The role of natural killer cells in adenovirus-mediated p53 gene therapy.

Adenovirus-mediated gene therapy is a promising new approach for treatment of ovarian cancer. In animal models, complete elimination of cancer cells is often achieved, although the therapeutic gene has not been delivered to all these cells. This is referred to as a bystander effect, because tumor cells near those that receive the therapeutic gene are also eliminated. Several mechanisms have been proposed for the bystander effect, including intercellular communication within the tumor via gap junctions, apoptosis, antiangiogenesis, cytokines or other soluble mediators, and immunological mechanisms. There are two well-documented antitumor effector cell populations in athymic nude mice: macrophages and natural killer (NK) cells. We hypothesize that peritoneal populations of NK cells in nude mice treated with adenoviruses are involved in the observed bystander effect in this in vivo model. We investigated the role of NK cells as immunological mediators for the bystander effect using the p53 tumor suppressor as the therapeutic anticancer gene. Most ovarian cancer cell lines tested were sensitive to lysis by NK cells, although different ovarian cancer cell lines exhibited different sensitivities to NK cell-mediated lysis. To determine the importance of NK cells in the overall efficacy and in the bystander effect of gene therapy, NK cells were depleted in mice by administration of anti-NK1.1 monoclonal antibodies. To study the efficacy of NK depletion, C57BL/6 (nu/nu) mice were given injections i.v. by a single tail vein injection or i.p. with increasing doses of anti-NK1.1 IgG. All doses of anti-NK1.1 antibody, from 100-500 micrograms, essentially eliminated cytotoxic NK activity. To assess the duration of depletion after a single dose of anti-NK1.1 IgG, a time-course experiment was performed. NK 1.1 antibody was effective in completely depleting cytotoxic NK cell activity in the mice for up to 7 days, whether given as 500 micrograms (i.p.) or 200 micrograms (i.v.). Flow cytometric analysis performed on peritoneal cell populations confirmed depletion of NK cells by approximately 80%. Finally, a survival study was performed, in which animals were depleted of NK cells. In this experiment, NK cell-depleted mice were injected with anti-NK1.1 IgG, and control mice were mice were treated with normal saline. Two days later, all mice were inoculated with a lethal i.p. dose of NIH:OVCAR-3 ovarian cancer cells. After 3 days, the mice were divided into two treatment groups; one treatment group received three consecutive daily i.p. injections of Ad-CMV-p53 (SCH58500), and the second treatment group received three consecutive daily i.p. injections of control adenovirus construct, rAd-null. All of the NK cell-depleted animals, whether treated with rAd-null or with Ad-CMV-p53 (SCH58500) were dead of disease by 116 and 138 days, respectively, after initiation of adenovirus treatment, and no statistically significant difference in survival was observed (P = 0.349). A significant survival advantage was seen in control (NK-competent) mice treated with rAd-null (P = 0.04), although all were dead of disease by day 184. Importantly, control NK-competent mice treated with Ad-CMV-p53 (SCH58500) showed no tumor growth or ascites production, and all animals survived. These results indicate that immunological mechanisms involving natural killer cells play an important role in the bystander effect involving adenovirus-p53 gene therapy for ovarian cancer.

Quantitation of proteins in the femtomole range by hemagglutination using an electronic particle counter.

By using an electronic particle counter equipped with two counting channels, it is possible to simultaneously count total particles and agglutinated particles in an erythrocyte population. Using these two parameters, an empirically useful measure of the degree of agglutination in a sample (the agglutination index) can be generated. This method has been used to accurately and precisely determine human serum IgG concentrations in an inhibition of hemagglutination assay. As little as 1 microgram/ml of IgG could be measured in this way. Agglutination and inhibition of agglutination assays with other systems indicate that this method will be widely applicable.

Circadian variations of human lymphocytes are not responsible for contradictory or variable results in studies of IL-2 production.

Efforts to correlate various immunological disorders with alterations in IL-2 production in vitro have led to highly variable and often contradictory results. One possible contributing factor to these results could be the existence of circadian variations in the capacity of lymphocytes to produce IL-2 in culture. Although there were reasons to suspect that such variations might exist, the present study revealed no significant variations in 6 of 10 normal subjects in three experiments or in the mean values for all subjects at each of three sample acquisition times. In addition, no subject exhibited circadian variations in all three experiments. Since it was possible that failure to detect variations was due to selection of an inappropriate mitogen or inappropriate culture conditions, 2 mitogens and various culture conditions were used. Circadian variation was noted for only one subject out of eight under only one set of conditions. This suggests that circadian variations are not responsible for variable or contradictory results which have been reported. Therefore, the recently reported circadian variations in T helper to T suppressor ratios do not seem to significantly affect mitogen-induced IL-2 production.

