Coculture of porcine cumulus-oocyte complexes with porcine luteal cells during IVM: effect on oocyte maturation and embryo development.

Coculture with somatic cells is an alternative to improve suboptimal invitro culture conditions. In pigs, IVF is related to poor male pronuclear formation and high rates of polyspermy. The aim of this study was to assess the effect of a coculture system with porcine luteal cells (PLCs) on the IVM of porcine cumulus-oocyte complexes (COCs). Abattoir-derived ovaries were used to obtain PLCs and COCs. COCs were matured invitro in TCM-199 with or without the addition of human menopausal gonadotrophin (hMG; C+hMG and C-hMG respectively), in coculture with PLCs from passage 1 (PLC-1) and in PLC-1 conditioned medium (CM). In the coculture system, nuclear maturation rates were significantly higher than in the C-hMG and CM groups, but similar to rates in the C+hMG group. In cumulus cells, PLC-1 coculture decreased viability, early apoptosis and necrosis, and increased late apoptosis compared with C+hMG. PLC-1 coculture also decreased reactive oxygen species levels in cumulus cells. After IVF, monospermic penetration and IVF efficiency increased in the PLC-1 group compared with the C+hMG group. After invitro culture, higher blastocysts rates were observed in the PLC-1 group. This is the first report of a coculture system of COCs with PLCs. Our model could be an alternative for the conventional maturation medium plus gonadotrophins because of its lower rates of polyspermic penetration and higher blastocysts rates, key issues in porcine invitro embryo production.

Expression of GnRH receptor and 3ßHSD during meiosis and foliculogénesis in Columba livia (Aves: Columbiformes): Histological and immunohistochemical analysis.

During the ovarian ontogeny in birds, five fundamental events can be recognized: migration and colonization of the primordial germ cells, differentiation and proliferation of oogonies, an organization of germinal nests, beginning of the meiotic process and folliculogenesis. The knowledge of these events is fundamental for the interpretation of the processes involved in the differentiation of female gametes. However, there are only references for some model species such as Gallus gallus domesticus and Coturnix coturnix. In a previous study, the histological structure of embryonic ovaries of Columba livia was revealed. Therefore, the objective of this work is to characterize the processes of meiosis and folliculogenesis C. livia from the analysis of the expression of the GnRH receptor, the 3ßHSD enzyme and the cell proliferation protein PCNA in embryonic and postnatal ovaries. Therefore, the expression of GnRHR, 3ßHSD, and PCNA was revealed in histological testicular and ovarian preparations in embryos (stages 41-43) and neonates (2, 5, 7, 10 and 75 days post-hatching). The present study demonstrates that the fate of germline cells is dictated by their location during gonadal development. Thus, the germline cells located in the cortex of the left gonad enter meiosis, while those in the right gonad and those in the medulla of the left ovary fail to go into meiosis. This indicates that somatic signals, instead of an autonomous cellular mechanism, regulate the entry of the germline cells into meiosis in the C. livia embryo. Future studies will be focused on the analysis of proteins associated with meiotic events and folliculogenesis in embryonic and neonatal ovaries of C. livia, to evaluate the regulation of meiosis in vitro.

The antioxidant dimethylthiourea improves IVF efficiency and decreases cumulus cell apoptosis in pigs.

Abattoir ovaries, which are the main source of oocytes for reproductive biotechnologies, arrive at the laboratory under ischaemic conditions. Reoxygenation generates reactive oxygen species (ROS) in ischaemic tissues, which could affect oocyte quality. The aim of this study was to evaluate the effect of supplementation of media with dimethylthiourea (DMTU) during the collection and washing of cumulus-oocyte complexes (COC) on ROS levels, COC apoptosis and oocyte nuclear and cytoplasmic maturation. Thus, the collection (TCM-199) and washing (TCM-199 with 10% porcine follicular fluid, sodium pyruvate and antibiotics) media were supplemented with 1 and 10mM DMTU. In the control group, the media were not supplemented with DMTU. Intracellular ROS levels decreased significantly in the DMTU-treated groups (P<0.05). Although no effects on rate of nuclear maturation were observed, DMTU significantly increased sperm penetration rates without increasing polyspermy (P<0.05). The addition of 10mM DMTU to the collection and washing media enhanced IVF efficiency. DMTU did not modify the early or late apoptosis of oocytes. Both concentrations of DMTU significantly increased viability and decreased the apoptosis of cumulus cells (P<0.05). These results suggest that the addition of 1 or 10mM of DMTU to the media during the collection and washing of porcine COCs is useful for decreasing cumulus apoptosis mediated by ROS and for optimising the IVF of porcine oocytes.

