Percoll Gradient Research Articles

P-798 Fertility preservation in pre-pubertal boys with cancer: A three-dimensional prepubertal testicular organoid for in vitro spermatogonial stem cell propagation and spermatogenesis

Abstract Study question Can a three-dimensional (3D) prepubertal testicular organoid be formed and provide an in vitro microenvironment for spermatogonial stem cells (SSCs) maintenance and future spermatogenesis? Summary answer Primary cells extracted from immature testicular tissue (ITT) or SSCs can be grown long-term as 3D organoids, providing the potential for in vitro study. What is known already Aggressive cancer treatments, such as chemo- or radiotherapy, can leave young prepubertal boys infertile. Such patients are recommended to undergo the cryopreservation of testicular material to protect future fertility. Within the testes, the specialized 3D structure and direct cell-to-cell interactions play a critical role in the proliferation and development of SSCs. Over recent decades, 3D culture systems and organoids have been used to culture cells in vitro, however, a system that allows investigations into testicular organogenesis in vitro, and its impact on the SSC niche, has yet to be developed. Study design, size, duration This study aims to develop a 3D organoid culture system to support the proliferation of SSCs and spermatogenesis. Primary bovine ITT cells and enriched SSCs were isolated and 3D organoids were generated by in vitro culture for up to 40 days. Organoid formation was observed after using different foundation cells seeded at different densities and cultured in medium containing gonadotropic supplements. Participants/materials, setting, methods Post-thaw bovine ITTs (2 weeks-of-age) were dissociated using two-step enzymatic digestion. Enriched SSCs were selected by Percoll gradients and differential plating. Viability and apoptosis were evaluated by trypan blue staining and TUNEL assays, respectively. SSCs were evaluated immunocytochemically for germ-cell markers (PGP-9.5, PLZF) and Sertoli cell markers (Vimentin, Sox9). Expression levels of SSCs and spermatogenesis-related genes (Plzf, Gfrα-1, Nanog, Oct4, Stra8, Thy1) were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Main results and the role of chance The viability of digested cells from thawed ITTs was 78.667% ± 2.03. Total testicular cells (<10% SSCs) and enriched SSCs(>50% SSCs) were observed to self-assemble into structurally complex organoids recapitulating the cell type compartmentalization of the testis, in a 3D Matrigel-based culture system with 10% knockout serum replacement (KSR) culture medium, but not with 10% fetal bovine serum(FBS) medium. Testicular organoids were found to exhibit either a grape-like structure and a round-shape structure. Cytoplasmic extensions of spermatogonia/Sertoli cells were in contact with each other within a forming colony. Organoids were formed faster and larger when seeded at a final concentration of 1.5 × 106 cells/ml, compared to 5 × 105 cells/ml and 1.5 × 105 cells/ml. Organoids grew to a diameter of 400 μm within 10 - 15 days and were passaged by mechanical disruption at a ratio of 1:3 every 7 - 10 days. Immunocytochemistry results showed that clusters of PGP9.5 and PLZF-positive cells were present within the organoids. The expression of selected germ cell and spermatogenesis markers in the testicular organoids closely resembled that of primary testicular cells for up to 20 days of culture. Limitations, reasons for caution We used calves (2 weeks-of-age) as an animal model to study testicular organoids. This tissue may act differently than human tissues and may not fully represent prepuberty. Furthermore, we only evaluated gene expression levels for selected markers that may not represent the full functional capability of germ cells. Wider implications of the findings Testicular organoids, as an in vitro bioengineering testicular model, could potentially be used to study testicular tissue development, cellular interactions, endocrinology, and spermatogenesis, in the laboratory but may also be applied for clinical purposes in the future. Trial registration number University of Oxford

P–798 Fertility preservation in pre-pubertal boys with cancer: A three-dimensional prepubertal testicular organoid for in vitro spermatogonial stem cell propagation and spermatogenesis

