Ficoll Gradient Method Research Articles

Immune Cell Alterations and PI3K-PKB Pathway Suppression in Patients with Allergic Rhinitis Undergoing Sublingual Immunotherapy.

Our prior clinical study assessed the efficacy and safety of sublingual immunotherapy (SLIT) with standardized Dermatophagoides farina drops on patients with allergic rhinitis (AR) while analyzing the characteristics of adverse reactions. This study was conducted to evaluate the immune cell composition alterations in AR patients before and after SLIT, and to comprehensively investigate the role and changes of antigen-specific immune cells associated with treatment efficacy. A total of 68 AR patients who completed 12months of SLIT were included in the study. Before the trial's initiation and after 1year of SLIT, 10ml of venous blood was collected. Peripheral blood mononuclear cells were isolated using the Ficoll gradient method. The mRNA transcriptome was analyzed using an Affymetrix microarray. The proportions of 22 immune cell types were calculated via the CIBERSORTx platform. Correlations between each immune cell type and SLIT were analyzed. PI3K-PKB pathway dysregulation were analyzed using quantitative PCR and Western blot. Flow cytometry was utilized to assess the percentages of Th1 and Th2 cells. Mono-sensitized AR patients exhibited marked increases in plasma cells, activated memory T cells, regulatory T cells, and activated dendritic cells, while experiencing decreased neutrophils and resting dendritic cells. In poly-sensitized AR patients, the most notable change was an increase in regulatory T cells, coupled with decreased T follicular helper cells, resting dendritic cells, and activated mast cells. These findings indicated that SLIT reshaped immune cell profiles in AR patients, and, notably, the specific changes differed between mono-sensitized and poly-sensitized individuals. Furthermore, SLIT appeared to shift the immune response towards a Th2 decrease profile in both groups. Importantly, suppression of the PI3K-PKB pathway was evidenced as inhibition of PKB phosphorylation and the decrease of glycogen synthase kinase 3 β (GSKβ) and mammalian target of rapamycin (mTOR) expression after SLIT. Our study has demonstrated that SLIT treatment led to distinct changes in immune cell profiles between mono-sensitized and poly-sensitized AR patients. Furthermore, SLIT appeared to reduce a Th2 immune response, highlighting its efficacy in AR treatment. Importantly, the study revealed the suppression of the PI3K-PKB pathway, shedding light on the immunological mechanisms underlying SLIT's effectiveness.

High plasma levels of the TSLP cytokine in Saudi patients with chronic stable asthma

Allergic asthma is an inflammatory bronchial disease associated with the IgE response, allergic inflammation of the airways, recruitment of infiltrated inflammatory cells, increased mucus production, constriction of airways, and difficulty breathing. TSLP cytokine is derived mainly from airway epithelial cells and have been shown to play an essential role in the pathogenesis of asthma. This study was designed to determine the plasma protein concentrations of total IgE and TSLP cytokines in patients with chronic stable asthma. Furthermore, we examined TSLP-r in B cells and determined whether the expression of TSLP is correlated with increased total IgE. Thirty-one patients with chronic stable asthma and nineteen normal controls were enrolled in the study. Blood samples were separated using the Ficoll gradient method, and plasma and lymphocyte cells were collected. Plasma levels of total IgE and TSLP were quantified in patients with asthma and normal controls by specific ELISAs. Moreover, the surface expression of TSLP-r on B cells was analyzed using flow cytometry. Total IgE protein concentrations in the plasma of patients with chronic stable asthma (344 IU/ml, P < 0.002) were considerably higher than those in normal controls (mean 130 IU/ml). TSLP levels were significantly higher in the asthma group (78 Pg/ml, P < 0.02) than those in the normal control group (40 pg/ml). Additionally, the expression of TSLP-r was markedly higher in asthma patients (430 × 103 MFI, P < 0.0065) than in normal controls (280 × 103). Collectively, the findings indicate that measuring plasma levels of TSLP in patients with stable asthma can be used to assess asthma symptoms and possibly evaluate the efficacy of corticosteroid therapy.