Pedunculated liposarcoma of the esophagus.

Polypoid tumours of the esophagus present diagnostic and therapeutic problems. Liposarcomas are infrequent among them. We report a recent case. A 73-year-old male patient was seen in May 1995 in the Ear, Nose and Throat (ENT) Department, Clinica Modelo de Morón, with intermittent dysphagia and dyspnoea due to recurrent vomiting. A laryngeal lineal tomography showed a subglottic obstruction due to extrinsic compression. The patient was referred to the Gastroenterology Department, where an upper gastrointestinal (upper GI) series demonstrated mega-esophagus with abundant retained food. Endoscopy showed a large intraluminal mass covered by normal mucosa which arose on the posterior wall. Videofluoroscopy and chest CT diagnosed a probable polypoid lipoma due to its densitometric characteristics. The tumour was resected by left cervicotomy and left esophagotomy. The patient's progress to date is favourable. Pathology studies showed a well-differentiated liposarcoma. According to the literature, the first case was reported in 1983, and ours is only the seventh case in the world to be documented.

Fertilization defects in sperm from Cysteine-rich secretory protein 2 (Crisp2) knockout mice: implications for fertility disorders.

STUDY HYPOTHESIS: We hypothesize that fertility disorders in patients with aberrant expression of Cysteine-RIch Secretory Protein 2 (CRISP2) could be linked to the proposed functional role of this protein in fertilization. STUDY FINDING: Our in vivo and in vitro observations reveal that Crisp2-knockout mice exhibit significant defects in fertility-associated parameters under demanding conditions, as well as deficiencies in sperm fertilizing ability, hyperactivation development and intracellular Ca(2+) regulation. WHAT IS KNOWN ALREADY: Testicular CRISP2 is present in mature sperm and has been proposed to participate in gamete fusion in both humans and rodents. Interestingly, evidence in humans shows that aberrant expression of CRISP2 is associated with male infertility. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: A mouse line carrying a deletion in the sixth exon of the Crisp2 gene was generated. The analyses of the reproductive phenotype of Crisp2(-/-) adult males included the evaluation of their fertility before and after being subjected to unilateral vasectomy, in vivo fertilization rates obtained after mating with either estrus or superovulated females, in vitro sperm fertilizing ability and different sperm functional parameters associated with capacitation such as tyrosine phosphorylation (by western blot), acrosome reaction (by Coomassie Blue staining), hyperactivation (by computer-assisted sperm analysis) and intracellular Ca(2+) levels (by flow cytometry). MAIN RESULTS AND THE ROLE OF CHANCE: Crisp2(-/-) males presented normal fertility and in vivo fertilization rates when mated with estrus females. However, the mutant mice showed clear defects in those reproductive parameters compared with controls under more demanding conditions, i.e. when subjected to unilateral vasectomy to reduce the number of ejaculated sperm (n = 5; P< 0.05), or when mated with hormone-treated females containing a high number of eggs in the ampulla (n ≥ 5; P< 0.01). In vitro fertilization studies revealed that Crisp2(-/-) sperm exhibited deficiencies to penetrate the egg vestments (i.e. cumulus oophorus and zona pellucida) and to fuse with the egg (n ≥ 6; P< 0.01). Consistent with this, Crisp2-null sperm showed lower levels of hyperactivation (n = 7; P< 0.05), a vigorous motility required for penetration of the egg coats, as well as a dysregulation in intracellular Ca(2+) levels associated with capacitation (n = 5; P< 0.001). LIMITATIONS, REASONS FOR CAUTION: The analysis of the possible mechanisms involved in fertility disorders in men with abnormal expression of CRISP2 was carried out in Crisp2 knockout mice due to the ethical and technical problems inherent to the use of human gametes for fertilization studies. WIDER IMPLICATIONS OF THE FINDINGS: Our findings in mice showing that Crisp2(-/-) males exhibit fertility and fertilization defects under demanding conditions support fertilization defects in sperm as a mechanism underlying infertility in men with aberrant expression of CRISP2. Moreover, our observations in mice resemble the situation in humans where fertility disorders can or cannot be detected depending on the accumulation of own individual defects or the fertility status of the partner. Finally, the fact that reproductive defects in mice are masked by conventional mating highlights the need of using different experimental approaches to analyze male fertility. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by the World Health Organization (H9/TSA/037), the National Research Council of Argentina (PIP 2009-290), the National Agency for Scientific and Technological Promotion of Argentina (PICT 2011, 2023) and the Rene Baron Foundation to P.S.C. and by the MEXT of Japan to M.I. The authors declare that there are no conflicts of interest.

Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene-Expressing Embryos.

The use of vesicles co-incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co-incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx-egfp was injected circular (CP) at 3, 30 and 300 ng/µl and linear (LP) at 30 ng/µl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/µl (N = 105), 30 ng/µl (N = 95) and 300 ng/µl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/µl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/µl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co-incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.

Topical use of phytotherapic cream (Capilen® cream) to prevent radiodermatitis in breast cancer: a prospective historically controlled clinical study.

AIM: The purpose of this study was to evaluate the ability of a topical phytotherapic product (Capilen® cream) to limit acute radiodermitis and delay the use of corticosteroids in patients with breast cancer (BC). METHODS: From January 2012 to August 2012, 30 consecutive patients, undergoing radiotherapy with adjuvant intent, were invited to use Capilen® cream two times daily two weeks before and during radiotherapy. An historical group was used as an external control. Acute skin toxicity was scored weekly according to RTOG/EORTC criteria. Time of occurrence of acute skin toxicity was taken as endpoint. RESULTS: Compliance was good. Overall, no significative statistical difference was observed in rate of acute radiation dermatitis, 46.7% in experimental arm versus 63.3% in the historical control group, although only 3.3% of Capilen® cream treated patients had a G3 acute radiation dermatitis versus 10% of the control group. A delay in the onset of radition dermatitis in patients treated with Capilen® cream (P=0.04) was showed. CONCLUSION: Our findings suggested that Capilen® cream plays a role in reducing acute radiation dermatitis in breast cancer patients treated with adjuvant radiotherapy. Further evidence is needed to confirm these results.

Coculture with porcine luteal cells during in vitro porcine oocyte maturation affects lipid content, cortical reaction and zona pellucida ultrastructure.

CONTEXT: In pigs, in vitro fertilisation (IVF) is associated with high polyspermy rates, and for this reason, in vitro embryo production (IVP) is still an inefficient biotechnology. Coculture with somatic cells is an alternative to improve suboptimal in vitro maturation (IVM) conditions. AIM: This study was conducted to test a coculture system of porcine luteal cells (PLC) and cumulus-oocyte complexes (COC) to improve oocyte metabolism. METHODS: COC were matured in vitro with PLC. Oocyte lipid content, mitochondrial activity, zona pellucida (ZP) digestibility and pore size, cortical reaction and in vitro embryo development were assessed. KEY RESULTS: Coculture reduced cytoplasmic lipid content in the oocyte cytoplasm without increasing mitochondrial activity. Although ZP digestibility and ZP pore number were not different between culture systems, ZP pores were smaller in the coculture. Coculture impacted the distribution of cortical granules as they were found immediately under the oolemma, and more of them had released their content in the ZP. Coculture with porcine luteal cells during IVM increased monospermic penetration and embryo development after IVF. CONCLUSIONS: The coculture of COC with PLC affects the metabolism of the oocyte and benefits monospermic penetration and embryo development. IMPLICATIONS: The coculture system with PLC could be an alternative for the conventional maturation medium in pigs.

Morphometric analysis of llama (Lama glama) sperm head.

Llama production in Argentina has increased, as the international interest in breeding this type of animals has grown in the last years. Considering the great polymorphism that llama spermatozoa present at evaluation using light microscopy, the aim of this study was to objectively evaluate llama sperm head morphometry using digital morphometric analysis. Five ejaculates from each of eight males were obtained to evaluate morphometric parameters of 8000 sperm heads stained with Tinción 15(®). The following average results were obtained for each parameter: size parameters: area 20.09 µm(2), length 6.60 µm, width 4.14 µm, equivalent circle diameter 5.06 µm, curve length 5.79 µm and curve width 3.48 µm; boundary parameters: perimeter 18.54 µm and convex perimeter 17.34 µm; and shape parameters: roundness 1.28 and elongation 1.59. Morphometric parameters of sperm head were compared between ejaculates of the same male and between males. Significant differences between ejaculates of the same male were found for all parameters evaluated (P < 0.01). Significant differences between males were found for all morphometric parameters (P < 0.01) except for curve length, curve width and perimeter. The differences detected would indicate that there is not a single morphometric pattern for Lama glama sperm head, because parameter values cannot be standardised.

