The effect of X-rays and neutrons on lymphocyte death and transformation.

The effects of X-rays and neutrons on human lymphocytes in vitro has been tested. Radiation sensitivity of untransformed lymphocytes was assessed by the appearance of pyknotic cells, and the response of cells after stimulation by phyto-haemagglutinin was tested (a) morphologically and (b) by changes in DNA synthesis, using a labelled thymidine analogue. The data obtained for interphase cells suggest that lymphocytes are a mixed cell population with an insensitive component forming about 20 per cent of the population. The percentage of normal cells observed after both X-ray and neutron irradiation lie on the same dose--effect curve giving an r.b.e. of one. A biphasic response is seen after PHA stimulation with both tests of damage indicating at least two sub-populations of lymphocytes and these give r.b.e. values in the range 1.95 to 2.45. Providing the in vivo response is similar to that in vitro the r.b.e. for damage to circulating lymphocytes will be small and the reduction in white cell count will not therefore be a major factor limiting dose in neutron therapy.     

Antitumor activity of a new platinum(II) complex with low nephrotoxicity and genotoxicity

Cisplatin is an important antineoplastic agent, but dose-limiting nephrotoxicity and the occurrence of cellular resistance prevent its potential efficacy. Moreover, cisplatin is known to be carcinogenic and genotoxic in mammalian cells and this feature is of a special interest due to the risk of inducing secondary malignancies. There is a great interest in developing new platinum agents that have broad spectrum of antitumor activity and reduced toxicity. We have recently synthesized a novel platinum(II) coordination complex containing a pyridine nucleus and a dithiocarbamate moiety as ligands, [Pt(ESDT)(Py)Cl], in order to obtain an agent with more favorable therapeutic indices than cisplatin. In this study, the new platinum(II) complex was tested for its cytotoxicity, by MTT assay, on various human cancer cell lines also including different cisplatin-resistant cells endowed with different mechanisms of resistance. On human peripheral blood lymphocytes we evaluated the genotoxic potential of [Pt(ESDT)(Py)Cl] via micronuclei and SCE detection. We also performed in vivo experiments with the purpose of investigating the antitumor and nephrotoxic effects of the new platinum(II) complex. The antitumor activity was studied in ascitic or solid Ehrlich carcinoma bearing mice while nephrotoxicity was monitored in male Wistar rats by means of histopathological findings of renal specimens and of biochemical investigation on urinary parameters (GS and NAG activities and of TUP excretion) of urine samples. The results reported here indicate that [Pt(ESDT)(Py)Cl] showed a remarkable in vitro antitumor activity (with IC50 values about twofold as low as those of cisplatin), moreover, it markedly circumvented the acquired cisplatin resistance in selected human cancer cells. The analysis of the cytogenetic damage in normal cells clearly attested that the new dithiocarbamate complex, tested at equitoxic concentrations, is less genotoxic than cisplatin. Chemotherapy in Ehrlich carcinoma bearing mice with [Pt(ESDT)(Py)Cl] was significantly better tolerated than that with cisplatin. Against the ascitic tumor, [Pt(ESDT)(Py)Cl], showed an activity noticeably higher than that of cisplatin in increasing the life span of treated animals (% T/C = 190 and 129, respectively). In solid-tumor-bearing mice, [Pt(ESDT)(Py)Cl] induced a tumor size reduction very close to that observed with the reference compound. Finally, our findings obtained from the nephrotoxicity studies demonstrated [Pt(ESDT)(Py)Cl] was not nephrotoxic, contrary to cisplatin which caused a notorious acute proximal tubular damage. In summary, [Pt(ESDT)(Py)Cl] may be considered as a new platinum(II) complex with remarkable antitumor activity and low nephrotoxicity and genotoxicity compared with cisplatin.     

Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP

Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man. The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-[Pt(AF)2Cl2]) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation. It was shown that cis-[Pt(AF)2Cl2] acts directly and is mutagenic for three strains of S. typhimurium: TA97a, TA98 and TA100. In comparison with cis-DDP this compound showed a weaker genotoxicity. Contrary to cis-DDP it has not shown toxic properties in the tester bacteria. The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro. The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared. In all test systems applied, cis-[Pt(AF)2Cl2] was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation. cis-[Pt(AF)2Cl2] has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound. These results provide important data on the genotoxicity of cis-[Pt(AF)2Cl2] and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.     