Stage-Specific Oligonucleotide Primers for the Diagnosis of Toxoplasmosis Among Iranian Pediatric Heart Transplant Recipients; Evaluation of Cotrimoxazole as a Preventive Therapy

Background: Toxoplasmosis is an opportunistic infection that affects solid organ transplant (SOT) recipients. The parasite transmission may be occurred from a Toxoplasma-seropositive donor to a Toxoplasma-seronegative recipient by organ transplantation. Objectives: In this study, a nested PCR was carried out using different primers targeting the B1, SAG4, and MAG1 genes to assess Toxoplasma infection in pediatric heart transplantation at Shahid Rajaei Heart Center in Tehran. Methods: Blood samples were collected from 46 pediatric heart transplant patients aged 1 - 17 years referring to Rajaei Cardiovascular Medical and Research Center from 2018 - 2019. All patients were on oral administration of Trimethoprim-sulfamethoxazole (cotrimoxazole). Blood samples were collected, and peripheral blood mononuclear cell (PBMC) isolation using the Ficoll gradient method was performed. DNA was extracted from PBMC, and nested PCR was carried out. Serologic tests were performed using ELISA to determine IgG and IgM anti - Toxoplasma gondii antibodies. Results: The results of serologic tests showed that all 46 patients had negative anti-T. gondii IgM antibody. Furthermore, 6 (13.05%) and 3 (6.5 %) out of the 46 patients were positive for IgG T. gondii antibody before and after transplantation, respectively. All 46 patients were evaluated using PCR using B1, MAG-1, and SAG-4 genes, and PCR results were negative. Conclusions: In general, due to the negative results of Toxoplasma with PCR using B1 and bradyzoite-specific genes (SAG-4 and MAG-1), it is possible that the results obtained in this study are because of prophylaxis with cotrimoxazole.

