Induction Protocol Research Articles

Optimization of in vitro micropropagation of ethnomedicinally important underexplored plant Maytenus emarginata

In vitro callus induction and shoot emergence protocols for Maytenus emarginata (Willd) Ding Hou. have been established using young leaves and stem explants of in vivo growing, one and half year-old plants. Plants were growing in the month of August 2016 at department of Botany, Ch. Charan Singh University, Meerut. However, explants were collected in the month of August, 2019. MS (Murashige and Skoog) medium and WPM (Woody Plant Medium), supplemented with different concentrations and combinations of plant growth regulators (PGRs) such as 0.5-2.0 mg/l Naphthalene acetic acid (NAA), 0.5-1.0 mg/l Benzyl Amino Purine (BAP), 2, 4-D (1.0-2.0 mg/l) and 1.0-1.5 mg/l Gibberellic acid (GA3) and temperature regimes were used for callus induction and shoot emergence. The presence of GA3 was found necessary for callus induction and shoot emergence from both explants (leaf and stem). The best response of callus formation from leaf explants was obtained at 24°C on WPM supplemented with 0.5 mg/l NAA+1.0mg/l BAP+1.5mg/l GA3 and MS medium supplemented with 2.0 mg/l NAA+ 0.5 mg/l BAP+ 1.0mg/l GA3. Stem explants exhibited best degree of callus induction only on MS medium supplemented with 0.5mg/l NAA+ 1.0 mg/l BAP+1.5 mg/l GA3. Light and temperature play very crucial role in shoot emergence because exposure of callus or nodal section of stem, to 12 hours dark period followed by 12 hours PAR (photosynthetically active radiation) at 27±2 °C induced shoot emergence on MS medium supplemented with 0.5mg/l NAA+1.0mg/l BAP+1.5 mg/l GA3 after 42 days. This is the first optimization report on tissue culture and medicinal use of callus of Maytenus emarginata.

UK protocol for induction of puberty with gonadotropins in males with hypogonadotropic hypogonadism

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The effect of hCG day progesterone in 1318 cycles on pregnancy outcomes: ongoing discussion.

To investigate the effect of human chorionic gonadotropin day progesterone (hCG-P) level on pregnancy outcomes in in vitro fertilization (IVF) cycles. This study is an analysis of a cohort of 1318 fresh IVF- embryo transfer cycles, including 579 agonists and 739 antagonists, performed at a single IVF center between 2007 and 2018. For fresh cycles, we performed Receiver Operating Characteristic analysis (ROC) to calculate the threshold value of hCG-P, which affects pregnancy outcomes. We divided patients below and above the determined threshold value into two groups, then, correlation analysis and we performed logistic regression analysis. According to ROC curve analysis of hCG-P,AUC was 0.537 (95% CI: 0.510-0.564, p < 0.05) for LBR, and the threshold value for P was 0.78. The hCG-P threshold value of 0.78 proved to be significant in relation to BMI, type of drug used during induction, the hCG day E2, the total number of oocytes, the number of oocytes and the subsequent pregnancy outcome between the two groups (p < 0.05). However, the model we built, which accounted for hCG-P, total number of oocytes, age, BMI, induction protocol, total dose of gonadotropin used in induction did not prove significant in terms of its effect on LBR. The threshold value of hCG-P that we found to have an effect on LBR was quite low compared with the P-value generally recommended in the literature. Therefore, further studies are needed to determine an accurate P-value that reduces success in managing fresh cycles.

Callus Production and Analysis of Isoflavonoids and Some Terpenes from Bituminaria bituminosa

Background: Bituminaria bituminosa is a long-day plant. The plant has a high pastoral value and palatability. It is grazed by cattle, goats and sheep. Leaves and stems contain four furanocoumarins’ psoralen, angelicin, xanthotoxin and bergapten. The seed is covered by the fruit. Germination was not observed in beaked fruits. A cut with a scalpel can increase the germination rate. According to the literature there are not enough studies about the culture media and convenient conditions. Methods: In our study, we tried six medium for callus production with different hormone concentrations including 2,4-D, BAP and kinetin. In this study, a protocol for callus induction and analysis of isoflavonoids and terpenes of Bituminaria bituminosa has been developed. Result: Maximum callus induction frequency was observed on Murashige and Skoog (MS) medium supplemented with 0.75 mg/L 2,4-D + 2.5 mg/L kinetin in young primary leaves. 0.75 mg/L 2,4-D+2.5 mg/L kinetin medium is more successful in the production ofses of flavonoids that can be produced in vitro and is more successful in producing phenols. In terpene production, it is seen that 1.25 mg/L 2,4-D+3.5 mg/L BAP, which is the best callus producing medium containing BAP, is the most successful medium among all tested mediums.