Phlebotomy--a minimalist approach.

For each patient, laboratories usually collect more blood than is needed for specific determinations. We reviewed the amount of blood collected for laboratory measurements for an entire hospital stay of 113 patients admitted during a 1-week period to a medical ward or to a medical intensive-care unit in our tertiary-care facility. The amount of blood obtained was also compared with the minimal amount needed for analysis for 18 of the most frequently ordered laboratory tests in our facility. For routine collections, a mean of 45 times the required volume of specimen (range, 2 to 102 times) was obtained. For optimal utilization of laboratory services, both the positive and the negative consequences of testing must be thoughtfully considered. Two potential adverse effects of withdrawal of blood for laboratory determinations are iatrogenic anemia and infection. Moreover, the cost of care is increased with additional analyses. Practical strategies for decreasing the amount of blood collected include an increased awareness of ordering practices, a thorough knowledge of the volume of blood needed for each laboratory test, experienced phlebotomy personnel, storage of blood specimens for potential subsequent use, and communication of accurate minimal volumes needed for specific measurements.

Accidental needlesticks in the phlebotomy service of the Department of Laboratory Medicine and Pathology at Mayo Clinic Rochester.

OBJECTIVE: To determine the change in accidental needlestick rates in the Phlebotomy Service at Mayo Clinic Rochester and to identify safety practices implemented from 1983 through 1996. MATERIAL AND METHODS: We retrospectively reviewed yearly Phlebotomy Service accidental needlestick rates from 1983 through 1996. Interviews were conducted with representatives of the Infection Control Committee and the management team for the Phlebotomy Service, and minutes of meetings of these two groups were reviewed to identify implemented safety improvements that may have had an effect on accidental needlestick exposures. RESULTS: Accidental needlestick exposures in the Phlebotomy Service declined from a high of 1.5/10,000 venipunctures to 0.2/10,000 venipunctures. Several safety improvements were made during that time, including the implementation of a one-handed recapping block, change to single-use evacuated tube holders, increased number and improved locations of disposal containers for needles, implementation of resheathing needles and retractable capillary puncture devices, discontinuation of the practice of changing needles before inoculation of blood culture bottles, increased emphasis on safety for new and experienced phlebotomists, and improved exposure reporting tools. CONCLUSION: We believe that the decrease in our accidental needlestick exposure rate is correlated with the changes in education, practices, and products that we have implemented.

Macroenzyme as a cause of unexplained elevation of aspartate aminotransferase.

Aspartate aminotransferase (AST) can exist as a macroenzyme by forming a complex with an immunoglobulin. This immunoglobulin-complexed macromolecule can cause an elevation in serum AST activity, which may be detected on routine blood chemistry analysis and erroneously considered to indicate the presence of liver disease. Clinicians should be aware of this phenomenon so patients are not subjected to unnecessary procedures. In patients with unexplained AST elevation, liver and muscle disease can be biochemically excluded by the finding of normal serum levels of alanine aminotransferase and creatine kinase. The presence of macro-AST can be determined by exclusion chromatography, electrophoresis, and activation assays with pyridoxal 5-phosphate. The elevated AST values can persist for many years.

The role of thiols in lymphocyte responses: effect of 2-mercaptoethanol on interleukin 2 production.

The effects of 2-ME on the production of IL2 in mitogen-stimulated mouse splenocyte cultures were examined. Since 2-ME could conceivably affect IL2 production and utilization equally, resulting in no apparent change in the IL2 concentration of treated cultures, 12-h cultures were used to minimize any effects of IL2 utilization on the IL2 concentrations observed. Utilization of IL2 in 12h splenocyte cultures was estimated by means of simulations in which IL2 from 12h culture supernates was utilized by CTLL-2 cells comparable in number to the lymphoblasts present in splenocyte cultures. These stimulations indicate that IL2 utilization is insignificant during the first 12h of culture under a variety of conditions. Thus, the IL2 concentration at 12h reflects predominantly the rate of production. No effect was observed on IL2 concentrations at 12h when cultures were treated with 2-ME under conditions in which the 2-ME enhanced cellular activation/proliferation. These results indicate that 2-ME does not enhance proliferation in Con A-stimulated splenocyte cultures by increasing IL2 production, but by some other mechanism(s).