Coculture of porcine luteal cells during in vitro porcine oocyte maturation affects blastocyst gene expression and developmental potential.

Oocyte maturation in culture is still the weakest part of in vitro fertilization (IVF) and coculture with somatic cells may be an alternative to improve suboptimal culture conditions, especially in the pig in which maturation takes more than 44 h. In the present study, we investigated the effect of a coculture system of porcine luteal cells (PLC) during in vitro maturation (IVM) on embryo development and gene expression. Cumulus-oocyte complexes were matured in vitro in TCM-199 with human menopausal gonadotrophin (control) and in coculture with PLC. IVF was performed with frozen-thawed boar semen in Tris-buffered medium. Presumptive zygotes were cultured in PZM for 7 days. The coculture with PLC significantly increased blastocysts rates. Gene expression changes were measured with a porcine embryo-specific microarray and confirmed by RT-qPCR. The global transcription pattern of embryos developing after PLC coculture exhibited overall downregulation of gene expression. Following global gene expression pattern analysis, genes associated with lipid metabolism, mitochondrial function, endoplasmic reticulum stress, and apoptosis were found downregulated, and genes associated with cell cycle and proliferation were found upregulated in the PLC coculture. Canonical pathway analysis by Ingenuity Pathway revealed that differential expression transcripts were associated with the sirtuin signaling pathway, oxidative phosphorylation pathway, cytokines and ephrin receptor signaling. To conclude, the coculture system of PLC during IVM has a lasting effect on the embryo until the blastocyst stage, modifying gene expression, with a positive effect on embryo development. Our model could be an alternative to replace the conventional maturation medium with gonadotrophins with higher rates of embryo development, a key issue in porcine in vitro embryo production.

Risk factors for invasive aspergillosis in ICU patients with COVID-19: current insights and new key elements.

Invasive pulmonary aspergillosis (IPA) has always been a challenging diagnosis and risk factors an important guide to investigate specific population, especially in Intensive Care Unit. Traditionally recognized risk factors for IPA have been haematological diseases or condition associated with severe immunosuppression, lately completed by chronic conditions (such as obstructive pulmonary disease, liver cirrhosis, chronic kidney disease and diabetes), influenza infection and Intensive Care Unit (ICU) admission. Recently, a new association with SARS-CoV2 infection, named COVID-19-associated pulmonary aspergillosis (CAPA), has been reported worldwide, even if its basic epidemiological characteristics have not been completely established yet. In this narrative review, we aimed to explore the potential risk factors for the development of CAPA and to evaluate whether previous host factors or therapeutic approaches used in the treatment of COVID-19 critically ill patients (such as mechanical ventilation, intensive care management, corticosteroids, broad-spectrum antibiotics, immunomodulatory agents) may impact this new diagnostic category. Reviewing all English-language articles published from December 2019 to December 2020, we identified 21 papers describing risk factors, concerning host comorbidities, ICU management, and COVID-19 therapies. Although limited by the quality of the available literature, data seem to confirm the role of previous host risk factors, especially respiratory diseases. However, the attention is shifting from patients' related risk factors to factors characterizing the hospital and intensive care course, deeply influenced by specific features of COVID treatment itself. Prolonged invasive or non-invasive respiratory support, as well as the impact of corticosteroids and/or immunobiological therapies seem to play a pivotal role. ICU setting related factors, such as environmental factors, isolation conditions, ventilation systems, building renovation works, and temporal spread with respect to pandemic waves, need to be considered. Large, prospective studies based on new risk factors specific for CAPA are warranted to guide surveillance and decision of when and how to treat this particular population.

Periprosthetic fractures about total knee arthroplasty.