Childbirth experience in induced labor: A prospective study using a validated childbirth experience questionnaire (CEQ) with a focus on the first birth.

ObjectiveFirst birth and labor induction are risk factors for negative childbirth experiences. As labor inductions are increasing, research into this high-risk group’s childbirth experiences is important. We aimed to investigate whether nulliparity or factors related to labor induction, labor, and delivery explain the association.MethodsThis was a prospective study of 711 women undergoing labor induction at Helsinki University Hospital, Finland, between January 1, 2019, and January 31, 2020. The participants answered the Childbirth Experience Questionnaire (CEQ) after delivery (response rate 69.4%). The patient characteristics and delivery outcomes were collected from patient records. We analyzed the results for nulliparous and parous women.ResultsThe mean CEQ scores were 2.9 (SD 0.5) for nulliparous women (n = 408) and 3.2 (SD 0.5) for parous women (n = 303), on a scale of 1–4; higher scores represent more positive experiences. However, 7.3% of the women had negative childbirth experiences (8.8% nulliparous; 5.3% parous, p = 0.08). Negative experiences were associated with a cesarean section (OR 6.7, 95% CI 1.8–9.3, p < 0.001) and a hemorrhage ≥ 1500 ml in vaginal delivery (OR 2.8, 95% CI 1.1–7.5, p = 0.03). In the separate CEQ domains analyses, nulliparity was associated with negative experiences in the “Own Capacity” domain (OR 1.6, 95% CI 1.0–2.4, p = 0.03). Cervical ripening, oxytocin use, and daytime delivery were associated with negative experiences in at least one domain, whereas epidural or spinal analgesia was regarded positively in two domains and negatively in one.ConclusionsNulliparous women undergoing labor induction risk negative childbirth experiences mainly due to labor and delivery-related factors, similar to parous women. Their perceptions of their capacity and preparedness for labor and delivery should be enhanced antenatally. An effective labor induction protocol promoting as high a rate of vaginal delivery as possible and preparedness to promptly respond to postpartum hemorrhage are key for avoiding negative childbirth experiences.

Predictive value of neutrophil-to-lymphocyte, lymphocyte-to-monocyte, and platelet-to-lymphocyte ratios in adult and pediatric acute lymphoblastic leukemia patients

Background Methods used for prognostication of acute lymphoblastic leukemia (ALL) are expensive; discovering low-cost prognostic factors is challenging. Objectives This study aimed to explore the prognostic role of baseline neutrophil-to-lymphocyte (NLR), lymphocyte-to-monocyte (LMR), and platelet-to-lymphocyte (PLR) ratios in predicting the response to end of induction chemotherapy in ALL patients. Patients and methods We included 44 adult patients and 47 pediatric patients who were newly diagnosed with ALL. All participants were subjected to a full history taking and a thorough medical examination. Laboratory investigations included complete blood count (CBC) with differential count analysis, with calculation of NLR, LMR, and PLR; bone marrow examination; conventional cytogenetic analysis; and immunophenotyping. Patients were followed until the end of the induction phase, and their response to treatment was assessed. Results Among the adult patients, 63.6% showed complete remission at the end of induction; their baseline CBC showed significantly lower NLR (P=0.001) and higher LMR (P=0.013). On the other hand, 66% of the pediatric patients showed good response to induction chemotherapy; their baseline CBC showed significantly lower NLR (P&lt;0.001), greater LMR (P=0.0134), and lower PLR (P=0.017). NLR more than or equal to 1, LMR less than or equal to 2.846, and PLR more than or equal to 39.1 were able to discriminate adult patients who will respond to induction chemotherapy, similarly NLR more than or equal to 1, LMR less than or equal to 3.286, and PLR more than or equal to 10 among pediatric patients. Conclusion Our research discovered that the rise in NLR and PLR, together with the decline of LMR at ALL diagnosis, could predict future resistance to the routinely used induction protocols, and the need for intensification regimens.