Quantitative aspects of the feeder cell phenomenon: direct assessment of enhanced cystine uptake by lymphocytes.

It has been suggested that feeder cells and 2-mercaptoethanol enhance the survival and growth of murine lymphocytes in culture by increasing cysteine availability. We previously reported that although feeder cells produce thiols, they support lymphocyte growth at densities too low for measurable thiol production. This suggested that increasing the availability of cysteine might not be the major mechanism of feeder cell action. In the present study, [35S] cystine was used to directly monitor cyst(e)ine uptake in lymphocyte-feeder cell co-cultures. The results demonstrate that feeder cells substantially increase cyst(e)ine uptake by lymphocytes, even in the absence of detectable free thiols. Data are presented which suggest an explanation for this unexpected observation.

Quantitative relationships between the suppression of selected immunological parameters and the area under the corticosterone concentration vs. time curve in B6C3F1 mice subjected to exogenous corticosterone or to restraint stress.

The neuroendocrine response to stressors increases the concentration of several endogenous mediators, some of which are immunosuppressive. However, quantitative aspects of these effects have been overlooked. Although it should be possible to predict the degree of suppression of particular immunological functions by measuring the concentrations of stress-related mediators such as corticosterone, this cannot be done with data presently available. This study was designed to develop regression models to predict the relationship between the area under the corticosterone concentration vs. time curve (AUC) and two immunological parameters. Models were developed using mice treated with exogenous corticosterone and mice subjected to various periods of restraint stress. The latter treatment was included to determine if the effects of corticosterone were different from those of corticosterone in association with the other mediators induced in a restraint-stress response. Models relating corticosterone AUC to expression of MHC class II proteins on splenocytes were very similar, whether the corticosterone was exogenous or produced as part of a restraint-stress response. This was also true for splenic natural killer (NK) cell activity. However, MHC class II expression was more sensitive to the effects of corticosterone or restraint than was NK cell activity. The corticosterone and restraint models predicted the previously published effect of a chemical stressor (ethanol) on MHC class II expression, but neither model predicted the suppression of NK cell activity by ethanol. These results have mechanistic implications, which are discussed in the context of previous studies. The quantitative models described here should be useful in determining and predicting the stress-related portion of chemical-induced immunosuppression. In addition, these models provide quantitative data essential for a complete understanding of stress-induced immunosuppression.

Modeling and predicting selected immunological effects of a chemical stressor (3,4-dichloropropionanilide) using the area under the corticosterone concentration versus time curve.

Many chemicals and drugs can induce a neuroendocrine stress response that can be immunosuppressive. Mathematical models have been developed that allow prediction of the immunological impact of such stress responses in mice on the basis of exposure to the important stress-related mediator corticosterone. The area under the corticosterone concentration vs. time curve (AUC) has been used as an indicator of cumulative corticosterone exposure in these modeling studies. In the present study, an immunotoxicant known to induce a stress response, 3,4-dichloropropionanilide (propanil), was evaluated to determine if corticosterone AUC values are related to suppression of immunological parameters in mice treated with this chemical. Linear relationships between corticosterone AUC values and suppression of the following parameters were noted in B6C3F1 female mice: thymus cellularity and thymus subpopulation percentages, splenic subpopulation percentages, natural killer cell activity, MHC class II protein expression, and IgG1 and IgG2a antibody responses to antigen. Linear models derived in previous studies using mice treated with exogenous corticosterone or with restraint stress effectively predicted the immunological effects of 3, 4-dichloropropionanilide on the basis of corticosterone AUC values. The models derived using immobilization stress were more effective (r(2) for observed vs. predicted = 0.90) than the models derived using mice treated with exogenous corticosterone (r(2) for observed vs. predicted = 0.65). This was expected, because most stressors induce a variety of immunomodulatory mediators, not just corticosterone. These findings have implications for risk assessment in immunotoxicology.

Evaluation of multivariate statistical methods for analysis and modeling of immunotoxicology data.

