Use Of Supernatant Research Articles

Molecular yield and cytomorphologic assessment of fine needle aspiration specimen supernatants.

Cytology samples are frequently relied upon for the diagnosis of advanced cancer such as lung cancer. As the recommendations for solid malignancies biomarker testing continue to expand, it becomes increasingly important to efficiently utilize limited specimens to minimize the need for additional sampling and its associated risks and costs. We performed molecular testing on fresh or CytoLyt-fixed supernatants derived from fine needle aspirates (FNAs) and compared its performance against the clinical specimen (including formalin-fixed paraffin-embedded cell blocks, residual PreservCyt and fresh samples). Supernatants were assessed for cellularity using Field-stained Cytospin (CS) preparations. There was overall almost perfect agreement (41/45 cases, K = 0.822) and substantial to almost perfect agreement in molecular testing results of clinically actionable variants between fresh (20/23 cases, Κ = 0.742) and CytoLyt-fixed (21/22 cases, Κ = 0.908) and its clinical specimen counterpart. Interestingly, CS examination of the supernatants revealed viable tumor cells. Centrifugation for 1 minute at 300 rpm is optimal for overall or tumor cellularity recovery. Delayed molecular testing after 3, 4 and 7 days at 4 degrees Celsius showed identical molecular results. We validated the use of supernatants derived from FNA cytology samples as a substrate for molecular testing using next-generation sequencing and other molecular techniques.

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis.

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, etc.) of this bacterium have been lacking. A Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay was therefore developed as an easily accessible and unambiguous tool for high-resolution genotyping of recovered isolates. For the MLVA assay presented here, DNA is extracted from cultured Y. ruckeri samples by boiling bacterial cells in water, followed by use of supernatant as template for PCR. Primer-pairs targeting ten Variable-number tandem-repeat (VNTR) loci, interspersed throughout the Y. ruckeri genome, are distributed equally amongst two five-plex PCR reactions running under identical cycling conditions. Forward primers are labelled with either of three fluorescent dyes. Following amplicon confirmation by gel electrophoresis, PCR products are diluted and subjected to capillary electrophoresis. From the resulting electropherogram profiles, peaks representing each of the VNTR loci are size-called and employed for calculating VNTR repeat counts in silico. Resulting ten-digit MLVA profiles are thenused to generate Minimum spanning trees enabling epizootiological evaluation by cluster analysis. The highly portable output data, in the form of numerical MLVA profiles, can rapidly be compared across labs and placed in a spatiotemporal context. The entire procedure from cultured colony to epizootiological evaluation may be completed for up to 48 Y. ruckeri isolates within a single working day.

Isolation of Surfactants Synthesized by the Pseudomonas Bacteria and Study of Their Properties

Background. The important problem of biosurfactants production is biosynthesis optimization. But lack of effective isolation methods elaboration with simultaneous new costeffective products is the greatest weaknesses of existing technologies. Objective. The aim of the study is rational technology elaboration for isolation of biosurfactants obtained from strains Pseudomonas sp. PS-17 and P. fluorescens 8573. Investigation of the influence of different acids and temperatures on the efficiency of isolation of the surfactant products, study the properties of the obtained products. Determination of the possible directions of the use of supernatant obtained after precipitation of the biocomplexes (SPL) as a new inexpensive product. Methods. Rhamnolipid surfactant concentrate was precipitated from culture liquid supernatant (CLS) by acidification to pH 3-4 with acid solutions (HCl, H 2 SO 4 , H 3 PO 4 , HNO 3 , CH 3 COOH), kept at 100 °C for 25 min, cooled to room temperature, centrifuged before the phase separation, the supernatant was decanted. The surface tension of the SPL was determined by du Nouy method (with a platinum ring). The emulsification index of biosurfactants was determined regarding mineral oil and sunflower oil. The RLs were isolated from the SPL by extraction with a mixture of ethyl acetate and isopropanol, their composition was determined by thin layer chromatography. Influence on plants was assessed by their morphometric parameters after presowing seed treatment. Results. The rational technology for surfactants isolation from strains Pseudomonas sp. PS-17 and P. fluorescens 8573 was developed It was shown that the suitable method of the isolation of the biosurfactants of Pseudomonas sp. PS-17 and P. fluorescens 8573 is acidic precipitation from CLS with heating. As a result, the product yield was increased by 20%, and the duration of the process was reduced. The physico-chemical properties of the SPLs after the isolation of biosurfactants from the CLS were studied. SPLs have been shown to be effective oil emulsifiers, foaming and wetting agents for various surfaces. It was shown that SPLs (at dilutions 1:10) do not exhibit phytotoxic effects and stimulate the growth of watercress. Conclusions. The new wasteless technology for Pseudomonas strains biosurfactants isolation has been proposed, which provides for the elimination of the extraction stage with solvents, as a result, the yield of the target products has been increased. Thus, the technology has economic and environmental advantages. It was shown that SPLs, being inexpensive and effective products, can be used in environmentally friendly technologies: in agriculture (for stimulation of plant growth), for remеdiation of contaminated soils, production of detergent compositions.