ALL-312 Ig/T-Cell Receptor Clonality Testing as a Method for Minimal Residual Disease Monitoring in T-Cell Acute Lymphoblastic Leukemia Patients

Assessment of early response to therapy by minimal residual disease (MRD) monitoring in T-cell acute lymphoblastic leukemia (T-ALL) has been proven to be a fundamental tool for guiding further therapy. Polymerase chain reaction (PCR)-based immunoglobulin (Ig)-T-cell receptor (TCR) clonality testing is a standardized technique for routine diagnosis of lymphoproliferation. It is also widely applied for MRD detection in T-ALL and is most suitable for patients without additional rearrangements detected. To present the clinical significance and utility of a molecular-based approach for post-induction MRD detection in patients with T-ALL. Single-center, descriptive, retrospective study including 11 adult patients with T-ALL treated at the University Clinic for Hematology - Skopje from January 2018 to January 2022. TCR gene analyses were performed in 11 adults with T-ALL. TCR rearrangements in bone marrow samples were analyzed by multiplex fluorescent PCR amplifications of the TCRB-Vβ-Jβ, Dβ-Jβ, TCRG-Vγ-Jy, TCRD-Vδ-Dδ-Jδ, Dδ-Dδ, Vδ-Dδ, and Dδ-Jδ regions and subsequent capillary electrophoresis of PCR products by automatic DNA analyzer, following EuroClonality/BIOMED-2 guidelines. MRD status was assessed at the end of the induction cycle. Patients received a BFM-based protocol or Hyper-CVAD/high-dose methotrexate (HDM) and cytarabine (ARA-C) regimen. TCR rearrangements were detected in all 11 patients. All patients were negative for additional molecular prognostic markers. TCRB and TCRG gene rearrangements were slightly dominant over TCRD (64.3% vs. 50%). A total of 6 adolescents and young adults (mean age: 24 years) were treated with BFM. At the end of the induction regimen, MRD negativity was obtained in 5 patients (83.3%) and was sustained up to 3 months from stem cell transplantation (SCT) in 3 patients. The other 2 patients died from CNS infiltration. Hyper-CVAD/HDM-ARA-C was administered in 5 patients (mean age: 49 years). One patient died during the induction protocol due to intracerebral hemorrhage. Among the others, CR at the end of the first cycle was achieved in 3 patients, and a total of 4 cycles were applied. Two patients proceeded to SCT. The molecular technique for testing Ig/TCR clonality is a useful tool for post-induction MRD monitoring in patients with ALL, directing the further approach and clinical decisions.

A pilot study assessing effectiveness of a written shame induction protocol with and without a social evaluative threat manipulation.

A pilot study assessing effectiveness of a written shame induction protocol with and without a social evaluative threat manipulation.

Oral tolerance induction protocol to egg white – A report of three pediatric cases

Food allergies are the main cause of anaphylaxis in children. Cow’s milk and egg white are the most common agents and elimination diets are considered the current strategy for their management. Many children can tolerate increasing amounts of the known allergen, and thus, oral tolerance induction protocols seem to be a promising approach. The authors present a protocol drawn up in a pediatric allergic diseases’ outpatient clinic, at a level II hospital, with the aim of egg white oral tolerance induction. During the protocol there is an increase in the amount of egg white offered to children, at the end, tolerance is tested with the ingestion of an entire egg. The protocol was performed in three children with anaphylaxis to egg white. Two children completed the protocol successfully. Oral tolerance to egg white allows a less restrictive diet and improves the quality of life of the patients and their caregivers.

Co-Treatment of Erythroid Cells from β-Thalassemia Patients with CRISPR-Cas9-Based β039-Globin Gene Editing and Induction of Fetal Hemoglobin.

Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic hereditary diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9-based gene editing can be employed for the efficient correction of the β039-thalassemia mutation. On the other hand, robust evidence demonstrates that the increased production of fetal hemoglobin (HbF) can be beneficial for patients with β-thalassemia. The aim of our study was to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols. The gene editing of the β039-globin mutation was obtained using a CRISPR-Cas9-based experimental strategy; the correction of the gene sequence and the transcription of the corrected gene were analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; the relative content of HbA and HbF was studied by high-performance liquid chromatography (HPLC) and Western blotting. For HbF induction, the repurposed drug rapamycin was used. The data obtained conclusively demonstrate that the maximal production of HbA and HbF is obtained in GE-corrected, rapamycin-induced erythroid progenitors isolated from β039-thalassemia patients. In conclusion, GE and HbF induction might be used in combination in order to achieve the de novo production of HbA together with an increase in induced HbF.