In immunotoxicology, the critical functions of the immune system (host resistance to infection and neoplasia) cannot be measured directly in humans. It is theoretically possible to predict changes in host resistance based on changes in immunological functions known to mediate host resistance. However, quantitative predictive models of this type have not yet been achieved in humans or in animal models. Multivariate statistical methods were developed for analysis and modeling of the effects of several explanatory variables on a dependent variable, and they seem well suited for attempts to predict host resistance changes caused by changes in immunological parameters. However, these methods were developed with the assumption that all variables can be measured for each experimental subject. For a number of reasons, this generally cannot be done in comprehensive immunotoxicology evaluations. In the present study, the suitability of multivariate methods for analysis of variables measured in different experiments was examined, using a limited data set consisting of immunological parameters that could all be measured for each mouse. Analysis was done on the original data set and test data sets produced by randomizing data within dosage groups. This was done to simulate the random pairing of data that would occur if measurements were obtained from different sets of mice in different experiments. Statistical theory indicates that randomization will disrupt the correlation matrices that are central in multivariate analyses. However, the present results demonstrate empirically that for at least one immunotoxicant (dexamethasone), remarkably similar multivariate models were obtained for the original and 109 randomized data sets. In contrast, the randomized data sets produced substantially different multivariate models when data obtained with a different immunotoxicant (cyclosporin A) were analyzed. The major difference between the two data sets was that dexamethasone strongly and dose-responsively suppressed many more parameters than did cyclosporin A. Additional work is needed to determine whether there are consistent criteria that could be used to identify immunotoxicology data sets, which would be amenable to multivariate analysis.

Skull-base foramina of the middle cranial fossa: reassessment of normal variation with high-resolution CT.

PURPOSE: To evaluate by means of high-resolution CT the anatomic variations of the middle cranial fossa foramen. METHODS: We examined 123 CT studies of the temporal bone in patients with no evidence of disease that might alter foraminal anatomy. A checklist of known variants and suspected structures was used as each case was systematically examined for the presence or absence of these foramina; variations in size, shape, and location; and relationship of structures to each other. Inclusion criteria were established to eliminate error. RESULTS: The foramen rotundum had a constant appearance. We identified the inferior rotundal canal in 16% of patients and the lateral rotundal canal in 8%. The foramen of Vesalius was present, at least unilaterally, in 80% of our cases. Asymmetry of the foramen of Vesalius did not indicate disease in our patient group. We did not find an inverse relationship between the size of the foramen of Vesalius and that of the ipsilateral foramen ovale. We found variations in the size and shape of the foramen ovale and its confluence with the foramen spinosum (n = 2) and the foramen of Vesalius (n = 8). We did not find an inverse relationship between the size of the foramen ovale and that of the foramen spinosum. The canaliculus innominatus for the lesser superficial petrosal nerve was identified in 16.3% of our patients. Variations of the foramen spinosum that we found include a medial bony defect (26.8%) and absence (3.2%). CONCLUSION: Although it is unlikely that well-formed foramen will be misinterpreted as diseased, it is nonetheless important to recognize foraminal variants and associated neurovascular anatomy.

Quantitative aspects of stress-induced immunomodulation.

Recent studies indicate that neuroendocrine-immune interactions can cause sufficient immunosuppression to adversely affect human health, but quantitative relationships between stress-related hormones or neurotransmitters and immune function have not been well documented. The mechanisms of stress-induced immunomodulation cannot be fully understood solely by identifying the hormones, neurotransmitters, and cytokines involved. Quantitative relationships and interactions must also be understood. Depending on the nature and duration of the stressor and the immunological parameter under investigation, stress responses can enhance, have no effect, or suppress immunological parameters. These quantitative relationships have implications with regard to safety assessment of drugs and chemicals and with regard to potential development of pharmacological interventions to ameliorate some of the immunosuppressive effects of stress. This review describes selected studies that relate the quantity and duration of exposure to stress-related neuroendocrine mediators to modulation of the immune system. These studies provide a useful starting point, but they also illustrate how much work remains to achieve a fully integrated qualitative and quantitative understanding of stress-induced immunomodulation.

Bacterial DNA does not increase serum corticosterone concentration or prevent increases induced by other stimuli.

