Do chaperonins boost protein yields by accelerating folding or preventing aggregation?

The GroEL chaperonin has the ability to behave as an unfoldase, repeatedly denaturing proteins upon binding, which in turn can free them from kinetic traps and increase their folding rates. The complex formed by GroEL+GroES+ATP can also act as an infinite dilution cage, enclosing proteins within a protective container where they can fold without danger of aggregation. Controversy remains over which of these two properties is more critical to the GroEL/ES chaperonin's function. We probe the importance of the unfoldase nature of GroEL under conditions where aggregation is the predominant protein degradation pathway. We consider the effect of a hypothetical mutation to GroEL which increases the cycle frequency of GroEL/ES by increasing the rate of hydrolysis of GroEL-bound ATP. Using a simple kinetic model, we show that this modified chaperonin would be self-defeating: any potential reduction in folding time would be negated by an increase in time spent in the bulk, causing an increase in aggregation and a net decrease in protein folding yields.

N-acetyl cysteine protects pulp cells from resin toxins in vivo.

Potential risks of the use of resin-based restorative materials include direct damage to the pulp cells and the induction of hypersensitivity reactions in patients. In this study, we tested the hypothesis that N-acetyl cysteine (NAC) inhibits resin toxicity and restores the function of pulp cells. Analysis of our data demonstrates toxicity of composite resins on pulp cells in both an in vivo rat and an ex vivo human model system. Moreover, cells that survive after the placement of composites are weaker, and they are induced to undergo cell death when exposed to 2-hydroxyethyl methacrylate (HEMA). The toxic effect of composites on pulp cells is neutralized by NAC. Therefore, NAC protects the cells from damage induced by clinically relevant levels of restorative materials, in both rat and human model systems. The addition of N-acetyl cysteine prior to or concomitant with the application of restorative materials may be beneficial for the health and safety of dental patients.

N-acetyl cysteine (NAC)-assisted detoxification of PMMA resin.

Despite its proven cytotoxicity, poly-methyl methacrylate (PMMA) resin is one of the most frequently and extensively used materials in dental practice. This study hypothesized that an anti-oxidant amino acid, N-acetyl cysteine (NAC), has the potential to detoxify this material. Ten percent of the rat dental pulp cells were viable when cultured on the PMMA resin for 24 hours, while over 70% of the cells were viable on the NAC-added resin. Nearly all suppressed alkaline phosphatase activity, matrix mineralizing capability, and odontoblastic gene expression, such as dentin sialoprotein, on the untreated control resin was recovered by NAC in a concentration-dependent manner. A Ca/P ratio of 1.65 was found in the extracellular matrix of cultures on NAC-added resin, while that in the untreated resin culture was 0.70. The addition of NAC to PMMA resin significantly ameliorated its cytotoxicity to the dental pulp cells and restored their odontoblast-like cell phenotype to a biologically significant degree.

Restored viability and function of dental pulp cells on poly-methylmethacrylate (PMMA)-based dental resin supplemented with N-acetyl cysteine (NAC).

This study examines cytotoxicity of poly-methylmethacrylate (PMMA)-based dental temporary filling resin to dental pulp cells, and the potential amelioration of the toxicity with an anti-oxidant amino-acid, N-acetyl cysteine (NAC). Dental pulp cells extracted from rat maxillary incisors were cultured on the resin material with or without NAC incorporation, or on the polystyrene. The cultures were supplied with osteoblastic media, containing dexamethasone. Forty five percent of cells on the PMMA dental resin were necrotic at 24h after seeding. However, this percentage was reduced to 27% by incorporating NAC in the resin, which was the level equivalent to that in the culture on polystyrene. The culture on the untreated resin was found to be negative for alkaline phosphate (ALP) activity at days 5 and 10 or von Kossa mineralized nodule formation at day 20. In contrast, some areas of the cultures on NAC-incorporated resin substrates were ALP and von Kossa positive. Collagen I and dentin sialoprotein genes were barely expressed in day 7 culture on the untreated resin. However, those genes were expressed in the culture on the resin with NAC. These results suggest that the decreased cell viability and the nearly completely suppressed odontoblast-like cell phenotype of dental pulp cells cultured on PMMA dental resin can be salvaged to a biologically significant degree by the incorporation of NAC in the resin.

Folding on the chaperone: yield enhancement through loose binding.

