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The first paper in this series reported that it was possible to reconstitute the response of low concentrations of mouse spleen lymphocytes to concanavalin A (Con A) by adding small numbers of peritoneal exudate cells (PEC) to the cultures. In this paper it is shown that the role of the PEC in this system can be partially replaced by conditioned medium (CM) prepared from PEC cultures or completely replaced by CM taken from lymphocytes cultured at optimal concentration. These CM were inactive unless fresh Con A was added to the assay cultures. Activity was present in CM which was incubated with lymphocytes or PEC for the shortest possible time but maximal activity was found after 24 hr of incubation. Activity was also found in CM prepared in the absence of Con A. Only in the case of lymphocytes cultured at optimal concentration for 24 hr was there substantially more activity in the CM thus prepared if Con A was present. PEC preparations depleted of T lymphocytes produced as much activity in CM as the untreated control. CM produced by PEC was less sensitive to heat treatment or to freezing and thawing than that produced by lymphocyte cultures. [ABSTRACT FROM AUTHOR]

Human allergic serum has been fractionated by: Chromatography on diethyl amino ethyl cellulose columns. Ultracentrifugation in buffered sucrose gradients. Passive transfer tests with the fractions showed skin sensitizing activity to horse dandruff allergen to be associated with an electrophoretic gammax­globulin component possessing a 7S sedimentation constant. The difficulties encountered in the use of the chromatographic procedure for obtaining potent reagin preparations are discussed. [ABSTRACT FROM AUTHOR]

A total of 233 primary alloreactive T-cell clones have been tested for the production of interleukin-2 (IL-2), interleukin-3 (IL-3), immune (gamma) interferon (IFN) and granulocyte-macrophage colony-stimulating factor (CSF-2), S-cell growth factor I and II (SCGFI, BCGFII), and eosinophil differentiation factor (EDF). EDF was assayed by means of the eosinophil differentiation assay (EDA). Two principal correlations were observed: IL-3 was shown to be the major lymphokine detected in the bone marrow proliferation assay (BMPA) used to detect CSF-2, and there was a high correlation between the EDA and SCGFII. Subsequent work has suggested that this latter correlation is because a single factor is responsible for both activities. Apart from these two exceptions, and low level correlations probably due to the fact that different assays detect more than one lymphokine, there was no evidence for co-ordinate expression of lymphokines. There was a large variation in amounts of individual lymphokines produced. More clones produced multiple lymphokines than would be expects from independent control. Taken together, this pattern of regulation is consistent with the hypothesis that antigen stimulation of T cells results in the activation of all the lymphokine genes, but the amount of each produced is determined by secondary controlling mechanisms. [ABSTRACT FROM AUTHOR]

(1) Oil-elicited guinea-pig peritoneal macrophages (MPs) cultured for 2-3 days in medium containing supernatant of concanavalin A-activated lymphocytes (lymphokine, LK) generated large amounts of hydrogen peroxide (H2O2), as detected by the horseradish peroxidase (HRP)dependent oxidation of phenol red, in the absence of further stimulation. (2) H2)2 production increased with the duration of exposure to LK and was evident at high dilutions of supernatant (1/64). (3) Parallel cultures of MPs in medium or a supernatant of non-activated lymphocytes also increased their H2O2 production during cultural but levels at all time intervals were significantly lower than those measured in LK treated cultures. (4) The marked increase in H2O2 production was associated with only a moderate increment in superoxide (O2-) liberation and this was not specific for LK treated cells. (5) Detection of LK-dependent H2O2 production was dependent on ongoing pinocytosis during the assay. This and other arguments suggest that the HRP-phenol red assay, as applied here, detects H2O2 generation occurring at the level of intracellular vesicles and it is concluded that LK elicits H2O2 production that is limited to the intracellular compartment. (6) H2O2 is, apparently, derived by non-enzymatic dismutation of Of taking place within the cell; (7) LK treatment of MPs also resulted in a significant reduction in catalase activity. [ABSTRACT FROM AUTHOR]

