Evidence for formaldehyde antibodies and altered cellular immunity in subjects exposed to formaldehyde in mobile homes.

Eight symptomatic individuals chronically exposed to indoor formaldehyde (HCHO) at low concentrations (0.07-0.55 ppm) were compared to 8 nonexposed subjects with respect to: (1) presence of IgG and IgE antibodies to HCHO conjugated to human serum albumin (F-HSA); (2) the percentage of venous blood T and B cells by E and EAC-rosetting; and (3) the ability of T and B cells to undergo mitogen (PHA, PWM) stimulated blastogenesis as measured by the incorporation of tritiated thymidine. Anti-F-HSA IgG, but no IgE, antibodies were detected in the sera of the 8 exposed subjects; none were found in 7 of the unexposed controls. T lymphocytes were decreased in the exposed (48 +/- 11.5%) compared to the control (65.9 +/- 4.97%) subjects (p greater than .001 less than .01). B cells were 12.6 +/- 1.6% (HCHO group) and 14.75 +/- 2.1% (controls) (p greater than .02 less than .05). The incorporation of labeled thymidine by T cells (PHA) was decreased: 17,882 +/- 2,293 cpm (HCHO group) and 28,576 +/- 3,807 cpm (p greater than .001 less than .01). T and B cell blastogenesis (PWM) was 9,698 +/- 1,441 cpm (HCHO group) and 11,279 +/- 1,711 (controls) (p greater than .05 less than .1). Exposure to HCHO appears to stimulate IgG antibodies to F-HSA and decrease the proportion of peripheral T cells.

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.

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.

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.

IgG is not the only inhibitor of IgE in the RAST test.

Blocking phenomenon of the RAST test was studied in the sera of 50 different patients. Specific levels of IgE and IgG against 11 common foods were evaluated by ELISA. Results showed that decreases in IgG levels by protein-A were not always followed by IgE increases. In this regard, we found three different combinations in the levels of IgE and IgG: (1) an IgG decrease followed by IgE increase (29%); (2) an IgG increase followed by IgE decrease (18%); and (3) both IgG and IgE decrease (28%). From these results we concluded that IgG is not the only inhibitor of the RAST test.

Two biochemically distinct populations of histaminocytes separated by isokinetic sedimentation of dispersed rat gastric cells.

Two populations of histaminocytes, with different sedimentation rates (SR), were separated by a computer developed isokinetic gradient using dispersed rat gastric mucosal cells. Histamine content, histidine decarboxylase (HDC) activity and incorporation of radiolabelled histidine metabolites were used to assess the migration of specific cells throughout the gradients. One histaminocyte population, with cells of lower SR, contained high HDC activity and undetectable levels of histamine, whereas the other population, with cells of higher SR, contained lower HDC activity and high concentration of histamine. Both types of histaminocytes incorporated 3H-histidine metabolites. Electron microscopy showed that the fractions containing histaminocytes with lower SR had 3.5 times more endocrine ECL cells than the original population of dispersed fundic cells and lacked A and D cells, whereas the fractions with histaminocytes of higher SR were associated with a 2.7 times higher concentration of A and D cells and with a 7.7 times higher ratio of a variety of partial cells with a distinct mitochondrial morphology. These results are consistent with prior novel information regarding the separation of two populations of rat histaminocytes using different sedimentation techniques.

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.

Alterations in cell-mediated immune functions induced in mouse splenic lymphocytes by polycyclic aromatic hydrocarbons.

The effects of varying doses of three polycyclic aromatic hydrocarbons, 3-methylcholanthrene (MCA), benzo(a)pyrene (BaP), and benzo(e)pyrene (BeP), on cell-mediated immune functions of in vivo mitogen-activated splenic lymphocytes were measured. Inbred mice (C57, C3H, and DBA) were given injections i.p. with phytohemagglutinin to activate splenic lymphocytes and were subsequently treated, via the same route, with polycyclic aromatic hydrocarbon compounds. Isolated and T-cell-enriched mononuclear cell populations were assayed for aryl hydrocarbon hydroxylase activity, blastogenesis, antigen-specific cell-mediated cytotoxicity, and the percentage of macrophages. Under optimal conditions of phytohemagglutinin injection (1000 micrograms/20-g mouse) for 96 hr and MCA treatment (50 mg/kg of body weight) for 24 hr prior to sacrifice, aryl hydrocarbon hydroxylase was induced 5-fold. Tumorigenic doses of MCA (10 to 50 mg/kg of body weight) suppressed blastogenesis 40 to 60% in C57 and C3H lymphocytes but had no effect on DBA splenic lymphocytes. BaP and BeP had little or no effect on blastogenesis. MCA and BaP were clearly separated from BeP in the suppression of cell-mediated cytotoxicity. MCA and BaP suppressed cell-mediated cytotoxicity 40 to 80% in T-cells from all three strains, while BeP had no effect. MCA reduced the percentage of macrophages in a dose-dependent fashion compared to a stimulatory action by BaP and BeP. These results suggest that mitogen-activated and aryl hydrocarbon hydroxylase-induced splenic lymphocytes metabolize MCA and BaP to immunocytotoxic metabolites. A suppression of monocyte-macrophage function would account for the inhibition of blastogenesis. These early alterations in cell-mediated immune functions produced by tumorigenic doses of MCA and BaP may result in defective immunosurveillance mechanisms and enhance the development of polycyclic aromatic hydrocarbon-induced tumors in responsive mice.