Injectable progesterone for estrus and ovulation induction in seasonal anestrous ewes

We evaluated, for the first time, the effects of long-acting injectable progesterone (iP4) for estrus induction in ewes during non-breeding season. To evaluate the serum progesterone (P4) curve induced by the application of iP4 in sheep and the effect of its use prior to the cyclicity induction protocol, for Experiment 1, 39 ewes were allocated into three groups: no-treatment, control (n = 14); 37.5 mg of iP4 im (iP4 37.5, n = 13), and 75 mg of iP4 im (iP4 75, n = 12). P4 dosage in a subset of animals (5 per group) showed the highest P4 level at 48 h and reduction to basal levels 10 days after injection, when all females received an intravaginal device (IVD) containing 60 mg of medroxyprogesterone acetate (MPA) that remained for 11 days. At withdrawal the animals received 400 IU of eCG im, and rams remained for three days with ewes. On days 6 and 12 after IVD removal, blood samples were collected from a subset (n = 5 per group) of ewes that manifested estrous. iP4 75 group had lower luteal P4 concentration, and lower conception rate (35 days after mating) when compared to the other groups. To evaluate the possibility of replacing IVD with iP4, on experiment 2, 36 ewes were allocated into two treatments: control (n = 10), IVD for nine days and 300 IU of eCG at withdrawal; and iP4 75 (n = 26) 75 mg of iP4 im and, after 9 (n = 13) or 10 (n = 13) days, animals were injected with 300 IU of eCG. Estrus manifestation and conception rates were lower in groups treated with iP4. Eleven days after estrus, 93.3% of a subset of animals (5 from control and 10 from iP4 75) exhibited P4 concentrations above 1 ng/mL, indicating ovulations. In conclusion, the injection of 75 mg of long acting iP4 maintained levels above 1 ng/mL for at least 7 days, and negatively affected P4 luteal concentration and conception rate after a subsequent cyclicity induction protocol. Treatment with 75 mg of iP4 associated to eCG is suitable for inducing ovulations, although inhibits estrus manifestation.

Preliminary Results on the Preinduction Cervix Status by Shear Wave Elastography

The mechanical status of the cervix is a key physiological element during pregnancy. By considering a successful induction when the active phase of labor is achieved, mapping the mechanical properties of the cervix could have predictive potential for the management of induction protocols. In this sense, we performed a preliminary assessment of the diagnostic value of using shear wave elastography before labor induction in 54 women, considering the pregnancy outcome and Cesarean indications. Three anatomical cervix regions and standard methods, such as cervical length and Bishop score, were compared. To study the discriminatory power of each diagnostic method, a receiver operating characteristic curve was generated. Differences were observed using the external os region and cervical length in the failure to enter the active phase group compared to the vaginal delivery group (p &lt; 0.05). The area under the ROC curve resulted in 68.9%, 65.2% and 67.2% for external os, internal os and cervix box using elastography, respectively, compared to 69.5% for cervical length and 62.2% for Bishop score. External os elastography values have shown promise in predicting induction success. This a priori information could be used to prepare a study with a larger sample size, which would reduce the effect of any bias selection and increase the predictive power of elastography compared to other classical techniques.

Anesthetic effects of alfaxalone-ketamine-midazolam and alfaxalone-ketamine-dexmedetomidine administered intramuscularly in black-tailed prairie dogs (Cynomys ludovicianus).