Pilot-scale isolation and characterization of extracellular polymeric substances (EPS) from cell-free medium of Spirulina sp. LEB-18 cultures under outdoor conditions

This objective of this work was to monitor the EPS production during the growth of Spirulina sp. LEB-18, evaluate the productivity and to characterize the exopolymers obtained on pilot-scale under outdoor conditions. The production of crude EPS occurred in all phases of biomass growth and was approximately ten folds higher than that biomass concentration of Spirulina sp. LEB-18 at the end cultivation, demonstrating the importance of the use of supernatant after harvesting of Spirulina to obtain high value bioproducts. The EPS extracted by Spirulina sp. LEB-18 are typically heteropolymers with one high molecular weight fraction (polysaccharides) with potential to be utilized as an alternative bioflocculant and another fraction of lower molecular mass (proteins). The presence of uronic acids, pyruvates and acyl groups of carbohydrates or carboxylic groups of amino acids in protein moiety is the main responsible for overall negative charge of EPS, which is also of biotechnological importance. Moreover, due to the pseudoplastic behavior of the solutions and high thermal stability, the obtained EPS can be widely applied in several industrial sectors, thus determining its technological and market potentiality.

Studies on Conditioned Media in Human Cells: Evaluation Using Various Cell and Culture Conditions, Animal Disease Models

In the last several decades, cell therapy research has increased worldwide. Many studies have been conducted on cell therapy, and have revealed that transplanted cells did not survive for long, and implanted cells remained inactive causing immune rejection depending on the patient’s condition. Therefore, studies on cell-free therapy need to be conducted. To overcome these limitations, an alternative is the use of supernatant from cells, called “conditioned media (CM).” During in vitro cell culture, culture media supply nutrients to maintain cell characteristics and viability. In the culture, cells not only consume nutrients but also release beneficial proteins and substances, which are called “secretome.” CM from cells can be stored for a long time and is easy to handle. Moreover, secretome in CM can also be measured; exact amount of secretome is important to set the standard value for disease treatment. Here, we reviewed studies on CM and confirmed that various secretomes from CM were identified in these studies. Moreover, these findings could benefit cell and animal studies in future. In conclusion, CM could be a potential candidate for an alternative to cell therapy.

Can thrombin-activated platelet releasate compensate the age-induced decrease in cell proliferation of MSC?

Mesenchymal progenitor cells (MSCs) are promising for cell-based regeneration therapies. In elderly patients a reduced proliferation of MSCs has been described. Platelet-rich plasma (PRP) contains important factors necessary for osteogenic regeneration. The aim of this study was to find out whether the age-induced decrease in cell proliferation can be compensated by the use of supernatant of centrifuged, activated PRP (tPR). MSCs of donors of three age groups (A: young, 14-16 years, B: middle age, 36-46 years, C: older, 74-83 years) were expanded with 20% FCS alone or supplemented with thrombin-activated platelet releasate (tPR) (1%, 2.5%, and 5%) or platelet-poor plasma (PPP 5%). Cell proliferation and differentiation was measured on days 0, 3, and 7. Proliferation increased significantly in groups A and B with tPR, and non-significantly in group C. The generation times of MSCs of elderly patients were significantly increased in group C compared to groups A and B. Addition of 1% or 2.5% tPR significantly reduced population doubling times of all age groups. Adding tPR stimulates the proliferation rate of MSCs independent of donor age. For juvenile and middle-aged patients this influence was significant. Cells differentiation into osteoblasts was not influenced by addition of tPR.