We have developed a simple assay for human peripheral-blood suppressor cells induced by soluble protein antigens. Suppressor cells were obtained from 6-day cultures in the presence of tetanus toxoid or PPD and were assayed in co-cultures with fresh autologous PBLs. The proliferative response in this second culture showed significant suppression varying from 50% to 90%. The suppressor cells are OKT8-positive, resistant to X irradiation and nonspecific in their function. They do not impede either the function or production of TCOF in the secondary culture but we are unable to detect a non-specific suppressor factor made by these cells. [ABSTRACT FROM AUTHOR]

Using an homologous pig system, the lysis of African swine fever virus-infected cells by antibody and complement was investigated. The optimal conditions necessary for lysis are described, and it was found that the system was unique amongst reported virus infections in that infected cells were lysed by the classical complement pathway and not the alternative pathway. Development of antibody capable of initiating complement-mediated lysis was relatively late in the infected pig, although functional in vitro assays suggested that it might act as a significant effector mechanism. Investigations of sera taken from pigs infected with varying African swine fever isolates indicate that the assay may provide a means of discriminating between strains. [ABSTRACT FROM AUTHOR]

The CTLL-2 bioassay is used frequently to determine interleukin-2 (IL-2) concentrations in experimental samples, including samples that contain reagents which affect the CD28-B7 interaction. We therefore evaluated whether the CD28-B7 pathway plays a role in the growth of CTLL-2 cells. Flow cytometry demonstrated that CTLL-2 cells express both CD28 and B7-1. CTLA4-immunoglobulin (CTLA4-Ig) inhibited the growth of CTLL-2 cells over a range of IL-2 concentrations, suggesting that the CD28-B7 interaction plays an important role in the growth of CTLL-2 cells. Anti-B7-1 antibody also inhibited CTLL-2 proliferation at all concentrations of IL-2. These results indicate that the CTLL-2 bioassay may not be a reliable means of determining IL-2 levels in experimental samples containing reagents that affect the CD28-B7 interaction. They also suggest that co-expression of CD28 and B7 may contribute to the growth of malignant T cells. [ABSTRACT FROM AUTHOR]

A bioassay specific for human granulocyte colony-stimulating factor (G-CSF) was developed and used to measure G-CSF production in human pregnancy tissues. G-CSF was secreted by both foetal chorionic villous and maternal decidual tissues taken in the first trimester and at term. The level of G-CSF production by placental tissue was 6750 (1250-10,000) units of bioactivity per g of tissue in 48 hr in the first trimester and 104 (83-190) U/g at term. Bioactive G-CSF was also secreted by decidual tissue, more in the first trimester than at term. ELISA immunoassays measured 75 (10-820) ng/g/48 hr of G-CSF antigen from first trimester placenta, 15 (10-50) ng/g from first trimester decidua and less than 2 ng/g from term placenta. RNA isolated from decidual and chorionic villous tissue or from cells purified by flow cytometry, contained G-CSF mRNA in both tissues. In decidua, mRNA for G-CSF was confined to the macrophages, and cytotrophoblast from term amniochorion contained no detectable G-CSF mRNA. No G-CSF, measured as bioactivity or as mRNA, was detectable in choriocarcinoma cell lines. [ABSTRACT FROM AUTHOR]

A practical immunological method for the assay of clinical biological solutions, such as allergen extracts, in agar gel plates is presented. The method permits assay of individual antigens in antigenic mixtures. Application of the method to the assay of three ‘unknown’ dilutions of each of four grass pollen extracts gave results which were within—9 per cent and +6.7 per cent of the actual values. It is postulated that an immunological assay, since it indicates the antigenic state of the solution, is a more valid index to the probable clinical potency of an allergen extract than the Noon, total nitrogen, or protein nitrogen units currently in use. The quantitative gel plate assay method is quick and simple in application and suited to routine use. [ABSTRACT FROM AUTHOR]

A micro-immune adherence assay employing direct microscopic observation was used to detect the presence of thymus-derived cell antigenic specificities on guinea-pig lymph node cells, purified peritoneal exudate lymphocytes, and thymocytes using a heterologous burro anti-guinea-pig thyrnocyte serum. A method is described for the direct determination of thymus-derived cell specificity of anti-lymphocyte sera. [ABSTRACT FROM AUTHOR]