PAPP-A and osteoprotegerin, together with interleukin-8 and RANTES, are elevated in the peritoneal fluid of women with endometriosis

OBJECTIVE: The purpose of this study was to evaluate whether pregnancy-associated plasma protein A, glycodelin, osteoprotegerin, and soluble CD163 are possible peritoneal fluid markers for endometriosis and to compare them with the established chemokine markers interleukin-8 and regulated on activation, normal T-cell expressed and secreted. STUDY DESIGN: Determination of the concentrations of interleukin-8, regulated on activation, normal T-cell expressed and secreted, pregnancy-associated plasma protein A, glycodelin, CD163, osteoprotegerin, and progesterone in the peritoneal fluid collected from women undergoing laparoscopy. RESULTS: From a total of 132 women, 77 women were diagnosed with endometriosis, and 55 women were free of the disease and served as control subjects. Pregnancy-associated plasma protein A and osteoprotegerin showed significantly (P < 0.05) elevated peritoneal fluid concentrations as a function of the severity of the disease, together with interleukin-8 and regulated on activation, normal T-cell expressed and secreted (P < .001). Glycodelin and CD163 did not differ between cases and control subjects. Many of these marker concentrations were intercorrelated strongly. CONCLUSION: Pregnancy-associated plasma protein A and osteoprotegerin may play a role in the inflammation process of endometriosis, but interleukin-8 and regulated on activation, normal T-cell expressed and secreted are superior peritoneal fluid markers.

Carbapenems: past, present, and future

In this review, we summarize the current "state of the art" of carbapenem antibiotics and their role in our antimicrobial armamentarium. Among the β-lactams currently available, carbapenems are unique because they are relatively resistant to hydrolysis by most β-lactamases, in some cases act as "slow substrates" or inhibitors of β-lactamases, and still target penicillin binding proteins. This "value-added feature" of inhibiting β-lactamases serves as a major rationale for expansion of this class of β-lactams. We describe the initial discovery and development of the carbapenem family of β-lactams. Of the early carbapenems evaluated, thienamycin demonstrated the greatest antimicrobial activity and became the parent compound for all subsequent carbapenems. To date, more than 80 compounds with mostly improved antimicrobial properties, compared to those of thienamycin, are described in the literature. We also highlight important features of the carbapenems that are presently in clinical use: imipenem-cilastatin, meropenem, ertapenem, doripenem, panipenem-betamipron, and biapenem. In closing, we emphasize some major challenges and urge the medicinal chemist to continue development of these versatile and potent compounds, as they have served us well for more than 3 decades.

First trimester markers for pre-eclampsia: placental vs. non-placental protein serum levels

BACKGROUND/AIM: Parallel investigation, in a matched case-control study, of the association of different first-trimester markers with the risk of subsequent pre-eclampsia (PE). METHOD: The levels of different first trimester serum markers and fetal nuchal translucency thickness were compared between 52 cases of PE and 104 control women by non-parametric two-group comparisons and by calculating matched odds ratios. RESULTS: In univariable analysis increased concentrations of inhibin A and activin A were associated with subsequent PE (p < 0.02). Multivariable conditional logistic regression models revealed an association between increased risk of PE and increased inhibin A and translucency thickness and respectively reduced pregnancy-associated plasma protein A (PAPP-A) and placental lactogen . However, these associations varied with the gestational age at sample collection. For blood samples taken in pregnancy weeks 12 and 13 only, increased levels of activin A, inhibin A and nuchal translucency thickness, and lower levels of placenta growth factor and PAPP-A were associated with an increased risk of PE. CONCLUSIONS: Members of the inhibin family and to some extent PAPP-A and placental growth factor are superior to other serum markers, and the predictive value of these depends on the gestational age at blood sampling. The availability of a single, early pregnancy 'miracle' serum marker for PE risk assessment seems unlikely in the near future.

Novel mutation in OTC gene causes neonatal death in twin brothers

Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of the urea cycle. OTC locus is located in the short arm of X-chromosome. Authors report a case of a woman who gave birth to monozygotic male twins who later died because of severe neonatal-onset hyperammonaemic encephalopathy caused by a novel mutation of OTC gene. Post-mortem liver biopsy was taken from the second twin; afterwards, blood was drawn from the mother for examination. DNA sequence data showed that the mother was a carrier of the same novel mutation that was previously detected in the case of her son. In OTC deficiency, detection of female carriers is important for genetic counselling and eventual prenatal diagnosis.