To evaluate and compare the anesthetic effects of alfaxalone-ketamine-midazolam (AKM) and alfaxalone-ketamine-dexmedetomidine (AKD) in black-tailed prairie dogs (Cynomys ludovicianus). 9 male black-tailed prairie dogs. Prairie dogs were anesthetized with AKM (6 mg/kg alfaxalone, 30 mg/kg ketamine, and 1.5 mg/kg midazolam) and AKD (6 mg/kg alfaxalone, 30 mg/kg ketamine, and 0.15 mg/kg dexmedetomidine) in a prospective, complete cross-over study. Atipamezole (1.5 mg/kg) after AKD or flumazenil (0.1mg/kg) after AKM was administered 45 minutes after induction of anesthesia. Onset of general anesthesia, physiologic parameters, depth of anesthesia, and time to recovery after reversal administration were evaluated for each treatment. Both AKM and AKD produced a deep plane of anesthesia in black-tailed prairie dogs that varied in duration. The median induction times for AKM and AKD were 82 and 60 seconds, respectively. The median recovery times for AKM and AKD were 27 and 21 minutes, respectively. There were no significant differences between protocols for induction (P = .37) and recovery (P = .51) times. All measured reflexes were absent in all animals at 5 minutes postinduction, with hindlimb reflexes returning prior to forelimb reflexes. Heart rate was lower but respiratory rate was higher in the AKD treatment. Body temperature decreased significantly for both protocols (P < .001) and was significantly lower with AKM than AKD (P < .001). Both AKM and AKD produced a deep plane of anesthesia in black-tailed prairie dogs. For both protocols, heat support and oxygen support are indicated.

The Acute Effect Of A Laboratory Shame Induction Protocol On Endothelial Function

Shame is a self-conscious emotion that arises when one internalizes social devaluations from others. Increased shame in a laboratory setting is associated with increased cortisol and tumor necrosis factor alpha activity, however, no studies have investigated the acute effects of shame on important indices of cardiovascular disease risk such as endothelial function. The endothelium is a single layer of cells which lines the inside of all arteries and acts as a vasoprotective and vasoactive agent by inhibiting the development of atherosclerosis and adjusting vessel diameter in response to changes in blood flow associated shear stress (flow-mediated dilation). PURPOSE: The objective of this study was to examine the impact of a shame induction protocol on endothelial function. METHODS: Fifteen, young (23 ± 2 years), healthy participants (n = 7 men, n = 8 women) completed both a 20-min written shame induction and control protocol on two different experimental days. Pre- and post-protocol we assessed: 1) Endothelial function via a standard brachial artery reactive hyperemia flow-mediated dilation (RH-FMD) test which was characterized as the peak % and absolute change in diameter following release of 5 min of forearm occlusion, and; 2) Perceived shame via the Experiential Shame Scale (ESS). RESULTS: Shame increased in response to the shame induction protocol (pre: 2.9 ± 0.6 vs. post: 3.7 ± .5, p < 0.001) but not the control protocol (pre: 3.0 ± 0.5 vs. post: 2.8 ± 0.5, p = 0.15). %RH-FMD significantly decreased in response to the shame protocol (pre: 4.8 ± 1.9 vs. post: 3.2 ± 1.6, p < 0.001) but not the control protocol (4.2 ± 1.8 vs. post: 3.8 ± 1.5, p = 0.45). Covariation of the shear stress stimulus for RH-FMD did not alter the RH-FMD results. Increased shame was significantly associated with decreased absolute RH-FMD (r = -0.37, p = 0.046). CONCLUSIONS: The shame induction protocol caused a significant increase in shame and reduction in RH-FMD. This suggests that temporary increases in shame cause transient endothelial dysfunction which, if chronically repeated, could manifest as reduced vasoprotection against atherosclerosis. Supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grant.

Tetraploid induction through somatic embryogenesis in Panax vietnamensis Ha et Grushv. by colchicine treatment

• Somatic embryos regenerated from AgNPs-sterilized leaf explants of Ngoc Linh ginseng. • Tetraploid induction by colchicine in Panax vietnamensis Ha et Grushv. from somatic embryo. • Tetraploid plantlets had vigorous growth compared to the diploid plantlets. In the present study, the protocol of polyploid induction via somatic embryogenesis of the Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) (2n = 2x = 24) was developed. Ex vitro leaf explants were disinfected with AgNPs at concentrations of 0.05, 0.1, 0.2 and 0.5 g/L for 5, 10, 15, 20 and 30 min and cultured on MS medium supplemented with 1.0 mg/L 2,4-D, 0.5 mg/L NAA and 0.2 mg/L Kin for somatic embryogenesis. Globular embryos induced from the leaf explants were treated with colchicine by immersed method at different concentrations of 0, 0.1, 0.2, 0.3, 0.5 and 0.7% for 24, 36, 48 and 72 h. Then, these embryos were cultured on medium supplemented with 1.0 mg/L 2,4-D, 0.5 mg/L NAA and 0.2 mg/L Kin for secondary embryos induction. The ploidy level was identified by counting chromosomes of the root tip of plantlets derived from these secondary embryos. The results showed that the survival rate and somatic embryo induction the highest (65.56% and 29.89%, respectively) at 0.2 g/L AgNPs for 20 min. For colchicine treatment, the increase in the concentration and exposure time of colchicine decreased the survival rate of explants and the number of secondary embryos but increased the abnormal secondary embryo formation. Identifying the ploidy level showed that the treatments with colchicine concentrations from 0.3% to 0.5% for 48 h lead to tetraploid induction rates from 22.22% to 25.92%. The results also revealed that the tetraploids (2n = 4x = 48) had larger stomatal size, lower stomatal density, denser stomatal chloroplast density, and better growth than did the diploids.