Periprosthetic fracture after total knee arthroplasty presents a difficult complication for many orthopaedic surgeons. These fractures occur most frequently around the distal femur followed by the patella and then tibia. These fractures are frequently complicated by poor bone quality or compromised bone due to the presence of the implants. Surgical treatment is typically necessary and requires varied techniques of open fixation, intramedullary fixation, or revision arthroplasty. Outcomes of these injuries vary widely. This review aims to describe the epidemiology, classification, treatment options and outcomes for periprosthetic fractures following total knee arthroplasty.

Relationship between hospital size and teaching status on outcomes for reverse shoulder arthroplasty.

BACKGROUND: The use of reverse shoulder arthroplasty (RSA) continues to grow with expanding indications and increased surgeon awareness. Previous data for other lower extremity joint replacements indicate that high-volume centers have better outcomes, with lower complication rates, decreased length of stay, and complications for both hemiarthroplasty and total shoulder arthroplasty. The purpose of this study is to evaluate the effects of hospital size and setting on adverse events for RSA. MATERIALS AND METHODS: The National Inpatient Sample database was queried for RSA performed using ICD-9 codes. Primary outcomes included length of stay (LOS), total hospital charges, discharge disposition, and postoperative complications. Odds ratios were used to assess the risk of inpatient postoperative complications. RESULTS: A weighted national estimate of 24,056 discharges for patients undergoing RSA was included in the study. Patients at larger hospitals experienced higher total charges, increased average LOS, and slightly higher complication rates compared to those of small and medium hospitals. Patients in larger hospitals had significantly increased rates of genitourinary and central nervous system complications, while patients in small/medium hospitals experienced higher rates of hematoma/seroma. CONCLUSION: Results from this study indicate that large and non-teaching hospitals overall tend to burden the patients with higher hospital charges, longer hospital stay, and more frequent non-routine discharges. Also, larger hospitals are associated with higher risk of genitourinary and central nervous system complications rates, whereas non-teaching hospitals are associated with lower risk of infection and higher risk of anemia after RSA. With the growth in RSA in the USA, continued attention needs to be placed on improving outcomes and resource utilization for RSA patients even in larger hospitals.

Differential expression of E- and N- cadherins during gonadal histogenesis in Columba livia (Aves: Columbiformes).

The spatial organization of cells during tissue differentiation is a crucial process in the morphogenesis of vertebrates. This process involves the movement, separation, and connection of cells. It is essential to elucidate the molecular mechanisms involved in these processes for the understanding of animal morphogenesis. Cell-cell adhesion molecules, called cadherins, are involved in the selective adhesion of cells. In the case of birds, the expression of these molecules in various organ systems during embryonic development has been reported in Gallus gallus domesticus. In this work, we present the immunohistochemical analysis of the differential expression of E and N-cadherin binding molecules in Columba livia embryos at various stages of gonadal morphogenesis. The expression of E and N-cadherin in embryos corresponding to the stages 41, 43 and in neonates of 2, 5, 7 and 75 post-hatching days were assessed by immunohistochemistry. Results revealed the expression of N-cadherin in the plasma membrane and the perinuclear zone of germline cells in ovaries and testes. However, the expression of E-cadherin was noticed with similar immunoreactivity pattern, in Sertoli cells and in the cells of the follicular nests. The differential expression of follicular cells and Sertoli cells positive for E-cadherin and germline cell N-cadherin positive cells were evidenced in the present work at the cell-cell interaction level. Future studies will focus on determining the expression of E and N-cadherin molecules during the migration of the primordial germ cells and the colonization of the genital ridge.

Leptin and IGF1 receptors in alpaca (Vicugna pacos) ovaries.

Folliculogenesis and ovulation are regulated by gonadotrophins and other factors such as Insulin like growth factor 1 (IGF1) and leptin. In various species the presence of IGF1 receptor (IGF1R) and leptin receptor (ObR) has been detected in the ovary, but not in the alpaca. Thus, the aim of the present study was to evaluate the presence of these receptors in this tissue and analyze if the presence of these receptors in the ovary is related to the presence of a corpus luteum (CL) and if abundances, as determined by immunostaining intensity vary with follicle size. The IGF1R and ObR were identified in primary and secondary follicles, granulosa and theca interna cells of tertiary follicles and in CL. There were greater abundances of IGF1R in granulosa cells of tertiary follicles of ovaries without compared with those with CL. In both groups, the immunostaining of granulosa cells was greater than in theca interna cells. The abundance of ObR was greater in primary and secondary follicles, and theca interna cells of tertiary follicles in ovaries with than those without CL. Immunostaining of granulosa cells was greater than theca interna cells only in ovaries without CL. There were no differences in the abundance of ObR and IGF1R between primary and secondary follicles and granulosa cells of tertiary follicles, neither in ovaries with or without CL. The abundance of IGF1R was not correlated with abundance of ObR neither in ovaries with or without CL. These results indicate a possible role for IGF and leptin in ovarian function. Furthermore, these receptors could be regulated by ovarian steroid hormones because abundance of these receptors in ovaries varies depending on whether there is a CL present in the ovary.