RNA Editing By APOBEC3A Confers Dynamic Mutational Load in Monocytic Leukemias

Background: Although genomic mutations play an important role in the initiation or progression of myeloid leukemias, the role of mutations at the transcript level is less well understood. Myeloid neoplasms have recurrent somatic mutations in genes for pre-mRNA splicing, cohesin complex, histone modification, and DNA methylation. However, the number of somatic mutations identified in myeloid neoplasms (average 9-13) is usually lower than that of solid tumors. Recent analyses of baseline transcriptome sequences in various cancers have revealed abundant adenosine to inosine (A>I) RNA editing events, mainly at intronic and untranslated regions of genes. The role of condition-specific RNA editing in cancer, especially of the cytosine to uracil (C>U) type, however, remains essentially unknown. We recently discovered antiviral restriction enzyme APOBEC3A (A3A) cytidine deaminase as a novel cellular site-specific C>U RNA editing enzyme in monocytes. RNA editing by A3A is dynamically induced by interferons and/or severe hypoxia in monocytes, resulting in stop codon gains or missense changes in transcripts of scores of genes. The effect of hypoxia in inducing RNA editing by A3A can be mimicked by inhibition of mitochondrial respiration. Whether A3A-mediated RNA editing occurs in monocytic leukemias, including acute monocytic leukemia (AMoL) and chronic myelomonocytic leukemia (CMML), is unknown. We hypothesize that dynamic C>U RNA editing by A3Acontributes to mutational load of leukemic cells under stressful conditions of hypoxia and interferon exposure. In this study, we examined transcript sequences of monocytic leukemias for evidence of inducible RNA editing. We also examined the role of mitochondrial respiratory inhibition, which induces RNA editing by A3A, in growth of leukemic progenitors of CMML.Methods: Monocytic leukemia samples are obtained from the RPCI Leukemia Bank. Standard diagnostic information included classification (WHO 2008 and FAB), fraction of monocytic component (mature monocytes, promonocytes or monoblasts by flow cytometry and/or aspirate differential count), cytogenetic analyses (conventional karyotype and targeted FISH), and survival data. Leukemic BM mononuclear cells (BM-MNC) prepared by the Ficoll gradient method are thawed and cultured in hypoxia (1% O2) with interferon type 1 (IFN-1). DNA and RNA are isolated. AMoL samples are subjected to nextgen high throughput exome and RNA sequencing. Once variants from DNA and RNA of corresponding samples are obtained, the set difference of variants is extracted to keep those detected on RNA samples. We also examined 3 CMML bone marrow samples for evidence of hypoxia-inducible RNA editing by analyzing selected RNA editing sites. Colony forming unit (CFU) assays are also performed in CMML samples.Results: We find that the A3A gene is robustly expressed inboth in AMoL and CMML. Hypoxia (with or without IFN-1) induced A3A-mediated RNA editing in AMoL and CMML samples. We previously showed that several RNA edited genes by A3A in normal monocytes function in recurrently altered pathways in myeloid leukemias. Sanger sequencing confirmed RNA editing of such genes in monocytic leukemia samples as well (Table 1), suggesting that epitranscriptomic sequence changes may contribute to molecular defects driving leukomogenesis. RNA seq analysis of AMoL samples (n=3) showed that dozens of genes acquire nonsense or missense mutations at the transcript level under the conditions of severe hypoxia and IFN-1. CFU assays of CMML samples showed that inhibition of mitochondrial respiration, which triggers A3A-mediated RNA editing, inhibits the growth of progenitor cells (Figure 1).Conclusions: We show that A3A-mediated RNA editing is active in monocytic leukemias and targets genes functioning in pathways recurrently altered in myeloid leukemias. RNA editing by A3A may contribute to growth arrest and survival of leukemic progenitors under certain stress conditions. To our knowledge, these results provide the first experimental evidence that conditional RNA editing by A3A confers substantial mutational load in myeloid leukemias. Such RNA editing events will dynamically increase the total mutational load in monocytic leukemias, and may create neo-antigens for immune therapy by checkpoint inhibition. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cell Survival Effects of Autophagy Regulation on Umbilical Cord-Derived Mesenchymal Stem Cells Following Exposure to Oxidative Stress.

Background:Due to oxidative stress, hypoxia, and serum deprivation, a large percentage of mesenchymal stem cells (MSCs) die in the early stages of transplantation. The present study aimed to address whether induction or inhibition of autophagy would affect the viability of MSCs after exposure to oxidative stress. Methods:MSCs were isolated from umbilical cord tissue using the Ficoll gradient method. pCMV-GFP-LC-3 plasmid containing GFP-tagged LC3 was transfected into MSCs to assay autophagy level in these valuable cells. The four study groups were: MSC-LC3-Rapa, MSC-LC3-3MA, MSCs without any transfection, and MSC-GFP-LC3 (control groups). To induce autophagy, the MSC-GFP-LC3 was treated with different concentrations of Rapa for 24 hours and named MSC-LC3-Rapa. To inhibit autophagy in MSC-GFP-LC3, these cells were cultured in the presence of 3MA for 24 hours and named MSC-LC3-3MA. Non-treated MSC-GFP-LC3 and MSCs were considered as control groups. MSCs were exposed to lethal doses of H2O2 followed by cell viability evaluation with the water-soluble tetrazolium salt assay method. The data were analyzed with SPSS version 18.0 using one-way ANOVA test. P<0.05 was considered statistically significant. Results:The results revealed that the enhancement of autophagy in MSC-LC3-Rapa sensitized them against oxidative stress (P=0.0006) and inhibition of autophagy in MSC-LC3-3MA led to resistance against oxidative stress (P=0.0003). Conclusion:Inhibition of autophagy, as a non-genetic engineering method, in MSCs enhances cell viability following exposure to the oxidative stress. This may provide a novel strategy to promote the efficiency of MSC-based cell therapy for clinical applications.

Characterization and Isolation of Very Small Embryonic-like (VSEL) Stem Cells Obtained from Various Human Hematopoietic Cell Sources.