A variety of small cageless chaperones have been discovered that can assist protein folding without the consumption of ATP. These include mini-chaperones (catalytically active fragments of larger chaperones), as well as small proteins such as alpha-casein and detergents acting as "artificial chaperones." These chaperones all possess exposed hydrophobic patches on their surface that act as recognition sites for misfolded proteins. They lack the complexity of chaperonins (that encapsulate proteins in their inner rings) and their study can offer insight into the minimal requirements for chaperone function. We use molecular dynamics simulations to investigate how a cageless chaperone, modeled as a sphere of tunable hydrophobicity, can assist folding of a substrate protein. We find that under steady-state (non-stress) conditions, cageless chaperones that bind to a single substrate protein increase folding yields by reducing the time the substrate spends in an aggregation-prone state in a dual manner: (a) by competing for aggregation-prone hydrophobic sites on the surface of a protein, hence reducing the time the protein spends unprotected in the bulk and (b) by accelerating folding rates of the protein. In both cases, the chaperone must bind to and hold the protein loosely enough to allow the protein to change its conformation and fold while bound. Loose binding may enable small cageless chaperones to help proteins fold and avoid aggregation under steady-state conditions, even at low concentrations, without the consumption of ATP.

Emerging mechanisms of immunosuppression in oral cancers.

Mounting effective anti-tumor immune responses against tumors by both the innate and adaptive immune effectors is important for the clearance of tumors. However, accumulated evidence indicates that immune responses that should otherwise suppress or eliminate transformed cells are themselves suppressed by the function of tumor cells in a variety of cancer patients, including those with oral cancers. Signaling abnormalities, spontaneous apoptosis, and reduced proliferation and function of circulating natural killer cells (NK), T-cells, dendritic cells (DC), and tumor-infiltrating lymphocytes (TILs) have been documented previously in oral cancer patients. Several mechanisms have been proposed for the functional deficiencies of tumor-associated immune cells in oral cancer patients. Both soluble factors and contact-mediated immunosuppression by the tumor cells have been implicated in the inhibition of immune cell function and the progression of tumors. More recently, elevated levels and function of key transcription factors in tumor cells, particularly NFkappaB and STAT3, have been shown to mediate immune suppression in the tumor microenvironment. This review will focus on these emerging mechanisms of immunosuppression in oral cancers.

Resistance of AIDS-associated Kaposi's sarcoma cells to Fas-mediated apoptosis.

Escape of tumor cells from apoptotic-mediated stimuli results in tumor cell survival and resistance to cytotoxic mechanisms. Kaposi's sarcoma (KS) is the most common malignancy associated with AIDS, although its pathogenesis is not known. It is clinically important to determine whether AIDS-KS cells are resistant to apoptosis via the Fas system. Three isolates of AIDS-KS cells were studied. Although all KS cells express Fas on the cell surface, these cells were resistant to cytotoxic anti-Fas antibody (IgM, CH-11). Treatment of AIDS-KS cells with actinomycin D sensitized the tumor cells to anti-Fas cytotoxicity and apoptosis. Apoptosis was assessed by morphological changes and DNA fragmentation analysis. Three possible mechanisms related to AIDS-KS cells, resistance to anti-Fas cytotoxicity were examined. First, synthesis and secretion of soluble Fas by the tumor cells can neutralize antibody-induced cytotoxicity. However, none of the three types of KS cells expressed soluble Fas mRNA as determined by reverse transcription (RT)-PCR. Second, the expression of the proto-oncogene bcl-2 can protect cells from apoptotic signals. Analysis of bcl-2 mRNA by RT-PCR revealed that all three AIDS-KS cells express very low levels of bcl-2 mRNA. Third, the Fas-associated phosphatase-1 (FAP-1) is an antiapoptotic molecule reported to interact with Fas and can block transduction of the apoptotic signal. RT-PCR analysis revealed that all three types of AIDS-KS cells express high levels of FAP-1 mRNA, and treatment of KS cells with actinomycin D reduced the levels of FAP-1 mRNA significantly. These findings demonstrate that AIDS-KS cells are resistant to Fas-mediated apoptosis and suggest that FAP-1 may be involved in the acquisition of resistance of AIDS-KS to anti-Fas antibody-mediated apoptosis.

Effects of confinement in chaperonin assisted protein folding: rate enhancement by decreasing the roughness of the folding energy landscape.