Abstract Study question Can a three-dimensional (3D) prepubertal testicular organoid be formed and provide an in vitro microenvironment for spermatogonial stem cells (SSCs) maintenance and future spermatogenesis? Summary answer Primary cells extracted from immature testicular tissue (ITT) or SSCs can be grown long-term as 3D organoids, providing the potential for in vitro study. What is known already Aggressive cancer treatments, such as chemo- or radiotherapy, can leave young prepubertal boys infertile. Such patients are recommended to undergo the cryopreservation of testicular material to protect future fertility. Within the testes, the specialized 3D structure and direct cell-to-cell interactions play a critical role in the proliferation and development of SSCs. Over recent decades, 3D culture systems and organoids have been used to culture cells in vitro, however, a system that allows investigations into testicular organogenesis in vitro, and its impact on the SSC niche, has yet to be developed. Study design, size, duration This study aims to develop a 3D organoid culture system to support the proliferation of SSCs and spermatogenesis. Primary bovine ITT cells and enriched SSCs were isolated and 3D organoids were generated by in vitro culture for up to 40 days. Organoid formation was observed after using different foundation cells seeded at different densities and cultured in medium containing gonadotropic supplements. Participants/materials, setting, methods Post-thaw bovine ITTs (2 weeks-of-age) were dissociated using two-step enzymatic digestion. Enriched SSCs were selected by Percoll gradients and differential plating. Viability and apoptosis were evaluated by trypan blue staining and TUNEL assays, respectively. SSCs were evaluated immunocytochemically for germ-cell markers (PGP–9.5, PLZF) and Sertoli cell markers (Vimentin, Sox9). Expression levels of SSCs and spermatogenesis-related genes (Plzf, Gfrα–1, Nanog, Oct4, Stra8, Thy1) were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Main results and the role of chance The viability of digested cells from thawed ITTs was 78.667% ± 2.03. Total testicular cells (<10% SSCs) and enriched SSCs(>50% SSCs) were observed to self-assemble into structurally complex organoids recapitulating the cell type compartmentalization of the testis, in a 3D Matrigel-based culture system with 10% knockout serum replacement (KSR) culture medium, but not with 10% fetal bovine serum(FBS) medium. Testicular organoids were found to exhibit either a grape-like structure and a round-shape structure. Cytoplasmic extensions of spermatogonia/Sertoli cells were in contact with each other within a forming colony. Organoids were formed faster and larger when seeded at a final concentration of 1.5 × 106 cells/ml, compared to 5 × 105 cells/ml and 1.5 × 105 cells/ml. Organoids grew to a diameter of 400 μm within 10 - 15 days and were passaged by mechanical disruption at a ratio of 1:3 every 7 - 10 days. Immunocytochemistry results showed that clusters of PGP9.5 and PLZF-positive cells were present within the organoids. The expression of selected germ cell and spermatogenesis markers in the testicular organoids closely resembled that of primary testicular cells for up to 20 days of culture. Limitations, reasons for caution We used calves (2 weeks-of-age) as an animal model to study testicular organoids. This tissue may act differently than human tissues and may not fully represent prepuberty. Furthermore, we only evaluated gene expression levels for selected markers that may not represent the full functional capability of germ cells. Wider implications of the findings: Testicular organoids, as an in vitro bioengineering testicular model, could potentially be used to study testicular tissue development, cellular interactions, endocrinology, and spermatogenesis, in the laboratory but may also be applied for clinical purposes in the future. Trial registration number University of Oxford

A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue

SummaryThe importance of lysosomes in cardiac physiology and pathology is well established, and evidence for roles in calcium signaling is emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endolysosomal (EL) proteins. Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/mL); and EL with negligible contamination from SR or Mito (1.04 g/mL) were analyzed using Western blot, enzyme activity assay, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis (adapted discontinuous Percoll and sucrose differential density gradient). Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther, and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and data set of guinea pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

​Sex Ratio of Calves Resulted from Artificial Insemination Implementation using Sexed Semen with Percoll Gradient Density Centrifugation Method in Ongole Crossbred Cows

Background: Productivity of existing cattles in Indonesia is necessarily to be increase to balance meat consumption in this country. Determination of offspring of certain sex can be obtained from Percoll gradient density centrifugation.The purpose of this research was to elucidate the proportion of male calves that can result from artificial insemination using single and double doses of sexed semen in Ongole crossbred cows. Methods: The sexed semen samples were obtained through Percoll gradient density centrifugation performed by the Artificial Insemination Center. The artificial insemination method adopted here was deep insemination. As much as 10 ml of BIO ATP® (Rheinvet) was injected in each cow before immediately insemination. Further, as much as 3 kg/day of additional feed was given over three days after insemination, with a protein level of about 12%. Result: Our results showed the proportion of Y-bearing sperm among non-sexed semen was 52.77% and among sexed semen, was 80.79%. Further, 54.17% of the non-sexed semen, 42.11% of the single-dose sexed semen and 78.95% of the double-dose sexed semen treatments yielded male calves.

A method for the enrichment, isolation and validation of Mycobacterium smegmatis population surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin.

The bacterial populations surviving in the presence of antibiotics contain cells that have gained genetic resistance, phenotypic resistance and tolerance to antibiotics. Isolation of live bacterial population, surviving against antibiotics, from the milieu of high proportions of dead/damaged cells will facilitate the study of the cellular/molecular processes used by them for survival. Here we present a Percoll gradient centrifugation based method for the isolation of enriched population of Mycobacterium smegmatis surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin. From the time of harvest, throughout the enrichment and isolation processes, and up to the lysis of the cells for total RNA preparation, we maintained the cells in the presence of the antibiotic to avoid changes in their metabolic status. The total RNA extracted from the enriched population of live antibiotic-surviving population showed structural integrity and purity. We analysed the transcriptome profile of the antibiotic-surviving population and compared it with the orthologue genes of Mycobacterium tuberculosis that conferred antibiotic tolerance on tubercle bacilli isolated from the tuberculosis patients under treatment with four antitubercular antibiotics. Statistically significant comparability between the gene expression profiles of the antibiotic tolerance associated genes of M. smegmatis and M. tuberculosis validated the reliability/utility of the method.