Anatomical, histological and immunohistochemical study of testicular development in Columba livia (Aves: Columbiformes).

In this work, testicular ontogeny is analyzed at the anatomical, histological and immunohistochemical levels; the latter through the detection of GnRHR and PCNA in the testicles of embryos, neonates and juveniles of Columba livia. We analyzed 150 embryos, 25 neonates and 5 juveniles by means of observations under a stereoscopic magnifying glass and scanning electron microscope (SEM). The histological analysis was performed using hematoxylin-eosin staining techniques and the PAS reaction. For the immunohistochemical analysis, the expression of GnRHR and PCNA in embryos corresponding to stages 41, 43 and in neonates of 2, 5, 7 and 75 days post-hatch was revealed in testicular histological preparations. That gonadal outline is evident in stage 18. In stage 29, the testes are constituted of a medulla in which the PGCs are surrounded by the Sertoli cells, constituting the seminiferous tubules. From stage 37 a greater organization of the tubules is visualized and at the time of hatching the testicle is constituted of the closed seminiferous tubules, formed of the PGCs and Sertoli cells. The Leydig cells are evident outside the tubules. In the juvenile stages, the differentiation of germline cells and the organization of small vessels that irrigate the developing testicle begin to be visible. In the analyzed stages, the immunodetection of the GnRHR receptor and PCNA revealed specific marking in the plasma membrane and in the perinuclear zone for GnRHR and in the nucleus of the germline cells in juvenile testicles for PCNA. These results can be used as a basis for further study of endocrine regulation events during testicular ontogeny in avian species.

Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro.

Pancreatic carboxyl ester lipase (CEL) hydrolyzes cholesteryl esters (CE), triglycerides (TG), and lysophospholipids, with CE and TG hydrolysis stimulated by cholate. Originally thought to be confined to the gastrointestinal system, CEL has been reported in the plasma of humans and other mammals, implying its potential in vivo to modify lipids associated with LDL, HDL (CE, TG), and oxidized LDL (lysophosphatidylcholine, lysoPC). We measured the concentration of CEL in human plasma as 1.2+/-0.5 ng/ml (in the range reported for lipoprotein lipase). Human LDL and HDL3 reconstituted with radiolabeled lipids were incubated with purified porcine CEL without or with cholate (10 or 100 microM, concentrations achievable in systemic or portal plasma, respectively). Using a saturating concentration of lipoprotein-associated CE (4 microM), with increasing cholate concentration there was an increase in the hydrolysis of LDL- and HDL3-CE; at 100 microM cholate, the present hydrolysis per hour was 32+/-2 and 1.6+/-0.1, respectively, indicating that CEL interaction varied with lipoprotein class. HDL3-TG hydrolysis was also observed, but was only approximately 5-10% of that for HDL3-CE at either 10 or 100 microM cholate. Oxidized LDL (OxLDL) is enriched with lysoPC, a proatherogenic compound. After a 4-h incubation with CEL, the lysoPC content of OxLDL was depleted 57%. Colocalization of CEL in the vicinity of OxLDL formation was supported by demonstrating in human aortic homogenate a cholate-stimulated cholesteryl ester hydrolytic activity inhibited by anti-human CEL IgG. We conclude that CEL has the capability to modify normal human LDL and HDL composition and structure and to reduce the atherogenicity of OxLDL by decreasing its lysoPC content.

Isolation of human anti-idiotypic antibodies by phage display for clinical immune response assays.