Chaperonins, such as the GroE complex of the bacteria Escherichia coli, assist the folding of proteins under non-permissive folding conditions by providing a cavity in which the newly translated or translocated protein can be encapsulated. Whether the chaperonin cage plays a passive role in protecting the protein from aggregation, or an active role in accelerating folding rates, remains a matter of debate. Here, we investigate the role of confinement in chaperonin mediated folding through molecular dynamics simulations. We designed a substrate protein with an alpha/beta sandwich fold, a common structural motif found in GroE substrate proteins and confined it to a spherical hydrophilic cage which mimicked the interior of the GroEL/ES cavity. The thermodynamics and kinetics of folding were studied over a wide range of temperature and cage radii. Confinement was seen to significantly raise the collapse temperature, T(c), as a result of the associated entropy loss of the unfolded state. The folding temperature, T(f), on the other hand, remained unaffected by encapsulation, a consequence of the folding mechanism of this protein that involves an initial collapse to a compact misfolded state prior to rearranging to the native state. Folding rates were observed to be either accelerated or retarded compared to bulk folding rates, depending on the temperature of the simulation. Rate enhancements due to confinement were observed only at temperatures above the temperature T(m), which corresponds to the temperature at which the protein folds fastest. For this protein, T(m) lies above the folding temperature, T(f), implying that encapsulation alone will not lead to a rate enhancement under conditions where the native state is stable (T

Resin monomer 2-hydroxyethyl methacrylate (HEMA) is a potent inducer of apoptotic cell death in human and mouse cells.

Mechanisms by which the resin monomer 2-hydroxyethyl methacrylate (HEMA) induces hypersensitivity reactions in humans are not well-established, nor have the direct effects of HEMA on cell death been fully characterized. The objective of this study was to establish whether HEMA is capable of inducing apoptotic cell death, and whether differences exist in the levels of apoptotic death induced by HEMA in cells obtained from healthy individuals and from patients with established HEMA hypersensitivity. HEMA induced apoptotic death in Peripheral Blood Mononuclear Cells (PBMCs) obtained from both healthy and HEMA-sensitized patients and in the murine RAW cells in a dose-dependent manner. However, induction of cell death by HEMA was lower in PBMCs obtained from patients in comparison with healthy individuals. Studies reported in this paper demonstrate that HEMA induces apoptotic death, and that decreased susceptibility of lymphocytes to HEMA-mediated death might be an important mechanism for the generation and persistence of hypersensitivity reactions in patients.

Fusobacterium nucleatum apoptosis-inducing outer membrane protein.

The periodontal pathogen Fusobacterium nucleatum induces apoptosis in lymphocytes. We previously identified the autotransporter protein Fap2 in F. nucleatum strain PK1594 that induced apoptosis in lymphocytes when expressed in Escherichia coli. In this study, we identified protein homologs of Fap2 in the transformable F. nucleatum strain ATCC 23726, to determine their role in the induction of apoptosis in lymphocytes. We used a new gene-inactivation vector conferring thiamphenicol resistance (pHS31) to construct a mutant deficient in one of the homologs, aim1. Transcriptional analyses demonstrated disruption of aim1 expression, and phenotypic analyses revealed a 41% decrease in the ability of the mutant to induce apoptosis in Jurkat cells, as compared with the parental strain. These studies demonstrate, in the native host cell background, the contribution of aim1 to F. nucleatum induction of apoptosis and, to the best of our knowledge, represent the first report of a genetically defined and phenotypically characterized mutation in F. nucleatum.

Activation of c-Jun N-terminal kinase in the absence of NFkappaB function prior to induction of NK cell death triggered by a combination of anti-class I and anti-CD16 antibodies.

Addition of antibodies to major histocompatibility complex class I (MHC class I) and F(c) gamma RIII (CD16) antigens resulted in the synergistic augmentation of natural killer (NK) cell death, and the loss of NK cell cytotoxic function. The binding of anti-CD16 and anti-class I antibodies to the same population of NK cells is required for the synergistic augmentation of NK cell death. Moreover, the addition of antibodies to leukocyte function antigen-1 (LFA-1), which significantly inhibited the phorbol 12-myristate 13-acetate (PMA) and ionomycin mediated NK cell death, had no effect on NK cell death mediated by anti-CD16 and anti-class I antibodies. The increase in NK cell death was associated with an increase in tumor necrosis factor-alpha (TNF-alpha) secretion, and concomitant inhibition of nuclear factor kappa B (NFkappaB) activation and the induction of c-jun N-terminal kinase (JNK) activity in NK cells treated with the combination of anti-class I and anti-CD16 antibodies. Furthermore, the inhibition of NFkappaB activation in anti-CD16 and anti-class I antibody treated NK cells was paralleled with a significant increase in inhibitor of kappa B (IkappaB) protein expression. Overexpression of IkappaB super-repressor in YT, a NK cell line, caused significant up-regulation of TNF-alpha, PMA and ionomycin and Fusobacterium nucleatum mediated NK cell death. Overall, our studies suggest an important regulatory role for NFkappaB and JNK activities in MHC class I mediated NK cell death.