Cortisol response under low intensity exercise during cognitive-behavioral therapy is associated with therapeutic outcome in panic disorder-an exploratory study.

Patients with Panic Disorder (PD) show an abnormal stress-induced functioning of the Hypothalamic-Pituitary-adrenal (HPA)-axis. Different protocols for stress induction are of rather low relevance for the psychotherapeutic treatment. In practice, interoceptive exposure is often realized as Low Intensity Exercise (LIE), as compared to an incremental cycle exercise test to exhaustion. Currently, it is not known, whether LIE displays an effective interoceptive stressor 1.) leading to a significant anxiety induction; 2.) a comparable HPA- and Sympathetic-Adreno-Medullar (SAM)-axis response in both patients and healthy controls; 3.) stress responses under LIE are associated with treatment outcomes. N = 20 patients with PD and n = 20 healthy controls were exposed to ten minutes of LIE on an exercise bike. LIE was applied as part of the interoceptive exposure, during an intensive Cognitive-Behavioral Therapy (CBT) in a day clinic. Heart rate was monitored and salivary cortisol samples collected. Before and after the LIE, state anxiety/ arousal were assessed. In order to evaluate psychopathology, the Panic and Agoraphobia Scale, Mobility Inventory, Agoraphobic Cognitions Questionnaire and Body Sensations Questionnaire were applied, before (T1) and after five weeks (T2) of an intensive CBT. LIE led to a significant and similar heart rate increase in both groups. Cortisol decreased over time in both groups, especially in male patients. A higher psychopathology before, and after CBT, was associated with a significantly lower cortisol response under LIE. In the present study, LIE led to a divergent stress response: while there was a significant heart rate increase, cortisol decreased over time, particularly in male patients. A lower reactivity of the HPA-axis seems to be associated with a lower treatment outcome, which may affect extinction based learning. The findings suggest, that interoceptive stimuli should be designed carefully in order to be potent stressors.

Differential Aspects of Natural and Morphine Reward-related Behaviors in Conditioned Place Preference Paradigm.

Natural rewards are essential for survival. However, drug-seeking behaviors can be maladaptive and endanger survival. The present study was conducted to enhance our understanding of how animals respond to food and morphine as natural and drug rewards, respectively, in a conditioned place preference (CPP) paradigm. We designed a protocol to induce food CPP and compare it as a natural reward with morphine CPP in rats. The protocol for reward induction in both groups (foods and morphine) consisted of three phases: pre-test, conditioning, and post-test. In morphine groups, we injected morphine as a reward (5 mg/kg, SC). To induce natural reward, we used two different protocols. In the first one, the rats were deprived of food for 24 h. In the other method, the rats were restricted to food for 14 days. During the conditioning period, the animals received daily chow, biscuits, or popcorn as a reward inducer. Results revealed that CPP was not induced in food-deprived rats. A combination of food restriction (as a facilitator) and a biscuit or popcorn-induced reward using CPP. In contrast, food deprivation did not facilitate food CPP in response to regular food. Interestingly the CPP score of the group which received biscuits during a 7-day conditioning period was more than that of the morphine group. In conclusion, food restriction could be a better protocol than food deprivation to facilitate food reward.

Uncovering the origins and lineage markers of human heart fields.

In this issue of Cell Stem Cell, Yang etal. devise a protocol for induction and differentiation of human heart-field precursors, drawing on inspiration from invivo development. Intricate computational analyses uncovered conserved factors governing heart-field segregation, which facilitate enhanced study of human heart development and disease in the dish.