Stem cell transplantation is one of the available treatments for leukemia, lymphoma, hereditary blood diseases and bone marrow failure. Bone marrow (BM), peripheral blood progenitor cells (PBPC), and cord blood (CB) are the predominant sources of stem cells. Recently a new type of stem cell with a pluripotent potential has been identified. These cells were named "very small embryonic like stem cells (VSELs)". It is claimed that VSEL stem cells can be found in adult BM, peripheral blood (PB), CB and other body tissues. This study is designed to characterize and isolate VSEL stem cells from different human hematopoietic sources; CB, PB and apheresis material (PBPC). VSEL stem cells were isolated from MNC and erythrocyte layers for all materials by using centrifugation and ficoll gradient method. We determined embryonic markers by flow cytometry, immunofluorescence and western blotting methods. Results from western blotting and immunofluorescence show high level of NANOG and OCT4 protein expression in PB, apheresis material and CB. Immunofluorescence images showed cytoplasmic and nuclear presence of these proteins. Flow cytometry results exhibited a higher expression of VSELs markers on debris area than CD45- population and higher expression on CB than PB. As a result, these findings have shown that it is necessary to investigate the function of pluripotent stem cell markers in differentiated adult cells. We further conclude that erythrocyte lysis method had the highest cell recovery amount among erythrocyte lysis and ficoll gradient methods. Consequently, this study gives us new information and viewpoints about expression of pluripotent stem cell (PSC) markers in adult tissues.

Abstract PD4-01: A new additive diagnostic assay for breast cancer screening - Biochemical infrared analysis of immune cells and plasma

Abstract Background: Screening for early detection of breast cancer is currently based on breast imaging. Screening mammography, the most commonly used imaging modality, has limited sensitivity, especially in dense breast tissue, and a significant rate of false positive results. An adjuvant blood-test based assay may improve breast cancer detection irrespective of breast density. In previously reported studies, Total Biochemical Infrared Analysis (TBIA) of peripheral blood mononuclear cells (PBMCs) and plasma was able to differentiate between patients with breast cancer and healthy controls or patients with benign breast disease. The TM-B1TM assay is based on TBIA technology and is intended for breast cancer screening. In the present study, the performance of the TM-B1TM assay was evaluated for cancer detection across breast densities. Patients and Methods: A total of 220 women were recruited to this IRB approved study: 50 women with newly diagnosed breast cancer, and two control groups – 79 patients with benign findings and 61 patients with no detectable abnormality. Twenty-nine women were withdrawn from the study (for eligibility or technical reasons), and the data presented refer to 190 cases. Breast cancer patients included 6 cases of DCIS, 1 of LCIS, 42 of IDC and 1 of ILC. Ten milliliters of blood were drawn and separated by Ficoll gradient method into PBMCs and plasma. The samples were dried on a zinc selenide optical window and analyzed by a FTIR spectrometer. The spectra were analyzed by the proprietary software TodoSpectra to distinguish between infrared spectra of cancer patients vs. patients with benign findings and healthy controls. The influence of age and breast density on TM-B1TM results were evaluated. Results: The TM-B1 assay obtained a sensitivity of 86 % and specificity of 98 % for breast cancer detection. The positive predictive value (PPV) was 95.1% and the negative predictive value (NPV) was 93.5%. Dense breast tissue (BIRADS categories C&amp;D) was present in 55 % of the subjects. The specificity and sensitivity for patients with dense breasts was 99 % and 83 % respectively. No major differences in accuracy of TM-B1 were found due to patients' age. Conclusions: TM-B1 in conjunction with current imaging techniques may contribute to early detection of breast cancer by increasing sensitivity and reducing false positive results and unnecessary biopsies. Further studies with larger patient numbers are required and under way to establish the utility of adding the TM-B1 assay to current standard screening for breast cancer. Citation Format: Allweis TM, Schmitt J, Zelig U. A new additive diagnostic assay for breast cancer screening - Biochemical infrared analysis of immune cells and plasma [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD4-01.

