Aristo Wojdani

Agglutinins in the earthworm Lumbricus terrestris: naturally occurring and induced.

A naturally occurring hemagglutinin against rabbit and rat erythrocytes is contained in the coelomic fluid of the earthworm Lumbricus terrestris. The hemagglutinin reacts with some chicken and human erythrocytes, but not others, and does not react with the erythrocytes of seven other vertebrates. Hemagglutinins appear in increased amounts within 24 hr after injection of rat, rabbit, horse and sheep erythrocytes, and some chicken and human erythrocytes, and are the highest (approximately four- to sevenfold) with rabbit erythrocytes. The response is brief, and increased or more rapid responses do not occur after multiple injections. Cross reactivity and absorption data indicate a close or possibly identical relationship between agglutinins induced against different erythrocyte types. Effects of heating, enzyme and chemical treatment on induced anti-rabbit erythrocyte agglutinins indicate at least two and perhaps three different agglutinins. Two of the agglutinins are protein, one trypsin-sensitive and the other trypsin-resistant. Agglutinin activity is reduced in the absence of divalent cations. Sensitivity to heat varies with the type of agglutinin. The naturally occurring agglutinin is a protein, trypsin-resistant and unaffected by heating at 100 degrees C for 30 min. These hemagglutinins constitute one of the earthworms humoral factors that may participate in immune responses.

Effects of methylcholanthrene and benzanthracene on blastogenesis and aryl hydrocarbon hydroxylase induction in splenic lymphocytes from three inbred strains of mice.

The effects of polycyclic aromatic hydrocarbons (PAH) such as benzanthracene (BA) and methylcholanthrene (MCA) on 3H-thymidine incorporation and aryl hydrocarbon hydroxylase (AHH) induction were assayed in mitogen-activated and non-activated splenic lymphocyte cultures derived from three strains of mice (C57BL, C3H and DBA/2). Results of three separate experiments on blastogenesis and the substrate induction of AHH were statistically significant. In mitogen-activated and non-activated lymphocytes from C57BL and C3H mice (AHH responsive strains) the percentage of blastogenesis induced by BA or MCA was higher than in lymphocytes from the non-responsive strain (DBA/2). On the basis of the PAH concentrations used MCA and BA were similar as inducers of blastogenesis and of AHH activity. AHH induction was measurable only in mitogen-activated lymphocytes and showed a non-linear relation to blastogenesis. In responsive strains, 10 microM oF BA or 1.5 microM of MCA induced AHH 2-5 fold, while in non-responsive mice induced AHH was very close to the basal level. This difference between the level of induction of lymphoblast formation and AHH in responsive and non-responsive strains of mice may be related to different subpopulations of lymphocytes in spleen or other lymphatic organs.

Immunocytotoxicity effects of polycyclic aromatic hydrocarbons on mouse lymphocytes

The in vivo effects of 3 polycyclic aromatic hydrocarbons (PAH): 3-methylcholanthrene (MCA), benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP) on the ability of mouse lymphocytes to bind and kill target tumor cells in vitro were measured. C57 and C3H inbred mice were preimmunized with P815 tumor cells and then treated with a single i.p. injection of corn oil alone or with varying doses of the above PAH compounds (0.5-50 mg/kg body wt). At different post-injection times, antigen sensitized splenic lymphocytes (SL) and peritoneal exudate lymphocytes (PEL) were measured for binding and killing rates, using a single cell assay. MCA doses of 5 and 50 mg/kg inhibited SL: target cell binding 29-42% and PEL: target cell binding 23-60%. BaP had a similar significant dose dependent suppression on SL and PEL binding. Target cell killing rates by SL and PEL from MCA and BaP treated C57 and C3H mice were consistently suppressed at significant levels, compared to oil injected controls (P less than 0.05). On the other hand, binding and killing rates by SL and PEL from BeP treated mice showed an inconsistent and borderline significance at the above dose levels. When measured as a function of post-injection time of MCA, binding rates of SL from both mouse strains remained essentially unchanged after 10, 30 and 45 days. Target cell killing by SL from C3H and C57 mice, however, was suppressed 55-65% after 10-30 days post-injection. At 45 days post-injection, the capacity of SL to kill target tumor cells was restored to 64-70% of control values. The results suggest that binding is an early event that depends on dose, whereas target cell killing is a function of dose and post-injection time.

Mitogenic Effect of Earthworm (Lumbricus terrestris)Coelomic Fluid on Mouse and Human Lymphocytes

We have cultured mouse and human lymphocytes with earthworm (Lumbricus terrestris) coelomic fluid and measured their mitogenic responses. Normal fluid was collected from untreated worms, while induced fluid was harvested from worms injected 24 h earlier with rabbit erythrocytes. At low protein concentrations in the coelomic fluid, human and mouse lymphocytes were significantly activated, as measured by incorporation of 3HTdr. The activation index for induced fluid was approximately 2.5 times that for uninduced fluid. Separation of mouse lymphocytes into B- and T-cell populations revealed that primarily T cells were activated. Normal and induced coelomic fluid contains 12 and 28 electrophoretic bands and agglutinin titers of 32 and 1024, respectively. Addition of agglutinin inhibitors or absorption of agglutinins from coelomic fluid did not alter levels of mitogenic activity, thus the relation between earthworm agglutinins and mitogens is problematic. Techniques designed to separate and purify the agglutinins are in progress to elucidate this point.