Control of aromatase activity in breast tumours: the role of the immune system

Cytokines such as interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF), have been identified as important regulators of aromatase activity in fibroblasts derived from normal and malignant breast tissues, and may play an important role in controlling aromatase activity in breast tumours. The major source of such cytokines within breast tumours remains to be established but macrophages and lymphocytes, which can infiltrate tumours, have been identified as a potential source of aromatase stimulatory cytokines. To obtain further insight into the possible role played by the immune system in cancer development, and in particular its ability to regulate aromatase activity via cytokine production, we have obtained peripheral blood monocytes and lymphocytes from an immunosuppressed kidney transplant recipient, receiving cyclosporin A therapy, and a woman with breast cancer. Monocytes and lymphocytes were stimulated with lipopolysaccharide (LPS), and the conditioned medium (CM) collected from these cells was tested for its ability to stimulate aromatase activity in fibroblasts derived from normal breast tissue from a woman undergoing lumpectomy for the removal of a breast tumour. The white blood cell count was lower for the immunosuppressed patient, mainly because of the reduction in the number of monocytes and lymphocytes. The ability of CM from the monocytes and lymphocytes of the immunosuppressed patient to stimulate aromatase activity was significantly reduced (68% and 82% for monocytes and lymphocytes, respectively) compared with that of CM from the cells of the woman with breast cancer. It is possible, therefore, that immunosuppression, which has been found to be associated with a reduction in the incidence of de novo breast cancer in kidney transplant recipients, may exert its effect by inhibiting cytokine production by the cells of the immune system and thus oestrogen synthesis. In contrast to the stimulatory effects that TNF has on aromatase activity in breast fibroblasts, in MCF-7 breast cancer cells, which possess low aromatase activity, it reduced activity. However, the extent of inhibition of aromatase activity in these epithelial cells was much lower than the marked stimulation which it can induce in breast fibroblasts.     

Relative biological effectiveness of tritium oxide by the criterion of death of the germ cells in mice

It was shown that low-level beta-radiation of tritium is much more effective than gamma-radiation of 137Cs with respect to reduction of the mouse testis weight. The RBE coefficient increases from 1.8 at a dose of 1 Gy to 2.2-2.3 at 0.1 Gy. On the basis of the data obtained by the authors and those reported in the literature a quality factor is proposed for tritium: QF = 2. Using THO and Na36Cl labels a mean water content of the testis cells, necessary for the estimation of a tritium-radiation dose absorbed, has been determined: gamma ct = 0.70 +/- 0.02 ml/g.     

Effect of lymphocytes on the differentiation of in vitro transformed L-line mouse fibroblasts

Possible participation of immune system cells in the differentiation of non-lymphoid tissues has been examined. Lymphocytes were tested on transformed fibroblasts of L mouse strain. The presence of lymphocytes in L cell culture enhanced their differentiation, assessed by morphological, proliferative and biophysical criteria. The induction of L-cell differentiation took place during contact and short-distant interaction both with mouse and human lymphocytes. The phenomenon had a stable character, as it was revealed in L cells separated from lymphocytes during passaging. The results obtained indicate real and potential abilities of the immune system to affect differentiation of proliferating non-lymphoid tissues.     

Measurements of total body calcium by prompt-gamma neutron activation abalysis using a252Cf source

A clinical neutron activation instrument has been developed for in vivo elemental analysis. Utilizing the prompt-capture gamma ray technique, simultaneous total body (TB) measurements of primarily Ca, but also Cl, N, C, and H are routinely performed. This paper describes a technique for the measurement of TBCa (g) that relies on the use of TBCl as an internal standard. The method has been tested with four anthropomorphic phantoms covering a range of body habitus. The mean discrepancy between the measured and known Ca contents was 3.6%. The technique has been applied to two patient groups, and encouraging results were obtained.     