Human Intestinal Dendritic Cells Decrease Cytokine Release against Salmonella Infection in the Presence of Lactobacillus paracasei upon TLR Activation

Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, little is known about the signalling pathways that are engaged by probiotics. Dendritic cells (DCs) are antigen-presenting cells that are involved in immunity and tolerance. Monocyte-derived dendritic cells (MoDCs) and murine DCs are different from human gut DCs; therefore, in this study, we used human DCs generated from CD34+ progenitor cells (hematopoietic stem cells) harvested from umbilical cord blood; those DCs exhibited surface antigens of dendritic Langerhans cells, similar to the lamina propria DCs in the gut. We report that both a novel probiotic strain isolated from faeces of exclusively breast-fed newborn infants, Lactobacillus paracasei CNCM I-4034, and its cell-free culture supernatant (CFS) decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with Salmonella. Interestingly, the supernatant was as effective as the bacteria in reducing pro-inflammatory cytokine expression. In contrast, the bacterium was a potent inducer of TGF-β2 secretion, whereas the supernatant increased the secretion of TGF-β1 in response to Salmonella. We also showed that both the bacteria and its supernatant enhanced innate immunity through the activation of Toll-like receptor (TLR) signalling. These treatments strongly induced the transcription of the TLR9 gene. In addition, upregulation of the CASP8 and TOLLIP genes was observed. This work demonstrates that L. paracasei CNCM I-4034 enhanced innate immune responses, as evidenced by the activation of TLR signalling and the downregulation of a broad array of pro-inflammatory cytokines. The use of supernatants like the one described in this paper could be an effective and safe alternative to using live bacteria in functional foods.

Multikinase Inhibitor Sorafenib Induces Apoptosis, CD138-Downregulation, Actin Depolymerization and Inhibition of M210B4-Triggered Chemotaxis in Multiple Myeloma Cell Lines and Synergyzes with Proteasome Inhibitor Bortezomib

Abstract 2380 Introduction:Sorafenib is an oral multikinase inhibitor that targets several cancer-specific pathways and directly affects tumor cell proliferation, cell survival and neovascularization. The Ras/Raf/MEK/ERK pathway is particularly known to be critical for proliferation of multiple myeloma (MM) cells. Moreover, its blockage may not only compromise MM cell survival and proliferation, but also influence cell adhesion and migration. We sought to elucidate the effects of sorafenib on proliferation, phenotype, specific signalling pathways, actin polymerization and chemotaxis, as well as cytotoxic interactions when combined with other anti-MM agents, such as bortezomib. Methods:L363, U266 and RPMI8226 were cultured with RPMI1640, 10% FCS and 0.2% penicillin/streptomycin. On day 0, cells were treated with increasing concentrations of sorafenib and/or bortezomib. Cell viability and cytotoxicity were assessed on days 3 and 6, in addition to day 1 or 2 in previous analyses. The cytotoxic effect for sorafenib and bortezomib combined was evaluated using Calcusyn Software, whereby a combination index =1, <1 or >1 indicated additive, synergistic and antagonistic effects, respectively. CD138 expression and morphologic changes were evaluated via flow cytometry, immunocytochemistry and confocal microscopy. The effect of sorafenib on ERK1/2 phosphorylation was investigated by western blot. Actin polymerization was studied by flow cytometry after labeling with FITC-phalloidin. Chemokine receptor expression was assessed by flow cytometry and chemotaxis of L363 cells with various chemoattractants was studied using 96-well chemotaxis chambers. Results:Our MM-in vitro model confirmed potent cytotoxicity for sorafenib single use and synergistic effects when combined with bortezomib. With 10 and 100μM sorafenib in L363, we observed increased median PI+ cells (62% and 94% on d3, respectively) compared to the control (median PI+ d0: 11%), with similar increases on d6 (median 81% and 92%, respectively). Combined sorafenib and bortezomib use showed additive effects and synergism at 10μM and 10nM bortezomib (combination index: 0.80). Similar to PI-results, viable cells and CD138 expression by flow cytometry substantially decreased with sorafenib in a dose- and time-dependent manner. Regarding the effects on the MAPK pathway, after incubating L363 cells with 1 and 10μM sorafenib for 6 and 24 hours, a dose-dependent downregulation of ERK1/2 phosphorylation was observed. After 3 days of incubation with increasing concentrations of sorafenib, MM cells were stained with DAPI, Phalloidin-Alexa594 and CD138-FITC and analyzed via confocal microscopy. L363 cells highly expressed CD138 in the absence of sorafenib. Of note, sorafenib not only affected cell proliferation, but also phenotype, morphology, actin metabolism and chemotaxis of MM cells. With sorafenib concentrations as low as 1μM, CD138 was downregulated and impressive morphologic changes with a reduction in F-actin content were observed. We could show CXCL12-stimulated actin polymerization and after treatment with sorafenib with concentrations of 10μM and 100μM its inhibition, as confirmed via flow cytometry after labeling with phalloidin-FITC. L363 cells showed high expression of the chemokine receptors CCR4 and CCR5 and underwent chemotaxis to their common ligand CCL5. Chemotaxis of L363 cells was even more evident with the use of supernatant from M210B4 bone marrow stromal cells. This M210B4-induced chemotaxis also occurred in the presence of the specific CXCR4-inhibitor AMD3100, supporting the involvement of chemokines other than CXCL12 in M210B4-induced MM cell migration. M210B4-triggered chemotaxis was substantially inhibited after 3 days of incubation with increasing concentrations of sorafenib in a dose-dependent manner. Conclusions:To the best of our knowledge this is the first analysis of the effects of sorafenib on phenotype, morphology, actin polymerization and migration of MM cells. Sorafenib induced down-regulation of phospho-ERK appeared responsible for the observed actin depolymerization and reduction in M210B4-triggered chemotaxis. Hence, further analysis of sorafenib and other novel anti-MM agents, both in MM cells and their microenvironment, should enable greater progress in this hematopoietic disease. Disclosures:No relevant conflicts of interest to declare.