Bacterial DNA containing unmethylated CpG motifs (CpG DNA) and other microbial molecules such as lipopolysaccharide (LPS) have a broad range of immune stimulatory effects, which may include many shared cell signaling pathways leading to enhanced cytokine production. Some cytokines activate the hypothalamic-pituitary-adrenal (HPA) axis, and their production is downregulated by products of the HPA axis (glucocorticoids). Because such interactions have practical implications in the clinical use of CpG DNA, the present study was done to examine the effects of CpG DNA and LPS on serum corticosterone concentrations. In contrast to LPS, administration of CpG DNA (DNA from Escherichia coli) (30-300 microg) alone did not significantly increase serum corticosterone concentrations 1 or 4 h after administration. Administration of CpG DNA to mice prior to LPS caused a synergistic increase in serum tumor necrosis factor-alpha (TNF-alpha), indicative of an immune stimulatory effect. LPS and TNF-alpha, however, induced similar levels of corticosterone with or without concomitant CpG DNA. Increasing doses of LPS caused peak corticosterone levels similar to those induced by LPS in combination with CpG DNA. Exogenous TNF-alpha administered in vivo induced comparable concentrations of corticosterone with or without CpG DNA. An alternative stressor (restraint) yielded similar levels of corticosterone with or without CpG DNA. These results indicate that CpG DNA does not induce corticosterone release or alter its release by other stimuli, indicating biologically important differences in its immune effect compared to those of LPS, and possibly reduced toxicity.

Ethanol-induced activation of the hypothalamic-pituitary-adrenal axis in a mouse model for binge drinking: role of Ro15-4513-sensitive gamma aminobutyric acid receptors, tolerance, and relevance to humans.

A mouse model for binge drinking has been developed in this laboratory, and several aspects of this model have been characterized. Many of the immunosuppressive effects of ethanol (EtOH) in this model seem to be mediated by activation of the hypothalamic-pituitary-adrenal (HPA) axis and consequent increases in the concentration of glucocorticoids, catecholamines, and perhaps other immunosuppressive mediators. The purpose of the work described here is to examine three important issues regarding the EtOH-induced neuroendocrine response in this model: 1) Are Ro15-4513-sensitive gamma aminobutyric acid type A (GABA-A) receptors involved in activation of the HPA axis by EtOH? 2) Does daily administration of EtOH produce tolerance with regard to activation of the HPA axis or with regard to suppression of natural killer cell activity? 3) Is the HPA axis activated by similar blood EtOH concentrations in humans and in the mouse model? Ro15-4513, a partial inverse agonist of GABA-A receptors, did not affect EtOH-induced increases in blood corticosterone levels. This suggests that Ro15-4513-sensitive GABA-A receptors are not involved in EtOH-induced activation of the HPA axis and that inhibition of corticosterone production is not the mechanism by which Ro-15-4513 blocks EtOH-induced immunosuppression. To evaluate tolerance, mice were given a daily dose of EtOH (6.5 g/kg by gavage) or vehicle (water) for 10 days. Control groups received vehicle or EtOH only on the last day of the experiment. At the optimum time after EtOH administration serum corticosterone and splenic NK cell activity were measured. The results indicate no significant alterations in the response to EtOH of mice exposed to EtOH for 10 days compared to those exposed only once. To compare the HPA axis response of mice and humans, lower EtOH dosages than generally used in our model were administered to mice, and the corticosterone response was compared to published data for humans who had similar ranges of blood EtOH levels. The results suggest that humans and mice exhibit activation of the HPA axis only when blood EtOH levels exceed approximately 0.14%. Together these results further characterize a mouse model for binge drinking that seems to provide a reasonable representation of many aspects of binge drinking in humans.

Effects of paraoxon, p-nitrophenol, phenyl saligenin cyclic phosphate, and phenol on the rat interleukin 2 system.

Two organophosphorus compounds, paraoxon and phenyl saligenin cyclic phosphate, as well as p-nitrophenol and phenol which are structurally related to paraoxon, were tested for their effects on interleukin 2 (IL2) production and responsiveness by rat splenocytes in vitro. Three of the four compounds inhibited mitogen-induced lymphocyte proliferation as well as IL2 production and responsiveness. However, phenyl saligenin cyclic phosphate produced maximal inhibition at a much lower concentration (0.5 microM) than p-nitrophenol (200 microM) or paraoxon (200 microM). Phenol was not inhibitory at any concentration tested (up to 250 microM). Since the production of and response to IL2 are key events in immune responses, compounds which suppress these events can be identified as potential suppressors of host resistance to disease.

Bovine immunodeficiency virus: incidence of infection in Mississippi dairy cattle.