Correlation between symptoms of pain and peritoneal fluid inflammatory cytokine concentrations in endometriosis

Endometriosis affects 10-20% of women during reproductive age and is a common cause of infertility and pain leading to work absenteeism and reduced quality of life.The objective of this study was to investigate the association between the presence and concentration of interleukin-8 (IL-8), RANTES, osteoprotegerin (OPG), pregnancy-associated plasma protein A (PAPP-A), tumour necrosis factor-alpha (TNF-alpha), midkine and glycodelin in the peritoneal fluid (PF) and the intensity of pain reported by patients undergoing laparoscopy in our clinic. They rated their pain during menstruation, intercourse and lower abdominal using a visual analogue scale. During laparoscopy, PF was aspirated. Pain scores were correlated to the concentration of the above substances in the PF and to the stage of endometriosis. Endometriosis was histologically confirmed in 41 of 68 participating women; 27 without such evidence were considered as controls. TNF-alpha and glycodelin correlated positively with the level of menstrual pain. For IL-8, RANTES, OPG and PAPP-A no correlation between their PF concentration and the menstrual pain scores was observed. Patients with severe dysmenorrhoea had increased PF cytokine and marker levels; the difference was significant for TNF-alpha and glycodelin when compared with the other patients (no or moderate pain). TNF-alpha and glycodelin may thus play a role in endometriosis and the severity of menstrual pain.

Non-phenotypic tests to detect and characterize antibiotic resistance mechanisms in Enterobacteriaceae

In the past 2 decades, we have observed a rapid increase of infections due to multidrug-resistant Enterobacteriaceae. Regrettably, these isolates possess genes encoding for extended-spectrum β-lactamases (e.g., blaCTX-M, blaTEM, blaSHV) or plasmid-mediated AmpCs (e.g., blaCMY) that confer resistance to last-generation cephalosporins. Furthermore, other resistance traits against quinolones (e.g., mutations in gyrA and parC, qnr elements) and aminoglycosides (e.g., aminoglycosides modifying enzymes and 16S rRNA methylases) are also frequently co-associated. Even more concerning is the rapid increase of Enterobacteriaceae carrying genes conferring resistance to carbapenems (e.g., blaKPC, blaNDM). Therefore, the spread of these pathogens puts in peril our antibiotic options. Unfortunately, standard microbiological procedures require several days to isolate the responsible pathogen and to provide correct antimicrobial susceptibility test results. This delay impacts the rapid implementation of adequate antimicrobial treatment and infection control countermeasures. Thus, there is emerging interest in the early and more sensitive detection of resistance mechanisms. Modern non-phenotypic tests are promising in this respect, and hence, can influence both clinical outcome and healthcare costs. In this review, we present a summary of the most advanced methods (e.g., next-generation DNA sequencing, multiplex PCRs, real-time PCRs, microarrays, MALDI-TOF MS, and PCR/ESI MS) presently available for the rapid detection of antibiotic resistance genes in Enterobacteriaceae. Taking into account speed, manageability, accuracy, versatility, and costs, the possible settings of application (research, clinic, and epidemiology) of these methods and their superiority against standard phenotypic methods are discussed.

Regression of the inflammatory microenvironment of the peritoneal cavity in women with endometriosis by GnRHa treatment.

OBJECTIVE To investigate the effect of gonadotropin-releasing hormone analogues (GnRHa) on the peritoneal fluid microenvironment in women with endometriosis. STUDY DESIGN Peritoneal fluid was collected from 85 women with severe endometriosis (rAFS stage III and IV) during laparoscopic surgery during the proliferative phase. Prior to surgery clinical data were collected. The concentrations of specific markers for endometriosis in the peritoneal fluid were determined using an ELISA and a comparison between peritoneal fluid markers in women using GnRHa and no hormonal treatment was performed using a non-parametric Mann-Whitney U test. RESULTS The study included peritoneal fluid from 39 patients who had been administered GnRHa (Zoladex(®)) in the three months prior to surgery and 46 from women with no hormonal treatment in this period. Concentrations of IL-8, PAPP-A, glycodelin-A and midkine were significantly reduced in the GnRHa treatment group compared to women receiving no hormonal treatment. RANTES, MCP-1, ENA-78, TNF-α, OPG, IP-10 and defensin showed no significant change between the two groups. CONCLUSIONS GnRHa mediate a significant regression in the inflammatory nature of the peritoneal microenvironment in women with endometriosis.