A novel biopesticide production: attagel-mediated precipitation of chitinase from Beauveria bassiana SFB-205 supernatant for thermotolerance

Insect killing fungi have high potential in controlling agriculturally harmful pest, but their slow progress and high variation in killing insect are major impediments to successful industrialization. The present work describes the use of supernatant from the liquid culture of Beauveria bassiana SFB-205 to surmount this problem, particularly efficient production of thermotolerant chitinase, which is one of the major pathogenesis-related enzymes in the supernatant. The chitinase was precipitated using varying mineral precipitants and followed by lyophilization, which was compared with a salting out method using ammonium sulfate in effectiveness. Incorporating of the supernatant fraction of the Beauveria preparation with attagel at 0.5% (w/v) as a precipitant enabled this treatment to show the greatest chitinase-precipitation efficiency (93.4%), followed up with excellent insecticidal activity against cotton aphids when it was mixed with 0.01% (v/v) polyoxyethylene-(3)-isotridecyl ether (TDE-3) as a spreading agent in laboratory conditions. Consequently, lyophilized attagel-mediated precipitation pellet was superior to lyophilized salting out pellet in maintaining chitinase activity against a thermal stress at 50 degrees C. This finding provides that the attagel-mediated precipitation can be exploited to improve the thermotolerance of B. bassiana SFB-205 chitinase as a novel strategy for biopesticide production.

Biosynthesis of silver nanoparticles using aqueous extract from the compactin producing fungal strain

In the present study, an eco-friendly process for the synthesis of nanomaterials using a fungus, Penicillium brevicompactum WA 2315 has been attempted. The fungus has been previously utilized for compactin production. Supernatant of seed culture was used for the biosynthesis of silver nanoparticles. The aqueous silver ions were reduced to silver metal nanoparticles when treated with the fungal supernatant. After 72 h of treatment, silver nanoparticles obtained were in the range of 23–105 nm as obtained from TEM. The nanoparticles were characterized by UV, FTIR, SEM, TEM and XRD. The use of supernatant of the seed media of the said fungus opens up the exciting possibility of rational strategy of biosynthesis of nanomaterials.

An alkaline solution simplifies nucleic acid preparation for RT-PCR and infectivity assays of viroids from crude sap and spotted membrane

Formats of a simple protocol for the preparation of nucleic acids for infectivity and RT-PCR detection of viroids from minute amounts of plant material are described. The method consists of preparing crude extracts in a NaOH–EDTA solution and then testing the supernatant. The NaOH–EDTA extract can be used at four distinct stages of preparation depending upon the accuracy desired, namely: (1) incubation of extract for 15 min at room temperature and the use of the supernatant for RT-PCR; (2) the supernatant can be spotted onto a nitrocellulose membrane (NCM) without vacuum, and the water-eluted liquid is used for RT-PCR; (3) centrifugation of the extract and use of supernatant in RT-PCR; (4) for quantitative accuracy, spotting the centrifuged supernatant on NCM using a vacuum device and then using the water-eluted liquid for RT-PCR. The protocols are rapid, inexpensive and applicable to large-scale epidemiological survey of ornamental plants or crops. The membranes are easily transported long distances and can be stored at room temperature for several months while retaining the ability to detect viroids by RT-PCR and by infectivity assays.

