNS tumors such as gliomas is complex and both positive and negative relationships between MC activity and tumor progression have been reported. In summary, MCs and their secreted mediators modulate inflammatory processes in multiple CNS pathologies and can thereby either contribute to neurological damage or confer neuroprotection. This review intends to give a concise overview of the regulatory roles of MCs in brain disease.     

Effect of hypoxanthine, antioxidants and allopurinol on cholinesterase activities in rats

In the present study, we investigate the in vitro effect of hypoxanthine on acetylcholinesterase and butyrylcholinesterase activities in the hippocampus, striatum, cerebral cortex and serum of 15-, 30- and 60-day-old rats. Furthermore, we also evaluated the influence of antioxidants, namely α-tocopherol (trolox) and ascorbic acid, and allopurinol to investigate the possible participation of free radicals and uric acid in the effects elicited by hypoxanthine on these parameters. Acetylcholinesterase and butyrylcholinesterase activities were determined according to Ellman et al. (Biochem Pharmacol 7:88–95, 1961), with some modifications. Hypoxanthine (10.0 μM), when added to the incubation medium, enhanced acetylcholinesterase activity in the hippocampus and striatum of 15- and 30-day-old rats and reduced butyrylcholinesterase activity in the serum of 60-day-old rats. The administration of allopurinol and/or antioxidants partially prevented the alterations caused by hypoxanthine in acetylcholinesterase and butyrylcholinesterase activities in the cerebrum and serum of rats. Data indicate that hypoxanthine alters cholinesterase activities, probably through free radicals and uric acid production since the alterations were prevented by the administration of allopurinol and antioxidants. It is presumed that the cholinesterase system may be associated, at least in part, with the neuronal dysfunction observed in patients affected by Lesch–Nyhan disease. In addition, although extrapolation of findings from animal experiments to humans is difficult, it is conceivable that these vitamins and allopurinol might serve as an adjuvant therapy to avoid progression of brain damage in patients affected by this disease.