Canonical Inflammasome Formation in Monocytes of Sickle Cell Anemia Patients

Sickle cell anemia (SCA) is now recognized as a chronic vascular inflammatory disease, in which inflammatory mechanisms drive the vaso-occlusive process. The inflammasome complex, responsible for the maturation and release of the IL-1β and IL-18 cytokines, is formed by pattern recognition receptors (PRRs), such as the NLRP3 protein, which recognize damage (or pathogen) associated molecular patterns (DAMPs). Following recruitment of the ASC adapter protein by the PRR, caspase-1 is activated by proximity-dimerization on the inflammasome and processes pro-IL-1β and pro-IL-18 into their bioactive forms. Elevated plasma levels of IL-1β and IL-18 may play a significant role in driving SCA inflammatory processes and it is important to identify the cellular sources and molecular mechanisms that contribute to the processing of these cytokines in SCA. Elevated caspase-1 activity and IL-1β processing has previously been reported in the neutrophils of SCA individuals (Mendonça et al., ASH Abstract 2016) and we, herein, aim to determine whether inflammasome assembly also occurs in the SCA monocytes. Peripheral blood samples and monocytes, separated by percoll gradients, were obtained from healthy control individuals (CON, total N=20) and SCA patients (total N= 20, of which 10 patients were on 15-30 mg/kg/day hydroxyurea therapy). No significant differences in data obtained from patients on/off hydroxyurea therapy were observed; thus, findings from these two groups were combined. Caspase-1 activity was determined in phenotypically-characterized monocyte populations by flow cytometry (Fam-FlicaTM kit, Immunochemistry Technologies). IL-1β release was quantified by ELISA. The percentage of CD14+CD16+ inflammatory monocytes presenting caspase-1 activity was significantly augmented in SCA, when compared to CON individuals (10.6 ± 1.3, 6.5± 1.1 % caspase-1 positivity, N=20 and 13 respectively; P<0.05). Confocal immunofluorescence microscopy indicated an augmented expression of the ASC adaptor protein and ASC speck formation in unstimulated SCA monocytes (N=3), compared to CON monocytes (N=2). Accordingly, Western blotting showed a higher ASC expression in pooled SCA monocyte total cell extract (TCE, N=4), compared to that of pooled CON monocyte TCE (N=4); furthermore, increased cleaved caspase-1 (20 KDa) was identified in pooled SCA monocyte TCEs, compared to CON monocyte TCEs. Consistent with this observation, significant pro-caspase-1 co-precipitation, together with immunoprecipitated ASC, was achieved from pooled SCA monocyte lysates, while this pro-caspase-1/ASC association was almost non-detectable in CON monocyte lysates. Finally, IL-1β release by SCA monocytes (5x105 cell/ml, 37°C during 2h, N=8) was significantly abrogated by both NLRP3 inhibition (5µM MCC950) (decreased from 50.4± 14.4 to 30.3± 11.2 pg/ml released IL-1β; P<0.05) and caspase-1 inhibition (40 µM YVAD-FMK) (decreased to 20.8± 8.3 pg/ml IL-1β; P< 0.05). Interestingly, the inhibition of NFκB (10 µM BAY117082, an inhibitor of IκBα phosphorylation) also significantly reduced IL-1β release from SCA monocytes in 2h (9.64±2.66 pg/ml; N=8, P<0.01). As such, the inflammasome assembled in SCA monocytes is apparently of the NLRP3 type, and appears to involve NF-κB activated transcriptional regulation of IL-1β processing in these cells, most likely at the priming stage. Our data indicate that monocytes of SCA individuals present elevated ASC expression and oligomerization, mediated by the NLRP3 PRR sensor, culminating in augmented pro-caspase-1 recruitment by ASC and its cleavage to active caspase-1. As such, this canonical inflammasome assembly in SCA monocytes appears to contribute to the expressive quantities of IL-1β that are released by SCA monocytes; these cells may contribute to the elevated IL-1β processing that occurs in SCA, possibly representing a therapeutic target for the disease. DisclosuresConran:Bayer AG: Research Funding.

In Utero Injections of Nanoparticle Encapsulated Myelin Oligodendrocyte Glycoprotein Peptides Prevent Experimental Autoimmune Encephalopathy Via Central Tolerance

IntroductionIn utero hematopoietic cell transplantation (IUHCT) of allogenic bone marrow mononuclear cells (MNC) results in successful engraftment across immune barriers. This is secondary to immune tolerance that is acquired due to the presence of donor cells in the thymus prior to E17. We have demonstrated that immune tolerance to specific proteins is possible by injecting nanoparticle encapsulated peptides which are taken up by dendritic cells and presented in the thymus.The aim of this study is to show that in utero injections of nanoparticle encapsulated antigen can produce tolerance to that antigen. We demonstrate this by preventing the autoimmune disease Experimental Autoimmune Encephalopathy (EAE, the mouse equivalent of multiple sclerosis) by injecting NPs loaded with the peptide Myelin Oligodendrocyte Glycoprotein (35-55) (MOG35-55.)MethodsIn utero injections of nanoparticles were performed in C57BL/6 mice at E14 intravenously through the vitelline vein. Post-natal boosters were injected subcutaneously at 2, 4, 6, and 8 weeks of age. Disease inducing injections of MOG with Complete Freud's Adjuvant (CFA) was given at 8 1/7 weeks. Intraperitoneal pertussis toxin (PT) was given on the same day as the MOG/CFA and again 48 hours later. Positive controls got MOG/CFA and PT injections. Negative controls got CFA without MOG as well as the PT injections.Mice were monitored for paralysis using the 5-point EAE scoring system for 4 weeks after the MOG/CFA injection. Spines and brains were harvested 2 weeks after the disease inducing MOG/CFA injection. Spines and brains were filtered and then MNCs were isolated using a Percoll gradient. MNCs were stained for CD45, CD19, CD11b, CD3, CD4, CD8, and I-A(b) MOG35-55 tetramer. Cells were then analyzed on the FACSAria™.Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children's Hospital of Philadelphia.ResultsMice that received MOG nanoparticles prenatally along with boosters (Group A) developed disease with less frequency than mice that received MOG nanoparticles postnatally along with boosters (Group B) or positive controls (Group D) (52.0% vs 87.5% vs 90.0% p<0.005) supporting the theory that antigen needs to be expressed prior to E17 in the thymus to develop tolerance. Mice that were injected prenatally with MOG nanoparticles and did not receive booster injections (Group C) also did as poorly as positive controls (85.72% vs 90.0%) suggesting that continued antigen expression is necessary to maintain tolerance. Group A also had significantly lower EAE scores on days 15 to 28 compared to Group B, Group C, and the positive control group (p<0.005).Spinal cord and brain analysis of the Group A mice showed significantly fewer CD45 cells compared to the positive control but not the negative control (133.2±52.0 vs 6062.5±1166.9 vs 79.8±15.0 p<0.0001.) The same is seen with CD3 cells (45.6±17.0 vs 4727.3±1283.0 vs 21.3±2.8 p<0.0001) and CD4 cells (12.6±5.7 vs 3339.6912.1 vs 11.8±0.8 p<0.0001.) Finally, the same is seen with CD4+ Tetramer+ cells (0.7±0.3 vs 379.1±156.6 vs 2.9±1.0 p<0.0001) showing that there is central tolerance to the MOG peptide.ConclusionBy decreasing the frequency and average severity of EAE and demonstrating the lack of CD4+ Tetramer+ cells in the spine, we have shown that autoimmune disease can be prevented by central tolerance to the target antigen. We have demonstrated that this central tolerance is possible with prenatal injections of nanoparticle encapsulated peptide. We have also shown that continued antigen presentation is necessary to maintain central tolerance. We hope that nanoparticle encapsulated peptides could provide a possible pharmacological benefit for numerous autoimmune diseases in the future. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Plasmodium vivax Gametocytes Adherence to Bone Marrow Endothelial Cells.