Cellular microcytotoxicity in a human bladder cancer system: Analysis of in vitro lymphocyte-mediated cytotoxicity against cultured target cells

Summary The microcytotoxicity test (MCT) has been used to determine the cytotoxic effects of purified peripheral blood lymphocytes from patients with carcinoma of the urinary bladder (BT), tumor control patients (TC) (tested after therapy), and healthy donors (HD) against cultured bladder tumor cells, melanoma cells, and normal bladder cells. Lymphocytes from all three donor groups were tested in parallel. Disease-specific cytotoxicity (CTX) is defined as statistically significant and selective destruction of disease-related tumor target cells by the test lymphocytes in comparison with the baseline controls. Nonspecific CTX is defined as statistically significant destruction of a proportion (selective) or all (nonselective) disease unrelated target cells by the effector cells.Within the different donor groups, an enormous variation in non-disease related cytotoxic effects against the different cell lines was seen. It appeared that the selection of the baseline control influences the level of CTX and the specificity of the reaction.In order to determine whether a disease-specific cytotoxic effect was superimposed on the nonspecific cytotoxicity, the overall cytotoxic effects of the lymphocytes from the BT, TC patients and HD were compared statistically. The analysis of results revealed that effector cells from BT, TC patients and HD showed the same pattern of reactivity, but the CTX of lymphocytes from BT patients tested before therapy was stronger in comparison with the CTX of lymphocytes from the same group of BT patients after therapy and in comparison with the CTX of lymphocytes from HD and TC patients.     

Early electrochemical events in lectin-lymphocyte interaction

Concanavalin A, at extremely low concentrations, will produce significant increases in the electrophoretic mobility of murine splenic T lymphocytes. It has been established that the alteration in cellular surface charge is mediated by a factor produced by those lymphocytes that have reacted directly with con A. We originally conjectured that the mobility change might be the consequence of an alteration in the distribution of the charged moieties of membrane glycoproteins. The results of experiments conducted at low temperature raise some questions about this mechanism. Further experiments have been performed to establish the nature of the physicochemical alterations in the peripheral zone of the factor-stimulated lymphocytes that are manifest as changes in cellular surface charge. The results of these studies indicate that, subsequent to the interaction of T lymphocytes with con A, there is a reduction in the number of positively charged amino groups effective at the electrophoretic surface of the cells.     

Early electrochemical events in lectin-lymphocyte interaction. I. Mechanism of lectin-mediated changes in the electrophoretic mobility of lymphocytes

Concanavalin A, at extremely low concentrations, will produce significant increases in the electrophoretic mobility of murine splenic T lymphocytes. It has been established that the alteration in cellular surface charge is mediated by a factor produced by those lymphocytes that have reacted directly with con A. We originally conjectured that the mobility change might be the consequence of an alteration in the distribution of the charged moieties of membrane glycoproteins. The results of experiments conducted at low temperature raise some questions about this mechanism. Further experiments have been performed to establish the nature of the physicochemical alterations in the peripheral zone of the factor-stimulated lymphocytes that are manifest as changes in cellular surface charge. The results of these studies indicate that, subsequent to the interaction of T lymphocytes with con A, there is a reduction in the number of positively charged amino groups effective at the electrophoretic surface of the cells.     

Spontaneous cytotoxicity of human lymphoblast cell lines mediated by normal peripheral blood lymphocytes. I. Differential susceptibility of T-versus B-cell lines.

Human lymphoblast cell lines of B- and T-cell origin have been tested for their ability to serve as targets in a 4-hr ⁵¹Cr release microcytotoxicity assay using normal human peripheral blood lymphocytes as effector cells. Cell lines of T-cell origin were susceptible to lysis in this assay by effector lymphocytes from all normal donors tested. Cell lines of B-cell origin were repeatably lysed by normal lymphocytes from some, but not all donors. Spontaneous cytotoxicity of B-cell lines, when observed, was also quantitatively less than was obtained using T-cell lines as targets. One cell line (RPMI-7666), of B-cell origin, was not susceptible to spontaneous cytotoxicity by almost all of the normal lymphocyte effectors tested. Lymphocytes from patients with acute lymphoblastic leukemia in remission were less capable of effecting lysis in this assay.     

Evidence for an essential histidine in carboxypeptidase Y. Reaction with the chloromethyl ketone derivative of benzyloxycarbonyl-L-phenylalanine.