Separation of X spermatozoa from cauda of epididymis prior to selection of female embryos in local goat

The objective of this study is the selection of X epididymal spermatozoa of local buck by discontinuous ficoll density gradient and using these spermatozoa for in vitro fertilization and identify the ratio of produced female embryos after identification by polymerase chain reaction (PCR). Epididymal spermatozoa were harvested from the cauda epididymis by slicing, and then spermatozoa were estimated and submitted to in vitro maturation and capacitation prior to separation of X from Y bearing spermatozoa and use for in vitro fertilization. Modified discontinuous ficoll density gradient method used for the separation of X and Y-bearing sperm by using 3 layers of discontinuous ficoll (60, 40, and 20%) or 4 layers of discontinuous ficoll (80, 72, 64, and 50%). The centrifugation applied at 200×g or 300×g. In the protocol of 3 layers of discontinuous ficoll (60, 40, and 20%) and centrifugation at 200×g and 300×g the results showed that the mean of the sperms at the 3rd was 79.014 ± 3.12 and 79.39 ± 2.11 respectively and this show the sperms was not completely separated. In the protocol of 4 layers of discontinuous ficoll (80, 72, 64, and 50%) and centrifugation at 200×g showed that the mean of the sperms at the 4th layer was (42.79 ± 1.38) as compared with the first three layers and with the protocol centrifugation at 300×g. The mean of the sperm lost showed (7.066 ± 0.56) and a little percent from centrifugation at 300×g showed 11.28 ± 1.55. Oocytes collected from ovaries which obtained from the slaughter house then matured oocyte was applied. The maturation rate for grade A and B used for IVF by spermatozoa after ficoll density gradient it was 67.60% and 52.74%, while the fertilization rate for the grade A and B was 70.68% and 78.26% respectively. The sex of embryos was determined by polymerase chain reaction (PCR) using specific primers for detection of SRY gene. The percentage of goat embryos obtained by IVF by sperms selected with ficoll density techniques after centrifugation at 200×g at different stage of development was 70.13%. The percentage of the male embryos was 16.67% while the female embryos was 83.33%. The results showed that the best method for selection of female embryos was by using 4 layers of discontinuous ficoll gradient method (80, 72, 64, and 50 %) with centrifugation at 200×g.

Tu1038 Laparoscopic Hepatectomy of Segment 7 or 8 of the Liver With a Transthoracic Port

[Background and Aims] Sporadic cases of unexpected recurrence, such as intrahepatic dissemination, extrahepatic seeding, and rapidly growing recurrence have been described following radiofrequency ablation (RFA). Some reports speculated that RFA during energy application may increase intratumoral pressure and favor intravascular spread of the tumor. Therefore, we examined the existence of hepatocytes in the hepatic vein during RFA. [Subjects and Methods] A total of 3 pigs with a mean body weight of 58.8 ± 5.7 kg were intubated and general anesthesia were maintained. Using the 8-Fr sheath inserted into the left jugular vein, a guidewire was used to guide a 4-Fr catheter to the hepatic vein. After median laparotomy, needle electrodes were inserted inside the liver, and RFA was performed near the catheter. A cooled-tip RF needle electrode was used for RFA. Ablation was performed by increasing output by 10 W/min, starting at 40 W. The duration of ablation was 6 min. The blood was collected by placing the catheter 10mm from the needle electrode tip. Fifteen RFA sessions of 3 animals were examined to detect exfoliated tissues. In every RFA session, 30 ml of the down-streaming blood in the vessel attached to the irradiated part was collected to a heparinized syringe and separated by using Ficoll-gradient method. Cell fractions were incubated with the culture medium for 15 hours. The number of cell clusters attached on culture dishes were counted and examined immunohistochemically for albumin as a hepatocyte marker. [Results] The results of immunohistochemistry showed that albumin-positive cells were found in cell clusters those were attaching and spreading on the culture dish. The margin of the cluster showed membrane ruffling. That means the live hepatocytes being trapped in the outbound vessel of RFA treated site. Various sizes of clusters were found. The clusters were classified in four as S-1 (5 to 10 cells), S-2 (11 to 20 cells), S-3 (21 to 50 cells), and S-4 (more than 50 cells) according to the constituted cell amount. The mean number of cell clusters found in every RFA was S-1; 9 ± 7.1, S-2; 5.1 ± 5.7, S-3; 0.6 ± 0.8, S-4; 0.5 ± 0.8, respectively. Smaller sizes of clusters (S-1 and S-2) were more frequently found (P, 0.01 vs. S-3 and S-4). These clusters were never found in the blood before RFA. Therefore, it should be concluded that the cell cluster was derived from the liver and was artificially exfoliated tissue masses by RFA. [Conclusions] The present results demonstrate that hepatocytes are pushed out alive into the hepatic vein from the hepatic lobules by RFA. These results suggested that a risk of dissemination due to RFA exists.