In a Plasmodium vivax infection, it was shown a proportionally increased on gametocyte distribution within the bone marrow aspirant, suggesting a role of this organ as a reservoir for this parasite stage. Here, we evaluated the ex vivo cytoadhesive capacity of P. vivax gametocytes to bone marrow endothelial cells (HBMEC) and investigated the involvement of some receptors in the cytoadhesion process by using transfected CHO cells (CHO-ICAM1, CHO-CD36 and CHO-VCAM), wild type (CHO-K1) or deficient in heparan and chondroitin sulfate (CHO-745). Ex-vivo cytoadhesion assays were performed using a total of 44 P. vivax isolates enriched in gametocyte stages by Percoll gradient in the different cell lines. The majority of isolates (88.9%) were able to adhere to HBMEC monolayer. ICAM1 seemed to be the sole receptor significantly involved. CD-36 was the receptor with higher adhesion rate, despite no significance was noticed when compared to CHO-745. We demonstrated that gametocyte P. vivax adheres ex vivo to bone marrow endothelial cells. Moreover, P. vivax gametocytes display the ability to adhere to all CHO cells investigated, especially to CHO-ICAM1. These findings bring insights to the comprehension of the role of the bone marrow as a P. vivax reservoir and the potential impact on parasite transmission to the vector.

Nuclear heterogeneity is prevalent in high-quality fractionated human sperm cells typically used for assisted conception.

What is the nuclear heterogeneity of high-density purified human spermatozoa typically used for IVF purposes. The data show that while density gradient separation has improved the overall sperm population, there is still a large degree of nuclear heterogeneity within these cells. Chromomycin A3 (CMA3) is an important DNA binding fluorochrome for the assessment of male-factor fertility. It is typically used to predict IVF outcomes on entire sperm ejaculates with very high receiver operating characteristic. Here we used CMA3 to characterise typical populations of human spermatozoa that would be used for IVF purposes after density gradient separation. We compared the intensity of CMA3 binding within high-dense sperm populations obtained from men. Binding heterogeneity was confirmed through fluorescence microscopy and FACS analysis independently. We also looked at CMA3 staining directly with head morphology in this sperm population. Finally, we looked at electron micrographs of nuclear heterogeneity (vacuoles, chromatin compaction) of spermatozoa following density gradient sorting of CMA3-stained cells. We used sperm donors who had fathered one or more children. Semen was collected after 2 days abstinence and purified over Percoll gradients. Only the high-quality spermatozoa, the same used for assisted conception, were then used. Cells were stained with CMA3 and sorted using FACS. Following this, electron micrographs were used to assess nuclear heterogeneity of CMA3-dependent sorted spermatozoa. CMA3 staining occurs within morphologically normal as well as abnormal spermatozoa. High-intensity CMA3-stained sperm possessed large vacuoles that were not seen in the low-CMA3 population. In addition, the high-CMA3 stained cells possess higher amounts of nuclear granulation. The present study only describes the issues within the chromatin of these cells and does not suggest an alternate selection technique. CMA3 is one of the better reported prognostic assays in predicting pregnancy outcomes, especially in cases where the male is at fault. However, it is clear that even in fractionated populations of human spermatozoa, there are sperm cells that are morphologically normal yet possess high levels of CMA3 staining and chromatin granulation. The implication of this is that the embryologist, whom selects on the basis of sperm morphology, may choose a cell with poor chromatin, which may lead to poor embryo outcomes. The project was funded by the National Health and Medical Research council, APP1118943. The authors have no conflict of interest to declare. N/A.