The possible role of histidine residues in the catalytic function of carboxypeptidase Y from bakers' yeast has been investigated using site-specific reagents. Among the reagents tested, benzyloxy-L-phenylalanylchloromethane (Z-PheCH2Cl) was the most powerful inhibitor of the enzyme. It irreversibly inactivated both the peptidase and esterase activities with an apparent second order rate constant of 3.8 M-minus 1 S-minus 1; the D isomer caused essentially no effect on either activity. Inhibition by L-Z-PheCH2Cl, the reaction retarded by certain competitive inhibitors of the enzyme. Using radioactive L-Z-PheCH2Cl, the reaction with the enzyme was shown to be essentially stoichiometric. Diisopropylphosphorofluoridate (iPr2PF)-inactivated enzyme failed to react with Z-PheCH2Cl, and conversely, the Z-PheCH2Cl-inhibited enzyme failed to react with radioactive iPr2PF. Amino acid analyses of the Z-PheCH2Cl-inactivated enzyme revealed the loss of essentially 1 residue, with a concomitant yield of a 0.62 residue of N-t-carboxymethylhistidine. Since carboxypeptidase Y has a reactive serine at its active center, we concluded from these results that the mechanism involves a charge-relay system in the hydrolysis of peptide and ester substrates, as in chymotrypsin. An -SH group of carboxypeptidase Y was not affected during the reaction with L-Z-PheCH2Cl. The generic name "serine carboxypeptidase" has been proposed for carboxypeptidase Y and for the iPr2PF-sensitive carboxypeptidases from plants, molds, and animal tissues, in order to distinguish them from "metal carboxypeptidase" to which carboxypeptidase A (EC 3.4.12.2) and B (EC 3.4.12.3) belong.     

A model of non-electrogenic cation transport through bilayer lipid membranes

Quantitative relationship between the proton diffusion potential in the unstirred layers near BLM and NH4Cl was investigated. It has been found that in the range of low concentrations of NH4Cl the potential value depends on the difference of salt concentrations on different sides of the membrane. At higher concentrations the potential value is the function of the ratio of salt concentrations at different BLM sides. In the limiting case the potential value equals 58 mV with NH4Cl concentrations ratio equaling ten. A model is suggested which quantitatively describes the experimental data. It has been shown that the results obtained can be used in determining BLM permeability for weak acids and bases.     

The cytotoxic effect of fumonisin B<Subscript>1</Subscript> and ochratoxin A on human and pig lymphocytes using the Methyl Thiazol Tetrazolium (MTT) assay

Lymphocytes cell obtained from healthy human donors and pigs were exposed to fumonisin B₁ (FB₁) and ochratoxin A (OTA), which have been found to be immunosuppressive, carcinogenic and mutagenic, to ascertain their single and combined cytotoxic effects with time and to assess the suitability of animal lymphocytes as test agents in comparison to human cells. The main objectives of this work were to assess the use of animal lymphocytes, particularly pig lymphocytes, for their use in the Methyl Thiazol Tetrazolium (MTT) cytotoxicity test, making them more accessible to animal research-based institutes in comparison to human lymphocytes previously used, and to study the cytotoxic and synergism or antagonistic effects of FB1 and OTA. The MTT assay, which measures cell viability and proliferation based on reduction of MTT to a blue dye, also used the addition of phytohaemagglutinin (PHA) to stimulate the blood cells. The results showed a progressive decrease in lymphocytes viability with time of exposure to the toxins. It was also noted that FB1, as compared to OTA, had a lower cytotoxicity on both human and pig lymphocytes cells. In addition, when the two mycotoxins were combined, a synergistic decrease of cell viability in both human and pig lymphocytes was observed, with pig lymphocytes showing a greater sensitivity. This study has shown that the MTT assay can be used for the determination of cytotoxicity of mycotoxins using animal, and in particular pig, lymphocytes, which eliminates the use of human donors and other cell cultures.     