Irreversible cancer cell-induced functional anergy and apoptosis in resting and activated NK cells.

The interaction of natural killer (NK) cells with target cells, such as K562, results in NK functional inactivation and apoptosis. The role of NK-activating cytokines, IL-2, IL-12, and IL-15, in the regulation of NK inactivation and programmed cell death by target cells was examined. Purified natural killer cells were obtained from human peripheral blood and either co-incubated with K562 target cells and cytokines or the NK cells were pretreated with cytokines for 18 h prior to co-culture with K562 cells. Sorted NK cells were examined for cytotoxic activity and NK co-cultured with K562 were examined for cytokine secretion, phenotyping and DNA fragmentation. The cytotoxic activity was inhibited and was not alleviated by cytokine treatment. Whereas the cytokine treatment maintained NK cell viability for several days, NK cell viability was decreased significantly in the presence of K562 target cells. Downregulation of CD16 and upregulation of CD69 on NK cells were induced by K562 target cells and no modulation of these antigens was observed with cytokine treatment. A subpopulation of target-treated NK cells succumbed to cell death by apoptosis and cell death was not rescued by the activating cytokines. These findings demonstrate that target-induced functional inactivation and apoptosis of NK cells were not rescued by the activating cytokines IL-2, IL-12, and IL-15 regardless of whether the NK cells were pretreated with cytokines prior to exposure to K562 or the cytokines were added to the NK-K562 mixtures. These results also suggest that signals triggered by the target cells and resulting in NK cell anergy and apoptosis override cytokine-mediated signals for activation, cell proliferation, and survival.

Differential regulation of human NK cell-associated gene expression following activation by IL-2, IFN-alpha and PMA/ionomycin.

Natural killer (NK) cells are important in host-defense mechanisms against infection and cancer and also participate in regulation of the immune response. The functions of NK cells as well as their maturation and differentiation are regulated by various stimuli such as interleukin-2 (IL-2) and interferon-alpha (IFN-alpha). The mechanisms by which these stimuli regulate distinct NK functions are not known. This study compared the patterns of gene expression for several NK-associated genes namely perforin (PEF), granzymes A and B (GA or B), IL-1beta, IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), CD16 and NK-specific genes, NKG2A, NKG5 and NKG7 in both unstimulated and in IL-2-, IFN-alpha and PMA/Ionomycin (PMA/I)-stimulated NK cells purified from human peripheral blood. IFN-alpha enhanced mRNA expression for PEF, IFN-gamma, TNF-alpha and NKG2A, but did not affect NKG7 mRNA expression. IL-2 augmented mRNA expression for PEF, IFN-gamma, TNF-alpha, NKG2A and NKG7. PMA/I increased mRNA expression for IFN-gamma, TNF-alpha and NKG2A but did not affect mRNA expression for PEF and NKG7. Further, PMA/I inhibited the expression of CD16 mRNA. These findings demonstrate that the three NK-stimuli used share in common the regulation of several genes but each regulates specifically other genes. These findings suggest that stimuli-specific expression of NK-associated genes may underlie the molecular mechanisms responsible for distinct NK-mediated activities induced by different stimuli.

Lysis of uninfected HIV-1 gp120-coated peripheral blood-derived T lymphocytes by monocyte-mediated antibody-dependent cellular cytotoxicity.