Suppression of basal and Corynebacterium parvum-augmented NK activity during chemically induced tumor development.

C3H mice were injected subcutaneously (s.c.) with a tumorigenic dose (150 micrograms/mouse) of 3-methylcholanthrene (MC), followed by a 24-h injection and subsequent weekly injections of Corynebacterium parvum (CP) i.p. for a total of 100 days. Basal and CP-augmented NK cell activities were measured in controls and treatment groups during pre-tumor and tumor development stages. Basal NK activity in spleen, peripheral blood and lung tissue was enhanced by CP, but was suppressed by MC. A resulting transient MC induced suppression of splenic NK activity at 10 days was partially restored and sustained by CP treatment and immunosuppression was again observed in tumor-bearing compared to control mice. Mice treated with MC alone showed a higher tumor incidence than animals treated with MC + CP at 45-60 days, while there was no difference in tumor incidence in these two treatment groups at 100 days post injection. The mechanism of the observed transient immunosuppression induced by MC appears to be related to an early toxic effect on large granular lymphocytes (LGL) which was decreased at 10 days and again at 100 days in tumor-bearing mice. Although MC did not appear to exert an effect on effector:target cell conjugate formation, an early suppression in the lytic activity of LGL, may have predisposed the animal to malignant transformation of susceptible cells at the site of MC injection.

In vivo augmentation of natural killer cell activity by Candida albicans

We determined in vivo effects of Candida albicans (CA) on murine natural killer (NK) cell activity. C3H mice were treated with heat-killed CA and splenic NK cell activity assayed at 2, 7, 30 and 50 days post treatment. A single injection of CA caused enhancement of splenic NK activity as measured in a 4 h 51Cr-release assay. Peak NK activity was detected at day 7 and persisted for up to 30 days, after which it declined to control values at 50 days. Augmentation of NK activity by CA resulted from enhanced lytic effects of NK cells, which was independent of effector cell binding capacity. Moreover, enhanced NK activity was associated with an increase in the proliferative response to CA antigen and in splenic cellularity when compared to saline injected controls. Thus, CA seems to act as an immunomodulator causing an augmentation of NK cell activity. Since other biological response modifiers (BRMs) do not show the same strength of augmentation, CA could be used as a new BRM having possible anticancer effects.

In vitro effects of certain polycyclic aromatic hydrocarbons on mitogen activation of mouse T-lymphocytes: Action of histamine☆

T lymphocytes were isolated from the spleens, thymuses, and bone marrow of three inbred mice strains, and the effects of two carcinogenic polycyclic aromatic hydrocarbons (PAH) on the mitogen activation of these cells were assessed. Benzanthracene (BA) and 3-methylcholanthrene (MCA) enhanced mitogen activation of splenic T cells in a strain-related fashion: C3H greater than C57BL greater than DBA/2 (P less than 0.025). This pattern of strain relatedness was not observed in T cells from the other lymphoid organs. Mitogen activation was suppressed by histamine to a greater degree in T cells from PAH-responsive mice (C3H and C57BL) than in the nonresponsive strain (DBA/2). Histamine inhibited rosette formation between T cells and histamine-conjugated sheep red blood cells. A histamine suppressor factor (HSF), isolated from splenic lymphocytes grown in the presence of histamine or histamine plus MCA, was significantly higher in activity in culture supernatants from T cells derived from responsive mice than from nonresponsive mice. With the use of Lyt 1 and Lyt 2 monoclonal antibodies, it is shown that the baseline percentage of T helper and T suppressor cells was not significantly different in all three strains. Further, histamine and MCA had no effect on the expression of the Lyt 1 and Lyt 2 surface antigens on splenic lymphocytes. These results suggest that PAH-responsive mice may have more T-cell H2 receptors than T cells from nonresponsive mice. Histamine and PAH compounds may act on the same T-cell subsets, as evidenced by the fact that BA and MCA enhance blastogenesis, histamine suppresses mitogen activation, and these PAH compounds enhance histamine and HSF activity.

Effect of methylcholanthrene on human natural killer cell cytotoxicity and lymphokine production in vitro.

We have developed a simple culture assay system for measuring the in vitro effects of a chemical carcinogen, 3-methylcholanthrene (MCA), on certain activities of human peripheral blood lymphocytes: blastogenesis, cell-mediated cytotoxicity by natural killer cells, interleukin-2 production, lymphotoxin release and percent T-cell subpopulations. After 72 h of treatment with different doses of MCA, blastogenesis was suppressed 23-87% and cell-mediated cytotoxicity was inhibited 45-90%. Interleukin-2 and lymphotoxin production were decreased by 64% and 38%, respectively. On the other hand, MCA treatment at the same doses caused no significant change in the percent of T-cell subsets. We conclude that MCA exerts an inhibitory effect on T-cell functional activity such as interleukin-2 and lymphotoxin production which correlate with a suppression of blastogenesis and natural killer cell activity. This in vitro assay system could be important for future studies in explaining specific inhibitory effects of chemical carcinogens on lymphoid cell function relative to tumorigenesis.