Mengelola Batas dalam Konseling: Standar Menerima Hadiah

The purpose of this study was to describe the explanation of gifts in counseling based on ethical and cultural considerations. The method in this research is a literature study, namely by collecting reading material from various scientific articles and then comparing theories and research results, which are then analyzed descriptively. Based on the results of a literature study conducted that counselors can receive gifts from counselees by considering aspects of culture, time of giving, and types of gifts given.

SLOW GROWTH IN VITRO CULTURE FOR CONSERVATION OF HANCORNIA SPECIOSA GOMES

Hancornia speciosa Gomes is a fruit species endemic to the Cerrado and coastal plains of Northeast of Brazil, with great economic, nutritional, ecological, and medicinal potential. This study aimed to evaluate the effect of sorbitol and sucrose as osmotic regulators on the in vitro growth of mangabeira, aiming at conservation by slow growth. The explants were obtained from in vitro germinated seedlings and inoculated in MS medium supplemented with sucrose (15 and 30 g L-1) and sorbitol (0, 5, 10 and 15 g L-1). The experimental design was completely randomized with 20 repetitions in a 4 x 2 factorial arrangement (sorbitol x sucrose concentrations). The evaluations were performed at 30, 60 90 and 120 days of incubation. The analyzed variables were number of nodes/budding, number of leaves, leaf abscission, leaf color and survival of explants. The data were statistically analyzed by generalized linear model analysis. The results indicated a significant difference between the osmotic regulators and the culture time for all variables. Sorbitol showed a more pronounced growth-reducing effect than sucrose. The use of 30 g L-1 sucrose combined with 10 or 20 g L-1 sorbitol reduced the growth in a critical way, making it clear that the water stress caused was not tolerated by the plants, negatively interfering in its development. Treatment with 15 g L-1 sucrose combined with 5 g L-1 sorbitol promoted the best result, allowing the conservation of plants for 120 days.

Cartilage Regeneration Characteristics of Human and Goat Auricular Chondrocytes

Although cartilage regeneration technology has achieved clinical breakthroughs, whether auricular chondrocytes (AUCs) represent optimal seed cells to achieve stable cartilage regeneration is not clear. In this study, we systematically explore biological behaviors of human- and goat-derived AUCs during in vitro expansion as well as cartilage regeneration in vitro and in vivo. To eliminate material interference, a cell sheet model was used to evaluate the feasibility of dedifferentiated AUCs to re-differentiate and regenerate cartilage in vitro and in vivo. We found that the dedifferentiated AUCs could re-differentiate and regenerate cartilage sheets under the chondrogenic medium system, and the generated chondrocyte sheets gradually matured with increased in vitro culture time (2, 4, and 8 weeks). After the implantation of cartilage sheets with different in vitro culture times in nude mice, optimal neocartilage was formed in the group with 2 weeks in vitro cultivation. After in vivo implantation, ossification only occurred in the group with goat-regenerated cartilage sheet of 8 weeks in vitro cultivation. These results, which were confirmed in human and goat AUCs, suggest that AUCs are ideal seed cells for the clinical translation of cartilage regeneration under the appropriate culture system and culture condition.

Blood culture time to positivity in non-β-hemolytic streptococcal bacteremia as a predictor of infective endocarditis—a retrospective cohort study

Non-β-hemolytic streptococci (NBHS) cause infective endocarditis (IE) and a short blood culture time to positivity (TTP) is associated with risk of IE in bacteremia with other pathogens. In this retrospective population-based cohort study, we investigate if TTP is associated to IE or mortality. Of 263 episodes with NBHS bacteremia, 28 represented IE and the median TTP did not differ significantly between episodes with IE (15 h) and non-IE (15 h) (p=0.51). TTP was similar among those who survived and those who died within 30 days. However, TTP significantly differed when comparing the different streptococcal groups (p<0.001).

Teaching Theory and Space: Human Territoriality in Political Science

This article describes a technique that helps students to understand how a theory about human characteristics may impact behavior. I use a mini-simulation in which two volunteers engage in small talk, I point out that they engage with one another at a certain distance and angle that reflects social space. As the exercise progresses, students easily relate to the theory of human territoriality, which is defined as the symbolic and physical connection to a space considered as their own. This mini-simulation achieves the following learning objectives: understanding (1) that theories are relevant and help to explain human behavior; (2) the workings of the individual level of analysis; and (3) that theories are not universal and have limits to their application across culture, time, and space. This teaching technique does not require preparation time or resources, and students easily comprehend the expected learning outcomes.