Previous reports from our laboratory have demonstrated that peripheral blood monocytes (PBM) from HIV-1 infected individuals are de novo activated and are cytotoxic in vitro. Significant monocyte-antibody-dependent cellular cytotoxicity (ADCC) was obtained against HIV-1 inactivated CD4+ CEM target cells coated with HIV-1 in the presence of autologous seropositive serum. Based on these findings, we hypothesized that in HIV-seropositive individuals the monocytes may play an important role in vivo in the autodestruction of non-infected CD4+ T lymphocytes. The present study was designed to test this hypothesis. Monocytes from normal donors activated with M-CSF lysed CD4+ T cells (CEM) coated with gp120 sensitized by plasma from asymptomatic HIV-1+ individuals in a 8 h 51Cr release assay. ADCC cytotoxic activity varied from one individual to another and was a function of the dilution of the individual seropositive plasma used. We then used circulating CD3+ T lymphocytes as targets for ADCC following treatment with actinomycin D to facilitate the release of radioactive 51Cr. Like CEM, ADCC was obtained with CD3+ T cells coated with gp120 in the presence of HIV seropositive plasma and monocytes. Lysis was specific as T cells that were not coated with gp120 were not destroyed. These findings demonstrate that activated peripheral blood derived monocytes can destroy non-infected gp120-coated circulating T lymphocytes by an ADCC-mediated mechanism. Thus, these findings suggest that ADCC may be one mechanism operating in vivo for the destruction of non-infected CD4+ T lymphocytes.

Distribution and excretion of TEGDMA in guinea pigs and mice.

The monomer triethyleneglycoldimethacrylate (TEGDMA) is used as a diluent in many resin-based dental materials. It was previously shown in vitro that TEGDMA was released into the adjacent biophase from such materials during the first days after placement. In this study, the uptake, distribution, and excretion of 14C-TEGDMA applied via gastric, intradermal, and intravenous administration at dose levels well above those encountered in dental care were examined in vivo in guinea pigs and mice as a test of the hypothesis that TEGDMA reaches cytotoxic levels in mammalian tissues. 14C-TEGDMA was taken up rapidly from the stomach and small intestine after gastric administration in both species and was widely distributed in the body following administration by each route. Most 14C was excreted within one day as 14CO2. The peak equivalent TEGDMA levels in all mouse and guinea pig tissues examined were at least 1000-fold less than known toxic levels. The study therefore did not support the hypothesis.

Rapid and quantitative detection of Streptococcus mutans with species-specific monoclonal antibodies.

Streptococcus mutans is known to be a prime etiologic agent for the initiation and progression of human dental caries. Rapid, accurate, and quantitative detection of S. mutans will help us better understand the pathogenic mechanisms of dental caries and will help to develop methods for caries diagnosis and risk assessment. This study describes the development of three highly species-specific monoclonal IgG antibodies against S. mutans. The antibodies were used to develop a number of methods that quantitatively detect S. mutans in less <1 min and are sensitive enough to detect a single bacterial cell. These methods could be widely used in basic and clinical studies related to S. mutans and in the clinical diagnosis and treatment of caries in humans.

Successful Integration of Hepatitis C Virus Point-of-Care Tests into the Denver Metro Health Clinic.

Background. The Centers for Disease Control and Prevention (CDC) recommends testing and linkage to care for persons most likely infected with hepatitis C virus (HCV), including persons with human immunodeficiency virus. We explored facilitators and barriers to integrating HCV point-of-care (POC) testing into standard operations at an urban STD clinic. Methods. The OraQuick HCV rapid antibody test was integrated at the Denver Metro Health Clinic (DMHC). All clients with at least one risk factor were offered the POC test. Research staff conducted interviews with clients (three HCV positive and nine HCV negative). Focus groups were conducted with triage staff, providers, and linkage-to-care counselors. Results. Clients were pleased with the ease of use and rapid return of results from the HCV POC test. Integrating the test into this setting required more time but was not overly burdensome. While counseling messages were clear to staff, clients retained little knowledge of hepatitis C infection or factors related to risk. Barriers to integrating the HCV POC test into clinic operations were loss to follow-up and access to care. Conclusion. DMHC successfully integrated HCV POC testing and piloted a HCV linkage-to-care program. Providing testing opportunities at STD clinics could increase identification of persons with HCV infection.

Field-based performance of three pre-market rapid hepatitis C virus antibody assays in STAHR (Study to Assess Hepatitis C Risk) among young adults who inject drugs in San Diego, CA.