Immunological depression produced in vitro by the action of normal RNA on mice spleen cells

Macrophages and lymphocytes from normal mice spleens were treated separately with isologous RNA, pooled again and then stimulated with antigen (rat red blood cells). The number of rosette-forming cells was used as a measure of the antibody production. A low immunological response was obtained when macrophages were incubated with normal RNA. No effect was observed when macrophages were previously sensitized with the antigen and then incubated with normal RNA or when the antigen was pretreated with RNA. On the other hand, RNA had apparently no effect on lymphocytes. Macrophage phagocytosis was markedly diminished by the addition of RNA to the culture.It can be concluded that the effect of normal RNA is exerted on the macrophages by depressing their ability either to recognize or process the antigen or both. The capacity of macrophages to transfer the immunological information to lymphocytes after it has been acquired does not seem to be altered. It can be reasonably assumed, moreover, that RNA has no effect on the antigen.     

Regulation of hepatic gluconeogenesis in the guinea pig by fatty acids and ammonia.

Octanoate and L-palmitylcarnitine inhibited the synthesis of P-enolpyruvate from alpha-ketoglutarate and malate by isolated guinea pig liver mitochondria. A 50% reduction in P-enolpyruvate formation was obtained with 0.1 to 0.2 mM octanoate or with 0.06 to 0.10 mM L-palmitylcarnitine. At these concentrations, oxidative phosphorylation remained intact and only much higher concentrations of fatty acids altered this process. The addition of NH4Cl in the presence of malate and increasing concentrations of alpha-ketoglutarate (or vice versa) enhanced the formation of glutamate, aspartate, and P-enolpyruvate. The addition of increasing concentrations of NH4Cl in the presence of fixed amounts of malate and alpha-ketoglutarate had a similar effect. Furthermore, the inhibition of P-enolpyruvate synthesis by fatty acids and the reduction of the acetoacetate to beta-hydroxybutyrate ratio were reversed by the addition of NH4Cl. Cycloheximide, which blocks energy transfer at site 1 of the respiratory chain, decreased P-enolpyruvate formation. When cycloheximide and either octanoate or L-palmitylcarnitine were added together, there was an even greater reduction in P-enolpyruvate synthesis from either malate or alpha-ketoglutarate than was noted with either fatty acid alone. Since cycloheximide lowers the rate of ATP synthesis this may in turn reduce P-enolpyruvate formation by a mechanism independent of changes in the mitochondrial NAD+/NADH ratio caused by fatty acids. In the isolated perfused liver metabolizing lactate, the inhibitory effect of octanoate on gluconeogenesis was partially relieved by the addition of 1 mM NH4Cl, but remained unchanged in the presence of 2 mM NH4Cl, despite a highly oxidized NAD+/NADH ratio in the mitochondria. In contrast to glucose synthesis, urea formation was markedly increased during the infusion of 1 mM as well as 2 mM NH4Cl. After cessation of NH4Cl infusion, there was an increase in glucose production, to a rate as high as that observed in the absence of octanoate. This increase was accompanied by the disappearance of alanine, aspartate, and glutamate which had been stored in the liver during NH4Cl infusion. Urea synthesis also decreased progressively. These results indicate that gluconeogenesis in guinea pig liver is regulated, in part, by alterations in the mitochondrial oxidation-reduction state. However, the modulation of this effect by changing the concentrations of intermediates of the aspartate aminotransferase reaction indicates competition for oxalacetate between the aminotransferase reaction and P-enolpyruvate carboxykinase.     

Chromosomal damage induced by caprolactam in human lymphocytes

Caprolactam was tested in the in vitro human lymphocyte cytogenetic assay both in the presence and absence of S9 mix at dose levels up to 5500 micrograms/ml using lymphocytes obtained from a male donor and in the presence of S9 mix using lymphocytes obtained from a female donor. Statistically significant increases in chromosomal damage were observed at 5500 micrograms/ml dose level in cells from both donors. This positive response was enhanced by the inclusion of chromosomal gaps in the calculations. It was concluded that caprolactam induces chromosomal damage in human lymphocytes in vitro albeit at comparatively high dose levels.     