Materno-fetal nutrient transfer across primary human trophoblast layer.

MATERNO-FETAL NUTRIENT TRANSFER ACROSS PRIMARY HUMAN TROPHOBLAST MONOLAYER Objectives: Polarized trophoblasts represent the transport and metabolic barrier between the maternal and fetal circulation. Currently human placental nutrient transfer in vitro is mainly investigated unidirectionallyon cultured primary trophoblasts, or bidirectionally on the Transwell® system using BeWo cells treated with forskolin. As forskolin can induce various gene alterations (e.g. cAMP response element genes), we aimed to establish a physiological primary trophoblast model for materno-fetal nutrient exchange studies without forskolin application. Methods: Human term cytotrophoblasts were isolated by enzymatic digestion and Percoll® gradient separation. The purity of the primary cells was assessed by flow cytometry using the trophoblast-specific marker cytokeratin-7. After screening different coating matrices, we optimized the growth conditions for the primary cytotrophoblasts on Transwell/ inserts. The morphology of 5 days cultured trophoblasts was determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally transport studies were performed on the polarized trophoblasts in the Transwell® system. Results: During 5 days culture, the trophoblasts (>90% purity) developed a modest trans-epithelial electrical resistance (TEER) and a sizedependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ~400-70’000D). SEM analyses confirmed a confluent trophoblast layer with numerous microvilli at day six, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein ZO-1, and the membrane proteins ABCA1 and Na+/K+-ATPase. Vectorial glucose and cholesterol transport studies confirmed functionality of the cultured trophoblast barrier. Conclusion: Evidence from cell morphology, biophysical parameters and cell marker expressions indicate the successful and reproducible establishment of a primary trophoblast monolayer model suitable for transport studies. Application of this model to pathological trophoblasts will help to better understand the mechanism underlying gestational diseases, and to define the consequences of placental pathology on materno-fetal nutrient transport.

A computer-assisted microscopic analysis of bone tissue developed inside a polyactive plymer implanted into an equine articular surface

One of the most promising applications for the restoration of small or moderately sized focal articular lesions is mosaicplasty (MP). Although recurrent hemarthrosis is a rare complication after MP, recently, various strategies have been designed to find an effective filling material to prevent postoperative bleeding from the donor site. The porous biodegradable polymer Polyactive (PA; a polyethylene glycol terephthalate - polybutylene terephthalate copolymer) represents a promising solution in this respect. A histological evaluation of the longterm PA-filled donor sites obtained from 10 experimental horses was performed. In this study, attention was primarily focused on the bone tissue developed in the plug. A computer-assisted image analysis and quantitative polarized light microscopic measurements of decalcified, longitudinally sectioned, dimethylmethylene blue (DMMB)- and picrosirius red (PS) stained sections revealed that the coverage area of the bone trabecules in the PA-filled donor tunnels was substantially (25%) enlarged compared to the neighboring cancellous bone. For this quantification, identical ROIs (regions of interest) were used and compared. The birefringence retardation values were also measured with a polarized light microscope using monochromatic light. Identical retardation values could be recorded from the bone trabeculae developed in the PA and in the neighboring bone, which indicates that the collagen orientation pattern does not differ significantly among these bone trabecules. Based on our new data, we speculate that PA promotes bone formation, and some of the currently identified degradation products of PA may enhance osteo-conduction and osteoinduction inside the donor canal.