Production of donor-derived offsprings by allogeneic transplantation of oogonia in the adult Japanese flounder (Paralichthys olivaceus)

Germ cell transplantation (GCT) is a promising reproductive technology in animals, including teleost fish. Here, we established a GCT procedure in Japanese flounder (Paralichthys olivaceus). Two-year-old male Japanese flounder were treated with temperature (28 °C) and busulfan (two doses, 18 mg/kg) to deplete endogenous germ cells and were then used as recipients. The discontinuous Percoll gradient purified oogonia were used as donor germ cells and transplanted into the recipients' testes through the genital pore. Histological and fluorescent observations showed the colonization, proliferation, and differentiation of transplanted oogonia in the testes of recipient fish. Ten months after GCT, 16 of 17 recipients produced sperm, and among them, two recipients produced donor-derived sperm, which was confirmed by microsatellite genotyping. The sperm of these two recipients were used to fertilize the eggs of wild-type females, and the donor-derived germline transmission rates were 45.5% and 43.0%, respectively, based on the microsatellite paternity analysis of the hatched larvae. Our established protocol can be applied to accelerate the reproduction cycle and be used for generating valuable surrogate breeding material and conserving the germplasm resources of Japanese flounder.

Evaluation of in vitro production rates of bovine embryos using melatonin-supplemented culture medium

In this study, we aimed to evaluate the rate of bovine embryo production by using 50 ng/mL melatonin supplementation in in vitro culture medium. For this, oocytes from slaughterhouse ovaries were matured in vitro in TCM-199 medium with Earle’s balanced salt solution + 10% SFB, FSH, and LH in an atmosphere of 5% CO2. Twenty-four hours after IVM, the oocytes underwent in vitro fertilization in human tubal fluid under the same conditions as above, for 18 h. Semen was fractionated by Percoll gradient centrifugation and the concentration of sperm was adjusted to 1 × 106/mL. Probable zygotes were then divided into two groups: the control group grown in drops of 90 μL SOFaa medium + 0.6% BSA + 2.5% SFB, in an atmosphere of 5% CO2, 90% N2, and a melatonin group (Mel), similarly cultured in 90 μL drops of SOFaa medium + 0.6% BSA + 2.5% SFB + 50 ng/mL melatonin. Cleavage rates were assessed on day 3 (D3). On D7, blastocyst formation rates were evaluated. Eight routines were performed (320 oocytes per routine). Data were analyzed with ANOVA, followed by Tukey’s range test using a general linear model. The level of statistical significance was set at 5%. There were no differences in the rates of cleavage or blastocyst formation between the control and melatonin groups (P &gt; 0.05). Thus, under the conditions used in this study, supplementation with melatonin did not yield benefits in increasing the rate of in vitro bovine embryo production.

Influence of maggot excretions/secretions on the anti-Pseudomonas aeruginosa effect of neutrophils in patients with diabetic foot ulcer

Objective: To investigate the effects of medical maggot excretions/secretions (ES) on neutrophils phagocytosis and bactericidal effect in patients with diabetic foot ulcer (DFU). Methods: The experimental research method was used. Thirty DFU patients (16 males and 14 females, aged (64±7) years)who were admitted to the Diabetes Foot Center, the Department of Endocrinology of Air Force Hospital of Eastern Theater Command from June to December 2020 and met the inclusion criteria were recruited. Discontinuous percoll gradient centrifugation method was used to separate the neutrophils. Cells from each patient were enrolled into normal saline group and maggot ES group (30 wells in each group), respectively; sterile normal saline and ES with a final mass concentration of 357 μg/mL (the same as below) were added, respectively. After 1 and 2 hour(s) of culture, the phagocytosis rate and phagocytic index of cells were observed and counted under Wright's staining. Ten patients were selected, then the cells of each patient were enrolled into Pseudomonas aeruginosa+neutrophils group and Pseudomonas aeruginosa+neutrophils+maggot ES group (10 wells in each group) and were treated corresponding, respectively. Pseudomonas aeruginosa alone group and Pseudomonas aeruginosa+maggot ES group (10 wells in each group) were set up respectively; Pseudomonas aeruginosa+RPMI 1640 culture medium+sterile normal saline and Pseudomonas aeruginosa+RPMI 1640 culture medium+maggot ES were added, respectively. After 2 hours of culture, the number of viable bacteria colony was counted by plate colony number method. Six, six, and three patients were selected respectively, and the cells of each patient were respectively enrolled into maggot ES group and normal saline group (6, 6, and 3 wells in each group, respectively) and treated accordingly. After 6 hours of culture, real-time fluorescent quantitative reverse transcription polymerase chain reaction was used to detect the mRNA expressions of interleukin 1β (IL-1β), IL-6, and lysozyme in cells, the content of IL-1β and IL-6 in cell culture supernatant were determined by enzyme-linked immunosorbent assay, and the positive cells expressing lysozyme were observed with immunofluorescence method. Data were statistically analyzed with one-way analysis of variance, paired sample t test, least significant difference test, and Wilcoxon rank sum test. Results: After 1 hour of culture, the phagocytosis rate and phagocytic index of cells in maggot ES group (53.5% (49.7%, 58.0%) and 3.18 (2.96, 3.32)) were similar to 52.0% (47.5%, 55.2%) and 3.15 (2.96, 3.25) of normal saline group (Z=-1.701, -1.092, P>0.05). After 2 hours of culture, the phagocytosis rate and phagocytic index of cells in maggot ES group (70.0% (66.7%, 72.0%) and 4.47 (4.22, 4.96)) were significantly higher than 58.0% (55.0%, 60.0%) and 4.11 (3.52, 4.24) in normal saline group (Z=-4.786, -4.279, P<0.01). After 2 hours of culture, the number of viable bacteria colony in Pseudomonas aeruginosa+neutrophils group was significantly lower than that in Pseudomonas aeruginosa alone group (P<0.01), and the number of viable bacteria colony in Pseudomonas aeruginosa+neutrophils+maggot ES group was significantly lower than that in Pseudomonas aeruginosa+maggot ES group and Pseudomonas aeruginosa+neutrophils group (P<0.01). After 6 hours of culture, the mRNA expressions of IL-1β, IL-6, and lysozyme of cells in maggot ES group were significantly higher those in normal saline group (t=-3.279, -4.273, -4.763, P<0.05 or P<0.01); the concent of IL-1β and IL-6 in cell culture supernatant of maggot ES group were significantly higher than those of normal saline group (t=-9.526, -6.447, P<0.01); there were significantly more positive cells expressing lysozyme in maggot ES group than in normal saline group. Conclusions: Maggot ES can enhance the phagocytosis and bactericidal effect of neutrophils on Pseudomonas aeruginosa by promoting the production of neutrophils immune defense related cytokines and lysozyme in DFU patients.