Activation of Na+/H+ exchange and the expression of cellular proto-oncogenes in mitogen- and phorbol ester-treated lymphocytes

It has been suggested that an intracellular alkalinization, resulting from stimulation of Na+/H+ exchange, is a necessary step and perhaps the signal leading to cellular proliferation in cells stimulated by mitogens. This hypothesis was tested by measuring the early stages of the proliferative cascade in cells where antiport activity was precluded by omission of Na+ or by the addition of potent amiloride analogs. To circumvent possible nonspecific effects due to long incubations under these conditions, an early response to mitogens, the increased level of c-fos mRNA, was monitored. In rat thymic lymphocytes, the increase in the level of c-fos RNA induced by the combination of 12-O-tetradecanoylphorbol 13-acetate and ionomycin was unaffected by inhibition of the antiport with 5-(N-ethyl-N-propyl)amiloride. Increased c-fos RNA was also observed in the absence of Na+ and when alkalinization was prevented by means of nigericin. Similar results were obtained with phytohemagglutinin-stimulated human T lymphocytes. Moreover, although the lectin stimulated the antiport in these cells, an alkalinization was not observed, due to the concomitant occurrence of an acidifying process. It was concluded that the stimulation of the Na+/H+ antiport that accompanies the addition of mitogens is neither sufficient nor necessary for the initiation of cellular proliferation.     

Na(+)/K(+)/Cl(-) cotransporter activates mitogen-activated protein kinase in fibroblasts and lymphocytes.

In a previous work, we have shown that overexpression of the Na(+)/K(+)/Cl(-) cotransporter (NKCC1) induces cell proliferation and transformation. We investigate in the present study the role of the NKCC1 in the mitogenic signal transduction. We show that overexpression of the cotransporter gene (NKCC1) in stablely transfected cells (Balb/c-NKCC1), resulted in enhanced phosphorylation of the extracellular regulated kinase (ERK) to produce double phosphorylated ERK (DP-ERK). Furthermore, the level of DP-ERK was reduced by 50-80% following the addition of bumetanide, a specific inhibitor of the Na(+)/K(+)/Cl(-) cotransporter, in quiescent as well as in proliferating cultures of the Balb/c-NKCC1 clone. In order to explore further the role of the Na(+)/K(+)/Cl(-) cotransporter in mitogenic signal transduction, we measured the effect of the two specific inhibitors of the cotransporter; bumetanide and furosemide, on DP-ERK level in immortalized non-transformed cells. In Balb/c 3T3 fibroblasts stimulated with FGF, bumetanide, and furosemide inhibited 50-60% of the ERK 1/2 phosphorylation. The inhibitor concentration needed for maximal inhibition of ERK 1/2 phosphorylation was similar to the concentration needed to block the K(+) influx mediated by the Na(+)/K(+)/Cl(-) cotransporter in these cells. To analyze whether the Na(+)/K(+)/Cl(-) cotransporter has a role in the mitogenic signal of normal cells, we measured the effect of bumetanide on ERK phosphorylation in human peripheral blood lymphocytes. The phosphorylation of ERK 1/2 in resting human lymphocytes, as well as in lymphocytes stimulated with phytohemagglutinin (PHA) was inhibited by bumetanide. The effect of bumetanide on ERK 2 phosphorylation was much lower than that of ERK 1 phosphorylation. The finding that the Na(+)/K(+)/Cl(-) cotransporter controls the ERK/MAPK (mitogen-activated protein kinase) signal transduction pathway, support our hypothesis that Na(+) and K(+) influxes mediated by this transporter plays a central role in the control of normal cell proliferation. Exploring the cellular ionic currents and levels, mediated by the Na(+)/K(+)/Cl(-) cotransporter, should lead to a better comprehension of cell proliferation and transformation machinery.     

Mitogenic factor from inbred guinea pigs. I. Isolation of the factor

Methods are described for the reproducible elicitation of mitogenic factor (MF) from antigen-sensitive lymphocytes of inbred strains of guinea pigs. The use of inbred animals minimizes complications due to histocompatibility factors. Each of several antigens tested was effective. Mitogenic factor is released in vitro as early as 6 hr after stimulation of lymphocytes by antigen. It was obtainable from serum-free cultures in which medium RPMI-1640 was used; this should facilitate isolation of MF. The addition of 5 mMl-cysteine to cultures substantially improved the yield of MF. MF was obtained from cultures of lymph node cells of highly purified small lymphocytes, which indicates that the small lymphocyte is the source of MF in the guinea pig. It was shown that MF can induce mitosis as well as blast transformation in non-immune lymph node cells. MF from a given strain of guinea pig is capable of stimulating lymphocytes of another strain.