P05.01 Organoid-specific optimization of killing assays to test novel immunotherapies in a high-throughput system

BackgroundThe immunotherapeutic drug dinutuximab, which binds to disialoganglioside (GD2) and activates natural killer (NK) cells, is part of the standard regimen in high-risk neuroblastoma (NB) patients. However, dinutuximab only results in tumor reduction in a subset of patients, and survival rates of high-risk neuroblastoma patients are below 60%. Novel immunotherapies are therefore needed. Current in vitro models lack the ability to study novel immunotherapies with high-throughput screening (HTS). We aimed to optimize NB organoid-lymphocyte cocultures for HTS, and possibly personalized testing, of novel antibody-mediated and cellular immunotherapies.Materials and MethodsTwo patient-derived organoids (691B: GD2+MHC-I- and 691T: GD2-MHC-I+) were transduced with an endogenous luciferase construct to use D-luciferin-induced bioluminescence as readout for cell growth. The growth rate, optimal seeding density and optimal pre-culture time per organoid were determined by density curves, and the number of needed cells was downscaled to facilitate HTS. After pre-culture, the luciferase-transduced organoids were co-cultured with primary PBMCs from healthy donors, PRAME-TCR transduced T cells or CAR-T cells.1 Several effector:target (E:T) ratios and timepoints were tested to identify the optimal window for read-out of dinutuximab-induced antibody-dependent cytotoxicity (ADCC) and T-cell mediated cytotoxicity. The required number of immune cells per ratio was calculated based on the expansion rate of organoid cells after 48 and 72 hours.ResultsThe density screens showed an optimal seeding density of 5000-10.000 organoid cells per well, yielding a high luminescence signal while minimizing the number of cells needed. Already at the lowest E:T ratio (1:3), we observed killing of the MHC-I expressing 691T organoid, likely based on allogeneic recognition of the organoids by T cells. The killing efficacy increased with higher E:T ratios and co-culture time. Pre-culturing of organoids for 72 hours before addition of effector cells resulted in formation of larger 3D spheres, which reduced the killing efficacy for all E:T ratios. ADCC effects of dinutuximab were studied in GD2+MHC-I- 691B organoids. Addition of dinutuximab resulted in 25% increase of killing after 24 hours and reached up to 70% increase after 72 hours for 10:1 and 20:1 E:T ratios. Higher E:T ratios were likely needed because NK cells make up a smaller proportion of PBMCs than T cells. Dinutuximab did not increase killing of the GD2- organoid, confirming specificity of the antibody. T cell mediated killing was almost 100% for MHC-I+691T organoids after 24 hours of culturing with PRAME-TCR transduced T cells and CAR-T cells at a 1:3 E:T ratio, showing the high anti-tumor cytotoxicity of these cells and potential for HTS at very low E:T ratios.ConclusionsWe have developed a robust in vitro bioluminescence-based organoid/lymphocyte co-culture assay with a low cell input, to facilitate high-throughput screening of novel antibody-based or cellular immunotherapies, possibly combined with chemotherapeutic or targeted compounds. In the future this method may be applied for personalized drug screens.ReferenceAvital L Amir, et al. PRAME-Specific Allo-HLA-restricted T cells with potent antitumor reactivity useful for therapeutic T-cell receptor gene transfer. Clin Cancer Res 2011.Disclosure InformationF. van den Ham: None. W.M. Kholosy: None. K. Ober: None. A.M. Cornel: None. S. Nierkens: None. J. Anderson: None. J.J. Molenaar: None. J. Wienke: None.

Associative effects in diets composed of alfalfa and corn soybean concentrate fed to growing cashmere goats

The associative effects resulting from the proportions of neutral detergent fibre (NDF) and non fibre carbohydrate (NFC) were explored and assessed by in vitro gas production . Total mixed rations (TMR) composed primarily of alf a lfa and corn soybean concentrate were fed to growing cashmere goats. Treatments were defined by three proportions of NFC and NDF namely 2.00 ( TMR1), 2.35 ( TMR2), and 3.00 ( that were used to grow cashmere goats , and these TRMs were incubated for 48 h ours to evaluate their influence on associative effects. The results indicated that the associative influences of these treatments on gas production occurred within the cultures predominantly at 2 8 hours, and disappeared gradually as culture time was ex tended . TMR 2 and TMR3 incubation increased gas production compared with that observed in the other groups at all incubation times P > 0.05), and these groups exhibited positive associative effects, particularly during the early hours of incubation P <0.05 )). TMR 3 displayed the best associative effect.

P–582 High level of concordance between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) at day5 and day6–7