Evaluation of a Novel Screw Position in a Type III Distal Phalanx Fracture Model: An Ex Vivo Study

OBJECTIVE: Mechanical evaluation of a novel screw position used for repair in a type III distal phalanx fracture model and assessment of solar canal penetration (SCP). STUDY DESIGN: Experimental study. SAMPLE POPULATION: Disarticulated equine hooves (n = 24) and 24 isolated distal phalanges. METHODS: Hooves/distal phalanges cut in a sagittal plane were repaired with 1 of 2 different cortical screw placements in lag fashion. In group 1 (conventional screw placement), the screw was inserted halfway between the proximal border of the solar canal (SC) and the subchondral bone surface on a line parallel to the dorsal cortex, whereas in group 2, the screw was inserted more palmar/plantar, where a perpendicular line drawn from the group 1 position reached the palmar/plantar cortex. Construct strength was evaluated by 3-point bending to failure. SCP was assessed by CT imaging and macroscopically. RESULTS: Screws were significantly longer in group 2 and in forelimbs. Group 2 isolated distal phalanges had a significantly more rigid fixation compared with the conventional screw position (maximum point at failure 31%, bending stiffness 41% higher). Lumen reduction of the SC was observed in 13/52 specimens (all from group 2), of which 9 were forelimbs. CONCLUSIONS: More distal screw positioning compared with the conventionally recommended screw position for internal fixation of type III distal phalangeal fractures allows placement of a longer screw and renders a more rigid fracture fixation. The novel screw position, however, carries a higher risk of SCP

Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport

STUDY HYPOTHESIS Using optimized conditions, primary trophoblast cells isolated from human term placenta can develop a confluent monolayer in vitro, which morphologically and functionally resembles the microvilli structure found in vivo. STUDY FINDING We report the successful establishment of a confluent human primary trophoblast monolayer using pre-coated polycarbonate inserts, where the integrity and functionality was validated by cell morphology, biophysical features, cellular marker expression and secretion, and asymmetric glucose transport. WHAT IS KNOWN ALREADY Human trophoblast cells form the initial barrier between maternal and fetal blood to regulate materno-fetal exchange processes. Although the method for isolating pure human cytotrophoblast cells was developed almost 30 years ago, a functional in vitro model with primary trophoblasts forming a confluent monolayer is still lacking. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Human term cytotrophoblasts were isolated by enzymatic digestion and density gradient separation. The purity of the primary cells was evaluated by flow cytometry using the trophoblast-specific marker cytokeratin 7, and vimentin as an indicator for potentially contaminating cells. We screened different coating matrices for high cell viability to optimize the growth conditions for primary trophoblasts on polycarbonate inserts. During culture, cell confluency and polarity were monitored daily by determining transepithelial electrical resistance (TEER) and permeability properties of florescent dyes. The time course of syncytia-related gene expression and hCG secretion during syncytialization were assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. The morphology of cultured trophoblasts after 5 days was determined by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally, glucose transport studies were performed on the polarized trophoblasts in the same system. MAIN RESULTS AND THE ROLE OF CHANCE During 5-day culture, the highly pure trophoblasts were cultured on inserts coated with reconstituted basement membrane matrix . They exhibited a confluent polarized monolayer, with a modest TEER and a size-dependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ∼400-70 000 Da). The syncytialization progress was characterized by gradually increasing mRNA levels of fusogen genes and elevating hCG secretion. SEM analyses confirmed a confluent trophoblast layer with numerous microvilli, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein 1 (ZO-1) and the membrane proteins ATP-binding cassette transporter A1 (ABCA1) and glucose transporter 1 (GLUT1). Applying this model to study the bidirectional transport of a non-metabolizable glucose derivative indicated a carrier-mediated placental glucose transport mechanism with asymmetric kinetics. LIMITATIONS, REASONS FOR CAUTION The current study is only focused on primary trophoblast cells isolated from healthy placentas delivered at term. It remains to be evaluated whether this system can be extended to pathological trophoblasts isolated from diverse gestational diseases. WIDER IMPLICATIONS OF THE FINDINGS These findings confirmed the physiological properties of the newly developed human trophoblast barrier, which can be applied to study the exchange of endobiotics and xenobiotics between the maternal and fetal compartment, as well as intracellular metabolism, paracellular contributions and regulatory mechanisms influencing the vectorial transport of molecules. LARGE-SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This study was supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland, and the Swiss National Science Foundation (grant no. 310030_149958, C.A.). All authors declare that their participation in the study did not involve factual or potential conflicts of interests.