Avaliação do efeito do polieletrólito aniônico na clarificação do extrato aquoso de Stevia rebaudiana

A clarificação é uma etapa relevante no processo de obtenção de adoçantes a base de esteviosídeo. Neste trabalho, utilizou-se o polieletrólito aniônico (BETZDEARBORN TM F11) na decantação da suspensão do extrato cru, que possui cor escura, e avaliou-se sua aplicabilidade. O extrato utilizado era obtido a partir de plantas selecionadas e modificadas geneticamente, no Núcleo de Estudos em Produtos Naturais (NEPRON) no Universidade Estadual de Maringá (UEM), o qual mantém uma linha de pesquisa denominada Projeto Stevia e planta piloto em parceria com a iniciativa privada. Os ensaios foram realizados em aparelho Jar Test divididos em duas etapas, que se distinguiam na dosagem do polímero e na utilização do sobrenadante ou da suspensão total após tratamento com óxido de cálcio, o qual realizava o ajuste do pH. Realizou-se leituras de absorbâncias em 420 (cor) e 670etam (turbidez) e, fez-se cálculos de percentagem de despigmentação e de velocidade de decantação. Os melhores resultados na etapa 1, onde se aplicou baixas quantidades de polieletrólito aniônico e utilizou-se o sobrenadante, foram 72,2 e 97,0% para despigmentação em 420etam e 670etam, respectivamente e velocidade de decantação de 0,72cm/min. Já, na etapa 2, onde elevou-se a quantidade do polieletrólito e utilizou-se a suspensão total, os melhores resultados obtidos foram de 70,5% de despigmentação em 420etam, 85,6% de despigmentação em 670hm e velocidade de decantação de 0,71cm/min.

Epidemiology: Culture of bovine bone marrow progenitor cells in vitro

Summary In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400g). The use of 3% bovine leucocyte‐conditioned medium, produced by stimulation of blood lymphocytes with 4 pg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte‐conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose‐based medium requires different culture conditions, which are different from the culture conditions for human cells.

Polyriboinosinic Polyribocytidylic Acid (Poly(I:C)) Induces Stable Maturation of Functionally Active Human Dendritic Cells

For vaccination strategies and adoptive immunotherapy purposes, immature dendritic cells (DC) can be generated from adherent monocytes using GM-CSF and IL-4. Presently, the only clinically applicable method to induce stable maturation of DC is the use of supernatants of activated monocytes (monocyte-conditioned medium (MCM)). MCM contains an undefined mixture of cytokines and is difficult to standardize. Here we report that stable maturation of DC can be simply induced by the addition of polyriboinosinic polyribocytidylic acid (poly(I:C)), a synthetic dsRNA clinically applied as an immunomodulator. Poly(I:C)-treated DC show a mature phenotype with high expression levels of HLA-DR, CD86, and the DC maturation marker CD83. This mature phenotype is retained for 48 h after cytokine withdrawal. In contrast to untreated DC, poly(I:C)-treated DC down-regulate pinocytosis, produce high levels of IL-12 and low levels of IL-10, induce strong T cell proliferation in a primary allo MLR, and effectively present peptide Ags to HLA class I-restricted CTL. In conclusion, we present a simple methodology for the preparation of clinically applicable mature DC.

焼酎蒸留粕上澄液を連続的に再利用した米焼酎製造

焼酎蒸留粕上澄液を連続的に再利用した米焼酎製造

Electron microscopic investigation of lysogeny of Clostridium difficile strains isolated from antibiotic‐associated diarrhea cases and from healthy carriers

An electron microscopic investigation was performed on 28 Clostridium difficile strains isolated from 15 antibiotic-associated diarrhea cases and from 13 healthy infants. Through the use of supernatants of the cultures induced by mitomycin C (1 or 3 micrograms/ml), 18 of the 28 C. difficile strains proved to harbor phage particles with a different morphology and size. Most of the phages revealed belonged in phage group B1 with a non-contractile tail. Seven of the 11 toxin-producing, lysogenic C. difficile strains carried a defective phage structure (120 nm-long tail with an incomplete head capsule) alone or together with other normal phages. With different C. difficile strains as indicator, plaque formation could not be detected in any of the lysates.

The mechanisms of cytotoxicity to tumor cells by polymorphonuclear leukocytes stimulated with cytokines.