Bovine immunodeficiency virus (BIV), a lentivirus, was originally derived from a Holstein cow with persistent lymphocytosis and severe wasting. The virus is known to occur sporadically throughout the United States and perhaps across the globe, but epidemiological data concerning the incidence of BIV are meager and the virus was previously unreported in Mississippi animals. This study examined the seroepidemiology of BIV infection from two Mississippi dairy herds (Coastal Plains and MSU). Serology revealed a 38% incidence of BIV infection in Coastal Plains animals and a 58% incidence in MSU animals. A cumulative BIV seroprevalence of 50% was found in the Mississippi animals, and BIV seroprevalence increased with increasing age of the animals. Peripheral blood leukocytes of age matched BIV seropositive and seronegative animals were enumerated to assess any effect of BIV infection on leukocyte populations. No significant differences were found in total leukocyte populations or leukocyte subpopulations between BIV seropositive or seronegative animals. These data indicate that BIV infection is prevalent in Mississippi animals, but the role of BIV in bovine disease remains unclear.

Quantitative modeling of suppression of IgG1, IgG2a, IL-2, and IL-4 responses to antigen in mice treated with exogenous corticosterone or restraint stress.

Exposure to toxic chemicals often induces a neuroendocrine stress response leading to increased concentrations of a variety of potentially immunomodulatory mediators. Corticosterone is a major stress-induced mediator that can be immunosuppressive. However, the quantity of corticosterone exposure required to produce particular decrements in particular immunological parameters is not known. Mice treated with various dosages of exogenous corticosterone were compared to mice exposed to a psychogenic stressor (restraint). Cumulative corticosterone exposure in these mice, expressed as the area under the curve (AUC) of corticosterone concentration versus time, was used to develop quantitative models of the effects of corticosterone on the immunoglobulin (Ig) G1 and IgG2a responses to keyhole limpet hemocyanin (KLH) and sheep erythrocytes (sRBC). The production of interleukin (IL)-2 and IL-4 by splenocytes stimulated with KLH in culture was also evaluated. Linear regression models were derived that describe the relationship between the IgG1 and IgG2a responses to KLH. Restraint had a greater effect (at equivalent corticosterone AUC values) than exogenous corticosterone, suggesting that mediators in addition to corticosterone are important in suppression of the IgG1 and IgG2a response to KLH. The production of IL-2 and IL-4 by cultured splenocytes was mostly, but not always, consistent with the changes in IgG1 or IgG2a. For example, the regression lines for IgG2a (a Th1-driven response) and IL-2 (a Th1 cytokine) were not significantly different. The relationships between corticosterone AUC and the IgG1 and IgG2a responses to sRBC were nonlinear and characterized by enhanced responses at low to moderate AUC values. The quantitative models developed here have implications for risk assessment in immunotoxicology.

Immunotoxicological assessment of methyl parathion in female B6C3F1 mice.

Methyl parathion is a widely used agricultural insecticide, and the recent unlicensed use of this compound in homes has led to the evacuation of approximately 1100 persons in Mississippi. Although the primary concern in such cases of acute exposure is neurotoxicity, a few organophosphorus compounds apparently have immunotoxic effects at dosages that do not produce neurotoxic symptoms. The purpose of the present study was to determine if this is the case for methyl parathion. Female B6C3F1 mice were exposed to methyl parathion by gavage, daily for 7, 14, 2 1, or 28 d (at 6 mg/kg/d). Exposure for 14-28 d produced significant, dose-responsive inhibition of acetylcholin-esterase (the target molecule for methyl parathion-induced neurotoxicity) in brain or plasma, indicating that the compound was active. The following immunological parameters were evaluated: white blood cell counts and differentials, spleen and thymus weight and cellularity, splenic natural killer cell activity, nitrite production by peritoneal macrophages following activation in vitro, antibody response to sheep erythrocytes in vitro and in vivo, the cytotoxic T lymphocyte response to allogeneic tumor cells, and resistance to Streptococcus agalactiae and B16F10 melanoma cells. Methylparathion at 1 or 3 mg/kg/d significantly increased splenic natural killer cell activity. Nitrite production by macrophages was increased in mice treated with 1, 3, or 6 mg/kg/d. The antibody response to sheep erythrocytes in vitro was significantly suppressed, but the humoral response to sheep erythrocytes in vivo was not affected. The cytotoxic T-lymphocyte response to allogeneic tumor cells was not significantly affected. Host resistance was not significantly decreased. Although it remains possible that immunological parameters not tested here may be affected by methyl parathion, the present results do not suggest substantial immunotoxic potential for this compound.