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.

A chemical carcinogen, 3-methylcholanthrene, alters T-cell function and induces T-suppressor cells in a mouse model system.

The in-vivo effects of a polycyclic aromatic hydrocarbon (PAH), 3-methylcholanthrene (MCA), on in-vitro mitogen activation, cell-mediated lympholysis (CML) and T-cell subset distribution in mouse splenic lymphocyte populations were measured. Three inbred mouse strains were treated with a single intraperitoneal injection of corn oil alone or with different doses of MCA in oil (0.5-50 mg kg -1). One to ninety days after injection, splenic lymphocytes were isolated, and assayed for blastogenesis, CML and the percent T-helper and T-suppressor cells using monoclonal antibodies. High doses of MCA suppressed mitogen activation (15.2-53.6%) and CML (69-90%) within 24 hr in lymphocytes from PAH-responsive mice (C57 and C3H). Blastogenesis was stimulated and CML was suppressed to a lesser degree (5-45%) in lymphocytes from non-responsive mice (DBA). MCA induced an increase in T-suppressor cells in responsive mice, but there was no change in DBA mice. These studies suggest a correlation between immunocytotoxicity of PAH compounds on T-cell subsets and the responsiveness of mouse strains to these carcinogens.

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.

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.

Agglutinins and proteins in the earthworm, Lumbricus terrestris, before and after injection of erythrocytes, carbohydrates, and other materials.

Agglutinin activity and total protein concentration in earthworm coelomic fluid were determined 24 hours after injecting different erythrocyte types and carbohydrates. Agglutinin titers increased from 14 to 1216 after a single injection of erythrocytes. At the same time, protein concentrations increased by variable amounts, suggesting an indirect correlation between increased agglutinin titer and protein concentration. To understand the nature of erythrocyte determinants responsible for inducing agglutinins and other proteins, separate groups of earthworms were injected with different carbohydrates. At 24 hours, coelomic fluid was assayed for agglutinin titer and protein concentration. The injection of different carbohydrates, amino acids, nucleotides and protein (BSA), like erythrocytes, also increased protein concentration and agglutinin titers. Changes in the protein concentration and composition of coelomic fluid were studied by standard and bidimensional immunoelectrophoresis. Different patterns and numbers of components were found before and after injection. At least two different components were present after injecting carbohydrates or erythrocytes, which may be related specifically to the agglutinins. Clearly, earthworms can synthesize and shed these molecules at rapid rates into the coelomic fluid. These molecules may be involved in earthworm defense.

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 earthworm's humoral factors that may participate in immune responses.

Isolation and characterization of tetracycline resistance proteins from Staphylococcus aureus and Escherichia coli.

Immunoglobulin (adsorbed resistance antiserum) reacting specifically with antigens from tetracycline (Tc)-resistant Staphylcoccus aureus or Escherichia coli was produced by adsorbing immunoglobulin against cell envelopes of resistant strains with envelope extracts from the respective isogenic susceptible strains. Adsorbed resistance antiserum against S. aureus reacted with envelope extracts from 32 Tc-resistant strains and failed to react with similar extracts from 76 Tc-susceptible strains of S. aureus. An antigen (Tc resistance antigen [TRA]) found only in Tc-resistant strains was produced by adsorbing envelope extracts from these strains with immunoglobulin against envelopes from isogenic Tc-susceptible strains. On immunodiffusion no cross-reactivity between TRAs from S. aureus and E. coli was observed. The TRAs behaved as proteins. The molecular weight of TRA from S. aureus was determined to be 32,000 and from E. coli to be 50,000. Data obtained by preliminary amino acid analysis of the TRAs are presented.