The clinical development of therapeutic proteins requires assays that measure the pharmacokinetic (PK) profile of, and the potential immune response (IR) to, the protein agent. Each assay requires reagents that are highly specific for the therapeutic protein. For therapeutic monoclonal antibodies, anti-CDR-specific, or anti-idiotypic (anti-id), antibodies are an ideal class of reagents suitable for these assays because of their high specificity and affinity to the drug antibody. We generated anti-ids to two human antibodies by antibody phage display using the MorphoSys HuCAL GOLD Fab library. To selectively target the CDR regions, serum and a framework-matched mAb were included as competitors during the phage selection process. Panels of CDR-specific Fabs, with low to sub-nM affinities, were isolated against both targets. The CDR specificity of these Fabs was shown by their lack of binding to a framework-matched control mAb and by competition of this binding with the soluble antigens of the respective therapeutic mAb targets. The candidate anti-id Fabs were able to detect both immobilized and soluble target Ab without being affected by serum, a requirement for both PK assay and the IR bridging assay format. Combinations of the Fabs for PK detection assays were identified by pairwise binding studies, although the pair for one target mAb lacks the desired sensitivity for PK assays. To evaluate their potential as anti-drug antibodies (ADAs), the best Fabs for one of the targets were converted and produced as the required bivalent human mAbs. In comparison to rodent mAbs and primate polyclonal serum, the phage display derived human mAbs were equally effective as reference standards. Our results demonstrate that competition-based phage selection can be an effective method for the isolation of anti-idiotypic antibodies for PK and IR assay development, and in this latter case, overcome limitations of current methods using rodent derived anti-ids.

Extremity fractures associated with ATVs and dirt bikes: a 10-year national epidemiologic study.

BACKGROUND: Morbidity and mortality of all-terrain vehicles and dirt bikes have been studied, as well as the association of helmet use and head injury. HYPOTHESIS/PURPOSE: The purpose of this study is to compare and contrast the patterns of extremity fractures associated with ATVs and dirt bikes. We believe there will be unique and potentially preventable injury patterns associated with dirt bikes and three-wheeled ATVs due to the poor stability of these vehicles. STUDY DESIGN: Descriptive epidemiology study. METHODS: The National Electronic Injury Surveillance System (NEISS) was used to acquire data for extremity fractures related to ATV (three wheels, four wheels, and number of wheels undefined) and dirt bike use from 2007 to 2012. Nationwide estimation of injury incidence was determined using NEISS weight calculations. RESULTS: The database yielded an estimate of 229,362 extremity fractures from 2007 to 2012. The incidence rates of extremity fractures associated with ATV and dirt bike use were 3.87 and 6.85 per 1000 participant-years. The largest proportion of all fractures occurred in the shoulder (27.2%), followed by the wrist and lower leg (13.8 and 12.4%, respectively). There were no differences in the distribution of the location of fractures among four-wheeled or unspecified ATVs. However, three-wheeled ATVs and dirt bikes had much larger proportion of lower leg, foot, and ankle fractures compared to the other vehicle types. CONCLUSIONS: While upper extremity fractures were the most commonly observed in this database, three-wheeled ATVs and dirt bikes showed increased proportions of lower extremity fractures. Several organizations have previously advocated for better regulation of the sale and use of these specific vehicles due to increased risks. These findings help illustrate some of the specific risks associated with these commonly used vehicles.

Apoptosis of bovine granulosa cells: Intracellular pathways and differentiation.

Follicular atresia in granulosa and theca cells occurs by apoptosis through weak hormonal stimulation. We have previously proposed an in vitro model to study this process by inducing apoptosis in BGC-1, a bovine granulosa cell line, and in primary cultures from ovaries with or without corpus luteum (CPGB+ and CPGB-, respectively), with different doses of gonadotropin releasing hormone (GnRH) analogs (leuprolide acetate (LA) as agonist and antide as antagonist). BGC-1 represent immature granulosa cells, whereas CPGB represent different degrees of luteinization. Our aim was to evaluate the intracellular pathways involved in the GnRH regulation of apoptosis in BGC-1. Treatment with LA 100nM but not with antide led to an increase in BAX over BCL-2 expression, showing antagonism of antide. All treatments inhibited phospholipase-D (PLD) activity compared to control, implying agonist behavior of antide. Progesterone in vitro production and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) expression revealed different degrees of luteinization: BGC-1 were immature, whereas CPGB+ were less differentiated than CPGB-. We concluded that LA-induced apoptosis in BGC-1 occurs by activation of the mitochondrial pathway and by inhibition of PLD activity and that antide might work both as an antagonist of the intrinsic pathway and as an agonist of the extrinsic protection pathway by inhibiting PLD activity.