Tu1037 Outflow of Hepatocytes Into the Hepatic Vein During Radiofrequency Ablation in Porcine Liver

[Background and Aims] Sporadic cases of unexpected recurrence, such as intrahepatic dissemination, extrahepatic seeding, and rapidly growing recurrence have been described following radiofrequency ablation (RFA). Some reports speculated that RFA during energy application may increase intratumoral pressure and favor intravascular spread of the tumor. Therefore, we examined the existence of hepatocytes in the hepatic vein during RFA. [Subjects and Methods] A total of 3 pigs with a mean body weight of 58.8 ± 5.7 kg were intubated and general anesthesia were maintained. Using the 8-Fr sheath inserted into the left jugular vein, a guidewire was used to guide a 4-Fr catheter to the hepatic vein. After median laparotomy, needle electrodes were inserted inside the liver, and RFA was performed near the catheter. A cooled-tip RF needle electrode was used for RFA. Ablation was performed by increasing output by 10 W/min, starting at 40 W. The duration of ablation was 6 min. The blood was collected by placing the catheter 10mm from the needle electrode tip. Fifteen RFA sessions of 3 animals were examined to detect exfoliated tissues. In every RFA session, 30 ml of the down-streaming blood in the vessel attached to the irradiated part was collected to a heparinized syringe and separated by using Ficoll-gradient method. Cell fractions were incubated with the culture medium for 15 hours. The number of cell clusters attached on culture dishes were counted and examined immunohistochemically for albumin as a hepatocyte marker. [Results] The results of immunohistochemistry showed that albumin-positive cells were found in cell clusters those were attaching and spreading on the culture dish. The margin of the cluster showed membrane ruffling. That means the live hepatocytes being trapped in the outbound vessel of RFA treated site. Various sizes of clusters were found. The clusters were classified in four as S-1 (5 to 10 cells), S-2 (11 to 20 cells), S-3 (21 to 50 cells), and S-4 (more than 50 cells) according to the constituted cell amount. The mean number of cell clusters found in every RFA was S-1; 9 ± 7.1, S-2; 5.1 ± 5.7, S-3; 0.6 ± 0.8, S-4; 0.5 ± 0.8, respectively. Smaller sizes of clusters (S-1 and S-2) were more frequently found (P, 0.01 vs. S-3 and S-4). These clusters were never found in the blood before RFA. Therefore, it should be concluded that the cell cluster was derived from the liver and was artificially exfoliated tissue masses by RFA. [Conclusions] The present results demonstrate that hepatocytes are pushed out alive into the hepatic vein from the hepatic lobules by RFA. These results suggested that a risk of dissemination due to RFA exists.