Bisphenol A and S, but Not F, Alter Oxidative Stress Levels in Spermatozoa

Exposures to endocrine disrupting compounds (EDCs) have been linked to male reproductive dysfunction. The increase in prevalence of male infertility to date has been correlated with the widespread use of bisphenols, particularly bisphenol A and its analogs. Current literature suggests that exposure to EDCs is associated with abnormalities in spermatogenesis, as well as reduced sperm motility and morphology. At this time, the mechanism for bisphenols’ action in spermatozoa is still unknown, though it has been proposed that bisphenols alter sperm parameters by affecting their oxidative stress pathways. The aim of this project is to elucidate whether BPA and its analogs affect oxidative stress levels inducing decreased sperm quality and fertilization capability. An in vitro bovine model was used as the most appropriate translational model to humans. Motile spermatozoa were separated using Percoll gradient centrifugation of frozen-thawed sperm and a pilot experiment was conducted in a time-dependent manner to establish the ideal incubation time of 4 hours. Sperm was then incubated in five treatment groups: control, vehicle (0.01% ethanol), BPA, BPS, and BPF, at the physiological relevant dose of 0.05 mg/ml in 0.01% ethanol. Key reactive oxygen species (ROS) scavenger genes (superoxide mutase 1 [SOD1], superoxide mutase 2 [SOD2], catalase [CAT], glutathione peroxidase 1 [GPX1], glutathione peroxidase 4 [GPX4]) were quantified at the RNA level using qPCR. Of the five genes examined: BPA, BPS, and BPF exposure significantly decreased GPX4 expression (p<0.05) compared to control and vehicle groups. BPA and BPS exposure significantly increased SOD1 expression compared to BPF, control, and vehicle (p<0.05). No significant differences were found in GPX1 after bisphenols’ exposure. SOD2 and CAT expression was not detectable in all five groups suggesting a lack of expression in bovine sperm. Future investigations will involve experiments at the protein level and quantifying total ROS levels. Additionally, sperm morphology was evaluated using Spermac stain. Preliminary results indicate an increased number of abnormal spermatozoa with notably tail defects after 4 hours of bisphenols exposure. These initial findings support BPA and BPS’s ability to alter oxidative stress pathways in sperm via changes in antioxidant enzymes but suggest that BPF likely acts through different mechanisms. Overall, this research aims to understand the effects of bisphenols on male fertility to improve male factors during assisted reproductive biotechnologies.

Downregulation of AW112010 upon treatment with 6-formylindolo[3,2-b] carbazole protects mice from fulminant Concanavalin-induced T cell-mediated liver injury

Abstract Upregulation of long noncoding RNA (lncRNA) AW112010 has been observed in multiple inflammatory disease models. In line with implication of this lncRNA as a regulator of inflammation, it has also been shown that AW112010 expression decreases upon treatment with anti-inflammatory compounds. Since lncRNAs show abnormal expression in many inflammatory diseases and reactions, elucidating their role in such ailments is necessary. Data from our lab has shown that treatment with aryl hydrocarbon receptor (AhR) ligands induce epigenetic pathway dysregulation, so this study aimed to determine inflammatory genes dysregulated upon treatment with the endogenous AhR ligand, 6-formylindolo[3,2-b] carbazole (FICZ). A murine model of immune cell-mediated liver injury was employed by intravenously injecting 12.5 mg/kg Concanavalin A (ConA). One hour after challenge, mice were treated with 50 μg/kg FICZ via an intraperitoneal injection. Interestingly, analysis of serum collected twenty-four hours after challenge showed that FICZ was able to reduce the level of alanine transaminase (ALT) suggesting a decrease in disease severity. Single cell RNA sequencing was conducted on infiltrating liver mononuclear cells enriched via a percoll gradient. We observed a reduced percentage of T cells upon FICZ treatment and an increase in the percentage of neutrophils. Overall, we observed downregulation of AW112010 in the FICZ-treated group. Specifically, AW112010 was identified as one of the top 20 genes dysregulated in the NK cell cluster and was downregulated in the B cell, CD8+ T cell, and Kupffer cell clusters. Together, these data suggest AW112010 downregulation as a potential mechanism through which FICZ protects mice from severe liver injury.