The mechanisms of tumor cytotoxicity of rat polymorphonuclear leukocytes (PMN) activated with cytokine(s) were studied with the use of supernatants from a rat myelomonocytic leukemia cell line, WRT‐7, incubated in the presence of bacterial lipopolysaccharide (LPS) (LPS WRT‐7 sup) as a source of cytokine. Rat peritoneal PMN treated with LPS WRT‐7 sup showed cytostasis from 3 hr after the start of incubation, while significant cytolysis was first observed after 24 hr. When target tumor cells were separated from PMN at 6 or 12 hr after the start of the assay, 3H‐UdR release from the separated target cells comparable to that from the group incubated with PMN for the whole assay time of 40 hr was observed during the following incubation, which indicates that priming for subsequent lysis occurs at a relatively early stage of the assay. None of various scavengers of active oxygens, inhibitors of heme enzymes, and inhibitors of neutral proteinases inhibited cytolysis mediated by PMN stimulated with LPS WRT‐7 sup. Heparin inhibited PMN cytolysis only when it was added within 1 hr after the start of the assay. Fractionation of heparin by ion exchange chromatography showed a parallelism between the negative charge and the inhibitory effect of heparin on PMN cytotoxicity.

The use of supernatants from neuraminidase and galactose oxidase (NAGO)-treated lymphocytes as a source of T cell growth factor

Human peripheral blood or tonsil lymphocytes produce T cell growth factor (TCGF), when activated with neuraminidase (NA) and galactose oxidase (GO). Partial purification of NAGO-TCGF on Sepharose G-100 columns gave a TCGF-active fraction within the same molecular weight range as the conventional lectin-induced TCGF (approximately 15,000 Da). Human T cells, activated in mixed lymphocyte culture (MLC) with irradiated allogeneic EB-virus transformed B-cells (LCL) could be maintained in continuous culture for several months with retained functional activities. The cells showed similar growth patterns when cultured in the presence of either NAGO-TCGF or PHA-TCGF. The growing cells were characterized by means of monoclonal antibodies. After 4 weeks of culture 98% of these were OKT3+ and 87% were also OKT8+. The cytolytic activities of the cultures were tested in cell-mediated lympholysis (CML) against allogeneic LCL as target cells, in natural cytotoxicity (NK) against K562 cells and in antibody dependent cytotoxicity (ADCC) against bovine erythrocytes. Cultures displaying one or several of these functions were obtained. The results indicate, that TCGF obtained from supernatants of NAGO-activated lymphocytes is as potent as the T cell growth promoting factor obtained by lectin stimulation. One major advantage of using NAGO-generated TCGF is that contamination with lectin is avoided.

Functional differences between cells from MLR colonies grown in soft agar cultures.

This report describes a method of growing soft agar colonies of human T lymphocytes activated in the MLR. Two types of colonies were demonstrated: lower colonies grew within the agar layer, and upper colonies grew on the surface of the agar layer. Three days of priming the lymphocytes in the MLR and the use of supernatants of day-3 MLR cultures to provide T cell colony growth factor were necessary for optimal colony formation. Lymphocytes obtained from colonies were grown in long-term (2 to 4 weeks) cultures to generate sufficient numbers of cells to be tested in different functional assays. Cells from both types of colonies exhibited PLT activity. Upper colony cells showed considerably higher CML activity than lower colony cells (mean percent cytotoxicity 37 +/- 5 vs 6 +/- 3). Cells from both types of colonies contained radiosensitive suppressor cell activity that inhibited the primary MLR. The suppressor cell effect of lower colony cells was specific for the original stimulator, but upper colony cells displayed nonspecific suppressive effects. For both types of colony cells, it appeared that suppressive effects were unrelated to the CML activity of these cells. These data suggest that the soft agar colony assay offers a promising approach to separate subpopulations of lymphocytes activated in the MLR.

Conditions for in vitro haemolytic activity by Corynebacterium ovis exotoxin

A substance, concluded to be Corynebacterium ovis exotoxin on the basis of properties shared, was found to have haemolytic activity below pH 6 and red cell adhesion activity at neutral or slightly alkaline pH. An acidified, solid blood agar medium was used to demonstrate the extent of haemolysis that could be obtained at optimal pH and its inhibition by immune serum. Culture in a liquid medium giving rise to a suitably acidic pH allowed titration for haemolytic activity by the exotoxin and use of supernatant in a haemolysis-inhibition test to detect antitoxin.