The Lymphocyte Dependent Bactericidal Assay of Human Monocyte and Alveolar Macrophage forMycobacteria

Background : Though mononuclear phagocytes serve as the final effectors in killing intracellular Mycobacterium tuberculosis, the bacilli readily survive in the intracellular environment of resting cells. The mechanisms through which cellular activation results in the intracellular killing is unclear. In this study, we sought to explore an in vitro model of a low-level infection of human mononuclear phagocytes with MAC and and determine the extent of the lymphocyte dependent cytotoxicity of human monocytes and alveolar macrophages. Materials and Methods : The peripheral monocytes were prepared using the Ficoll gradient method from PPD positive healthy people and tuberculosis patients. The alveolar macrophages were prepared from PPD positive healthy people via a bronchoalveolar lavage. The human mononuclear phagocytes were infected at a low infection rate (bacilli:phagocyte 1:10) with MAC(Mycobacterium avium) and Mycobacterium tuberculosis . Non-adherent cells(lymphocyte) were added at a 10:1 ratio. After 1,4, and 7 days culture in , 5% CO2 incubator, the cells were harvested and inoculated in a 7H10/OADC agar plate for the CFU assay. The bacilli were calculated with the CFU/ of the cells and the cytotoxicity was expressed as the log killing ratio. Results : The intracellular killing of MAC and within the monocyte was greater in patients with tuberculosis compared to the PPD positive controls (p within the alveolar macrophage appeared to be greater than that within the monocytes of the PPD positive controls. There was significant lymphocyte dependent inhibition of intracellular growth of the mycobacteria within the monocytes in both the controls and tuberculosis patients and within the macrophages in the controls(p. Conclusion : This study is an in vitro model of a low-level infection with MAC and of human mononuclear phagocytes. The intracellular cytotoxicity of the mycobacteria within the phagocytic cells was significantly lymphocyte dependent. During the 7 days culture after the intracellular phagocytosis, the actual confinement of the mycobacteria was observed within the monocytes of tuberculosis patients and the alveolar macrophages of the controls as in the case of adding lymphocytes.

Effect of protein kinase C activators on the uptake of GABA by rat brain synaptosomes.

Synaptosomes were prepared from rat brain by a discontinuous Ficoll gradient method and used for studying the uptake of labelled GABA. Two GABA uptake components were evidenced, a high (Km = 3.13 microM) and a low (Km = 92.4 microM) affinity one. Preincubation of synaptosomes with two different activators of protein kinase C, phorbol 12, 13-diacetate (PDAc) and oleyl-acetyl glycerol (OAG), resulted in a change of GABA uptake. In particular, the low affinity component increased its Vmax by 58-74%, with no change in the Km. No statistically significant modification was detected for the high affinity component.

Recovery of lymphocytes and epithelioid cells from fluids used to flush the uteri and oviducts of superovulated cattle and preparation of monolayer cultures from epithelial cells of oviduct and uterus

A Ficoll-gradient method to recover separately lymphocytes and sedimented cells from uterotubal flush fluid of cattle is described. Lymphocytes could be cocultured for virus isolation or used for histopathological studies, or both. Epithelioid cells, most likely of oviductal origin, were grown in monolayer cultures to initiate cell lines and isolate viruses. This technique could be applied to other situations that require the growth of bovine oviduct epithelial cells in vitro.

Studies on the mechanism of erythrocyte aging and destruction I. Separation of rat erythrocytes according to age by ficoll gradient centrifugation

A 22–34% linear Ficoll gradient is described for use in separating red blood cells of different ages. Newly synthesized, 59Fe-labeled red cells were found in the top fractions of the gradient, and these cells moved down toward the lower part of the gradient as they became older. The Ficoll gradient method gave a higher resolution compared to existing published methods. Red blood cells with increasing density, increasing sphericity and decreasing volume were separated by the Ficoll gradient method. The protein content remained constant in red cells up to about two weeks of age, and then showed a trend to decrease with age. Changes in density, shape, volume, and probably protein content, are characteristics of aging red cells.

Contribution of cytoplasmic enzymes to apparent heterogeneity of rat brain mitochondria.

The results of this investigation indicate that there is sufficient contamination of washed primary rat brain mitochondrial preparations with cytoplasmic enzymes to cause possible misinterpretation of mitochondrial heterogeneity after isopycnic sucrose density gradient centrifugation, when based solely on enzyme activity measurements. Most of the contamination may be removed by a single 30 min centrifugation using the 3-6 % discontinuous Ficoll gradient method of Clark and Nicklas (1970).