Visualizing the Integrity of Chloroplast Envelope by Rhodamine and Nile Red Staining

Chloroplasts are essential organelles in plant cells with many important functions. Chloroplasts isolated by Percoll density gradient centrifugation are widely used in the study of chloroplasts. The intactness of isolated chloroplasts is necessary for many of the experiments. In the past, those isolated chloroplasts were either simply believed to be intact or had to be analyzed by indirect biochemical methods. Here we show a new method to check the intactness of isolated chloroplasts by staining their envelope with fluorescent dyes, Rhodamine or Nile red, and then observing them with a fluorescence microscope. With this method, broken chloroplasts and intact chloroplasts can be distinguished easily and their integrity can be checked in a few minutes. Results of this method agreed well with those of biochemical methods. Moreover, we have also found that sometimes the middle layer chloroplasts from the Percoll gradient centrifugation could be mostly broken, which could cause mistakes in the experiment. With our method, this problem can be easily found. This chloroplast envelope staining method can be used in the preparation of isolated chloroplasts to ensure the intactness.

Differential proteins associated with plasma membrane in X- and/or Y-chromosome bearing spermatozoa in indicus cattle.

The differential proteins associated with plasma membrane of spermatozoa are less known, identification of which shall help overcome limitations of currently used methods of sperm sexing, considered as a high priority for livestock sector of many countries. This study has reported plasma membrane proteomics of unsorted spermatozoa and differential expression of plasma membrane-associated proteins between X- and Y-chromosome bearing spermatozoa of indicus cattle (Bos indicus). Isolation of plasma membrane fraction using percoll gradient, relatively a rapid method, from bovine spermatozoa has been reported to enrich isolation of plasma membrane proteins. Significant enrichment for plasma membrane-associated proteins was observed in plasma membrane fraction (p<.05) as compared to the total cell lysate using LC-MS/MS. Furthermore, these experiments were conducted in flow cytometry sorted, sexed-semen samples. Thirteen proteins were identified as differentially abundant between X- and Y-sorted spermatozoa. Among these, two proteins were downregulated in Y-sorted spermatozoa compared to the X-sorted spermatozoa (p<.05), while four and seven proteins could be noted in X- and Y-sorted spermatozoa, respectively. Proteins that are presumed to support sperm capacitation and sperm migration velocity were found to be abundant in Y-sorted spermatozoa while those associated with structural molecule activity were identified as abundant in X-sorted spermatozoa in the present study. Our study provides better insight into the plasma membrane proteomics of spermatozoa of indicus cattle and furnishes data that might aid in design and development of alternate and open technology for sex-sorting of semen.

Sperm Nuclei Analysis and Nuclear Organization of a Fertile Boar-Pig Hybrid by 2D FISH on Both Total and Motile Sperm Fractions.

Simple SummaryMammalian hybrids frequently form in nature between closely related species, and the majority are sterile due to the production of chromosomally unbalanced gametes. In Italy, the widespread wild boar has had negative consequences for free-range pig farming, which is considered the best practice for pig welfare and is a common method of farming most autochthonous pig breeds. This study aimed to analyze the sperm DNA integrity, sperm meiotic segregation and nuclear spatial organization in a boar–pig hybrid, for the first time, to evaluate its fertilizing capacity. The results show that the hybrid presented a high frequency (64%) of motile spermatozoa with a regular chromosome composition and a specific spatial distribution. This study underlines how fertile boar–pig hybrids represent a growing problem for conserving autochthonous pig breeds.A wide range of mammalian hybrids has recently been found by chance or through population-screening programs, but studies about their fertilizing capacity remain scarce and incomplete. Most of them are assumed to be sterile due to meiotic arrest caused by the failure of chromosome pairings. In this study, we evaluated both sperm meiotic segregation, by 2D fluorescent in situ hybridization (FISH) analysis, and sperm quality (Sperm Chromatin Structure Assay) by flow cytometer in a fertile boar–pig hybrid (2n = 37,XY) originating from a Nero Siciliano pig breed (Sus scrofa domesticus) and a wild boar (Sus scrofa ferus). Spermatozoa were also separated by a dual-layer (75–60%) discontinuous Percoll gradient, resulting in two fractions with a significantly better overall quality in the motile sperm fraction. These data were confirmed by FISH analysis also, where the frequencies of spermatozoa with a regular chromosome composition were 27% in total sperm fraction and 64% in motile sperm fraction. We also evaluated the nuclear architecture in all counted spermatozoa, showing a chromatin distribution changing when chromosome abnormalities occur. Our results demonstrate that the chromosome pairing has a minimal effect on the sperm segregation and semen quality of a boar–pig hybrid, making it fertile and harmful for the conservation of autochthonous pig breeds.

Safety and feasibility of apheresis to harvest and concentrate parasites from subjects with induced blood stage Plasmodium vivax infection

BackgroundIn the absence of a method to culture Plasmodium vivax, the only way to source parasites is ex vivo. This hampers many aspects of P. vivax research. This study aimed to assess the safety of apheresis, a method for selective removal of specific components of blood as a means of extracting and concentrating P. vivax parasites.MethodsAn iterative approach was employed across four non-immune healthy human subjects in single subject cohorts. All four subjects were inoculated with ~ 564 blood stage P. vivax (HMP013-Pv) and subjected to apheresis 10 to 11 days later. Blood samples collected during apheresis (haematocrit layers 0.5% to 11%) were tested for the presence and concentration of P. vivax by microscopy, flow cytometry, 18S rDNA qPCR for total parasites, and pvs25 qRT-PCR for female gametocyte transcripts. Safety was determined by monitoring adverse events. Malaria transmission to mosquitoes was assessed by membrane feeding assays.ResultsThere were no serious adverse events and no significant safety concerns. Apheresis concentrated asexual parasites by up to 4.9-fold (range: 0.9–4.9-fold) and gametocytes by up to 1.45-fold (range: 0.38–1.45-fold) compared to pre-apheresis densities. No single haematocrit layer contained > 40% of all the recovered P. vivax asexual parasites. Ex vivo concentration of parasites by Percoll gradient centrifugation of whole blood achieved greater concentration of gametocytes than apheresis. Mosquito transmission was enhanced by up to fivefold in a single apheresis sample compared to pre-apheresis.ConclusionThe modest level of parasite concentration suggests that the use of apheresis may not be an ideal method for harvesting P. vivax.Trial Registration Australia New Zealand Clinical Trials Registry (ANZCTR) Trial ID: ACTRN12617001502325 registered on 19th October 2017. https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=373812.