215.9: Achievement of Persistent Mixed Chimerism in Recipients of Matched and Mismatched Living Donor Kidney Transplants in a Tolerance Induction Protocol

Purpose: The induction of tolerance to kidney transplants after development of specific unresponsiveness of recipient immune cells to donor alloantigens allows elimination of maintenance immunosuppressive (IS) drugs while preventing graft loss due to rejection. We have previously reported successful induction of mixed chimerism and tolerance in recipients of fully HLA-matched living donor kidney and hematopoietic cell (HCT) transplants. This work continues in HLA-mismatched transplants, wherein achieving persistent mixed chimerism is more challenging. Methods: Living donors underwent hematopoietic stem cell mobilization and leukopheresis 6 weeks before kidney donation. The recipient conditioning regimen for the HCT consisted of 10 doses of total lymphoid irradiation (TLI) and 5 doses of anti-thymocyte globulin (ATG) following kidney transplant, with HCT on day 11. The HCT consisted of CD34+ hematopoietic progenitor cells and CD3+ cells. Twenty-nine patients received fully HLA-matched living donor transplants. Twenty-five patients received HLA-mismatched (1-haplotype matched) living donor transplants with an escalating dose of CD3+ cells to enhance HCT engraftment. Six patients recently received HLA-mismatched living donor transplants with substitution of a dose of total body irradiation (TBI) for the final dose of TLI. Results: Of the 29 fully HLA-matched living donor transplants, complete IS drug withdrawal was achieved in 24, all of whom had at least 6 months of persistent mixed chimerism. Of the 24, 17 are alive, well and have been off IS drugs from 4 to 14 years without evidence of rejection. Two off IS drugs died due to cardiovascular events at 2 and 3 years. Two returned to IS drugs after loss of chimerism at 1 year followed by acute rejection at 4 years off IS drugs, and 3 after relapse of kidney disease at 1, 5 and 6 years off IS drugs. In the 25 1-haplotype matched living donor transplants, mixed whole blood chimerism persisted in 10 (mean 20% chimerism) at the end of the 1st year posttransplant while immunosuppression was reduced to tacrolimus monotherapy. Chimerism was lost during the 2nd year in the 5 who were withdrawn from monotherapy; they returned to IS drug(s). Mixed chimerism without rejection has persisted in 3 maintained on IS monotherapy, for up to 5 years. The recent addition of a dose of TBI markedly increased levels and stability of mixed chimerism during the 1st year posttransplant. Conclusions: The loss of chimerism during withdrawal of IS drug monotherapy in the HLA-mismatched transplants may have been due to the low level of whole blood chimerism, a contrast to the HLA-matched. A very low single dose of TBI added to the TLI/ATG conditioning regimen increased early chimerism. The stability of high levels of mixed chimerism after complete IS drug withdrawal during the 2nd year posttransplant is the subject of continuing investigation.

245.2: Evaluation of Methods to Obtain Peripheral Blood Mononuclear Cells From Deceased Donors Adjacent to Organ Procurement for Tolerance-Induction Protocols

Regulatory cell therapies have shown promise in tolerance-induction protocols in living donor organ transplantation. These protocols should be pursed in deceased-donor transplantation. Donor peripheral mononuclear cells (PBMCs) are an optimal source of donor antigens for the induction of donor-specific regulatory cells that are more effective than antigen non-specific regulatory cells. During the development of a regulatory cell tolerance induction protocol with organs from deceased donors, we compared three methods of obtaining PBMCs from deceased donors focusing on: cell yield, viability, and contamination of unwanted cell types. PBMC procurement methods: During organ procurement at the time of cold perfusion, blood was collected from the vena cava and placed into a 10-liter blood collection bag, and thereafter transported to Karolinska University Hospital, where leukapheresis was performed (BCL) n=7. Blood was collected via the vena cava into blood donation bags before cold perfusion. The bags underwent buffy coat separation and thereafter automated leukocyte isolation using SEPAX (BCS) n=2.To collect PBMCs, leukapheresis was performed via a central dialysis catheter on deceased donors in the ICU prior to the organ procurement procedure (LEU) n=9. Our results show that all methods were safe in relation to the intended organ procurement procedure. LEU is a feasible method to obtain PBMC from deceased donors. LEU tend to have higher yield of donor PBMC/kg compared to BCS and BCL and acceptable level of granulocyte contamination compared to BCS. Further investigations including the quality of the cell products that was manufactured with PBMCs from these methods will be needed to determine which approach is the most suitable for tolerance induction protocols.