Study question To explore ploidy concordance between invasive and non-invasive PGTA (niPGT-A) at different embryo culture time. Summary answer High level (&gt;84%) of concordance rate for ploidy and sex, sensitivity (&gt;88%), and specificity (76%) were obtained for both day6/7 samples and day5 samples. What is known already The analysis of embryo cell free DNA (cfDNA) that are released into culture media during in vitro embryo development has the potential to evaluate embryo ploidy status. However, obtaining sufficient quality and quantity of cfDNA is essential to achieve interpretable results for niPGT-A. More culture time is expected to be directly proportional to the release of more cfDNA. But embryo culture time is limited due to in-vitro embryo survival potential. Therefore, it is important to estimate the duration of the culture that will provide the maximum cfDNA that can be obtained without adversely affecting the development of the embryo. Study design, size, duration A total of 105 spent culture media (SCM) from day5-day7 blastocyst stage embryos have been included in this cohort study. The cfDNA of SCM samples were amplified and analyzed for niPGT-A by NGS analysis. The SCM samples were divided into 2 subgroups according the embryo culture hours (Day5 and Day6/7 group). The DNA concentration, informativity and euploidy results have then been compared with their corresponding embryos after trophectoderm biopsy (TE) and PGT-A analysis by NGS Participants/materials, setting, methods Embryos cultured until Day3 washed and cultured again in 20µl fresh culture media until embryo biopsy on Day5, 6, or 7. After biopsy SCM samples were immediately collected in PCR tubes and conserved at –20 °C until whole genome amplification by MALBAC® (Yicon Genomics). The TE and SCM samples were analyzed by next-generation sequencing (NGS) using Illumina MiSeq® System. NGS data analysis has been done by Bluefuse Multi Software 4.5 (Illumina) for SCM and TE samples Main results and the role of chance Only the SCM samples which have an embryo with a conclusive result were included in this cohort (n = 105). Overall 97.1% (102/105) of SCM samples gave a successful DNA amplification with a concentration ranging 32.4–128.5ng/µl. Non-informative (NI) results including a chaotic profile (&gt;5 chromosome aneuploidies) were observed in 17 samples, so 83.3%(85/102) of SCM samples were informative for NGS data analysis. Ploidy concordance rate with the corresponding TE biopsies (euploid vs euploid, aneuploid vs aneuploid) was 84.7% (72/85). Sensitivity and specificity were 92,8% and 76,7%, respectively with no significant difference for all parameters for day 6/7 samples compared with day 5 samples. The false-negative rate was 3.5% (3/85), and false-positive rate was 11.7% (10/85). Limitations, reasons for caution The sample size is relatively small. Larger prospective studies are needed. As this is a single-center study, the impact of the variations in embryo culture conditions can be underestimated. Maternal DNA contamination risk cannot be revealed in SCM, therefore the use of molecular markers would increase the reliability. Wider implications of the findings: Non-invasive analysis of embryo cfDNA analyzed in spent culture media demonstrates high concordance with TE biopsy results in both early and late culture time. A non-invasive approach for aneuploidy screening offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population. Trial registration number Not applicable

Time- and Stimulus-Dependent Characteristics of Innate Immune Cells in Organ-Cultured Human Corneal Tissue

<b><i>Purpose:</i></b> The pattern of immune cells infiltrating the corneal stroma has been extensively studied in mice, but data on human tissue have been far less elaborate. To further characterize the number and differentiation state of resident immune cells in organ-cultured human corneal tissue, we employed a comprehensive bioinformatic deconvolution (xCell) of bulk RNA-sequencing (RNA-seq) data, immunohistochemistry (IHC), and flow cytometry (FC). <b><i>Methods:</i></b> A transcriptome-based analysis of immune cell types in human corneal samples was performed. The results were validated by IHC, focusing on the identification of pro-inflammatory (M1) and regulatory (M2) macrophages. A protocol was established to identify these 2 different macrophage populations in human corneal tissue by means of FC. Subsequently, corneal samples in organ culture were differentially stimulated by IL-10, IL-4 &amp; IL-13, or LPS and macrophage populations were evaluated regarding their response to these stimuli. Furthermore, cell survival was analyzed in correlation with time in organ culture. <b><i>Results:</i></b> xCell-based mathematical deconvolution of bulk RNA-seq data revealed the presence of CD8 T cells, Th17 cells, dendritic cells, and macrophages as the predominant immune cell types in organ-cultured human corneal tissue. Furthermore, RNA-seq allowed the detection of different macrophage marker genes in corneal samples, including <i>PTPRC</i> (CD45), <i>ITGAM</i> (CD11b), <i>CD14</i>, and <i>CD74</i>. Our RNA-seq data showed no evidence of a relevant presence of monocytes in human corneal tissue. The presence of different macrophage subtypes was confirmed by IHC. The disintegration and subsequent FC analysis of human corneal samples showed the presence of both M1 (HLA-DR⁺, CD282⁺, CD86⁺, and CD284⁺) and M2 (CD163⁺ and CD206⁺) macrophage subtypes. Furthermore, we found that the total number of macrophages in corneal samples decreased more than the total cell count with increasing tissue culture time. Treatment with IL-10 led to higher total cell counts per cornea and to an increased expression of the M2 marker CD163 (<i>p</i> &#x3c; 0.05) while expression levels of various M1 macrophage markers were not significantly reduced by interleukin treatment. <b><i>Conclusions:</i></b> Regarding different macrophage populations, untreated human corneas showed more M1 than M2 macrophages. With increasing organ culture time, these macrophages decreased. In terms of cell dynamics, adding interleukins to the organ culture medium influenced the phenotype of macrophages within the cornea as detected by FC. Modifying the immunomodulatory properties of human grafts appears a promising approach to further reduce the risk of graft rejection in patients. In this context, treatment with interleukins was more effective in upregulating M2 macrophages than in suppressing M1 macrophages in corneal tissue.