68 Comparison of two Percoll gradients for selection of frozen semen for invitro production of ovine embryos

Sperm selection methods are routinely applied to prepare semen for IVF in animal species. These procedures are used to improve sperm quality characteristics as well as to remove other background material and debris. Percoll gradient is widely used in animal IVF laboratories. Different Percoll gradient concentrations and volumes can be used to improve the sperm sample motility percentage. The objective of this research was to evaluate the effect of 2 different Percoll concentrations for ovine IVF sperm selection and effects, if any, on invitro embryo production (IVP). Specifically, Percoll gradients consisted of Group 1 (G1) 40–80% and Group 2 (G2) 45–90%, 400µL each. The research was carried out in the reproduction laboratory at Palominos Ranch (Jalisco, México). The IVP was performed with a continuous invitro culture system. Ovaries (n=157) were collected from a slaughterhouse (León, México) and transported to the laboratory within 2h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IU mL−1) and streptomycin sulphate (100µg mL−1). For IVP, IVF Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For IVM, the cumulus–oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n=800, divided equally into 4 replicates) were divided into 2 groups, G1 and G2. The IVF process was conducted with semen selected through a mini-Percoll technique with gradients at a concentration of 45% (top layer) and 90% (bottom layer) or 40% (top layer) and 80% (bottom layer) for G1 and G2, respectively, using frozen–thawed semen from the same ram, at a concentration of 2×106 sperm mL−1, for 18h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The percentages of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated, based on the initial number of oocytes entering into IVM. The statistical analyses were carried out with the GLM procedure of SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of G1 versus G2 (α level=0.05). Cleavage rate was 47.8%±2.5 and 55.9%±2.5, respectively, in G1 and G2. The percentages of embryos with more than 6 cells were 38.1%±2.2 and 43.5%±2.2, respectively, in G1 and G2. The percentage of blastocysts on Day 7 was 27.4%±1.1 and 37.3%±1.1, respectively, in G1 and G2. There were no significant differences between groups (P&amp;gt;0.05) for percentage of cleavage and embryos with more than 6 cells, although the percentage of cleavage tended to be greater for G2 (P=0.06). Additionally, G2 had a larger percentage of blastocysts on Day 7 compared with G1 (P&amp;lt;0.05). In conclusion, under the conditions of this research, the use of a Percoll gradient at a concentration of 40–80% increased the percentage of blastocysts for ovine IVP.

99 Increased expression of microRNAs in sperm of Nelore Bulls with high invitro fertility

The spermatozoon is no longer known only as a cell that delivers the male genetic material to the oocyte, because it also provides molecules such as microRNAs (miRNAs) that play a significant role in fertilization and embryonic development. MiRNAs are small noncoding RNAs capable of modulating mRNA translation, thus affecting important biological processes. Sperm miRNAs may influence embryo development and therefore, might be related to invitro production of embryos (IVP), considering that individual bulls have different fertility rates when used for IVF. The aim of this work was to identify miRNAs expressed in semen of bulls with high and low IVP rates. The composition of groups was based on a retrospective database from a reproductive biotechnology company between the years of 2016 and 2018, generating around 7000 IVP manipulations of 430 Nelore bulls. We only considered IVP manipulations that used a minimum of 30 oocytes and conventional semen selection by Percoll gradient. A total of 87 Nelore bulls fit these criteria. We then ranked bulls based on cleavage rate (number of cleaved structures/number of oocytes), blastocyst rate (number of blastocysts/number of oocytes) and embryo development rate (number of blastocysts/number of cleaved structures). Considering these three rates, we allocated bulls to two groups. The top eight were considered to have high IVP fertility (HF) and the bottom eight were grouped together as low IVP fertility (LF). We performed the T TEST procedure (SAS 9.3 software; SAS Institute Inc.) to compare the groups for cleavage (P&amp;lt;0.0001), blastocyst (P=0.0006), and embryo development (P=0.0001) rates. For miRNA analysis, sperm were separated using Percoll gradient and subjected to RNA extraction and cDNA synthesis. First, quantitative real-time PCR was used to evaluate the abundance of 380 bovine-specific miRNAs in a pool of samples for each group, using QuantStudio 6Flex. Then, 48 miRNAs presenting at least a 3-fold change of normalized cycle threshold values between groups were selected: 23 highly detected in HF, 1 highly detected in LF, 18 exclusively detected in HF, and 2 exclusively detected in LF. Last, we evaluated the abundance of these selected miRNAs in each experimental unit. We identified four miRNAs highly abundant in sperm from HF bulls, bta-miR-10a, bta-miR-383, bta-miR-93, and bta-miR-449b. Our results suggest that these miRNAs could play important roles in bovine embryo development. This work was supported by FAPESP (grant# 2018/03871-6).