BACKGROUND: Approximately 4.1 million Americans are estimated to have been infected with hepatitis C virus (HCV), 45-85% of whom are unaware of their infection. Persons who inject drugs (PWID) account for 55.8% of all persons with HCV antibody (anti-HCV) in the U.S. PWID have limited access to healthcare and are infrequently tested for anti-HCV using conventional laboratory assays. OBJECTIVE: To evaluate performance characteristics (sensitivity and specificity) of three, pre-market rapid point-of-care tests (one oral fluid and two finger-stick assays) from two manufacturers (Chembio and MedMira) in settings providing services to young adult PWID in San Diego, CA. STUDY DESIGN: Behavioral risk assessment surveys and testing for HCV were conducted among persons who reported injection drug use (IDU) within the past 6 months as part of the Study to Assess Hepatitis C Risk (STAHR) among PWID aged 18-40 years in 2009-2010. Sensitivity and specificity of the rapid anti-HCV assays were evaluated among STAHR participants, using two commonly used testing algorithms. RESULTS: Variability in sensitivity (76.6-97.1%) and specificity (99.0-100.0%) was found across assays. The highest sensitivity achieved for the Chembio finger-stick blood, Chembio oral fluid and MedMira finger-stick blood tests was 97.1%, 85.4% and 80.0% respectively; the highest specificity was 99.0%, 100.0% and 100.0%, respectively. In multivariate analysis false negative anti-HCV results were associated with female sex for the MedMira blood assay. CONCLUSIONS: Sensitive anti-HCV rapid assays are appropriate and feasible for high-prevalence, high-risk populations such as young PWID.

Activation of the human immature natural killer cell subset by IL-12 and its regulation by endogenous TNF-alpha and IFN-gamma secretion.

Interleukin-12 (IL-12) is a newly described cytokine that can activate NK cell cytotoxic function. The present study investigated the activation pathway of human peripheral blood-derived immature NK cells by IL-12 and its comparison to activation by interleukin-2 (IL-2). Immature NK cells were prepared by flow cytometry cell sorting techniques whereby the nonbinding Free NK subset was separated from the NK target conjugate subpopulation. Coculture of Free cells with IL-12 overnight resulted in enhanced cytotoxicity and recruitment of cytotoxic Killer cells. The IL-12-mediated activation of cytotoxicity was dependent on endogenous TNF-alpha secretion and was suppressed by endogenous (interferon-gamma (IFN-gamma) secretion. However, IL-2-mediated activation was inhibited by anti-TNF-alpha antibody, but was not affected by endogenous IFN-gamma secretion. There was an upregulation of the expression of CD69 and CD25 surface antigens by IL-12-treated cells and this upregulation was diminished in the presence of anti-TNF-alpha antibody. IL-12 also upregulated the expression of the 75-kDa TNF receptor (TNF-R p75) and the upregulation was inhibited by anti-IFN-gamma antibody. The 55-kDa TNF receptor (TNF-R p55), however, was not affected by IL-12, but in the presence of anti-IFN-gamma antibody, its expression was enhanced. Thus, these results suggest that IFN-gamma selectively upregulates the expression of TNF-R p75 by IL-12 but inhibits the expression of TNF-R p55 by IL-12. Unlike IL-2, IL-12, does not stimulate the proliferation of Free cells. Furthermore, unlike IL-2, IL-12 does not stimulate the secretion of TNF-alpha by Free cells, but significantly stimulates IFN-gamma secretion. These findings demonstrate that IL-12 share some, but not all, properties of IL-2 in the activation pathway of immature NK Free cells. Furthermore, the pathway of activation of Free cells by IL-12 is under the regulation of both endogenous TNF-alpha and IFN-gamma secretion.

Activation of human peripheral blood-derived monocytes by OK-432 (Streptococcus pyogenes): augmented cytotoxicity and secretion of TNF and synergy with rIFN-gamma.

The biological response modifier OK-432 has been shown both to exert the enhancement of several immunological activities and to have a direct anti-tumor effect. The present study examines the immunopotentiating effect of OK-432 on peripheral blood monocytes (PBM) derived from normal humans. Monocyte activation was assessed by examining direct cell-mediated cytotoxic activity (CMC) and secretion of cytotoxic factors in the supernatant by the 51Cr release assay and secretion of tumor necrosis factor (TNF)-alpha detected by a sensitive radioimmunoassay. The OK-432-augmented activity was compared to that achieved by recombinant interferon-gamma (rIFN-gamma). Coculture of PBM with OK-432 overnight resulted in significant augmentation of CMC and secretion of cytotoxic factors and TNF in the supernatant. The effects observed were dose dependent and the resulting activity was much more pronounced than that achieved with an optimal concentration of IFN-gamma. The monocyte- and supernatant-mediated cytotoxic activities were in a large part attributed to TNF as both activities were inhibited by anti-TNF antibody. Several parameters of monocyte activation by OK-432 were examined. The kinetics of monocyte activation revealed that a short time exposure (2-6 hr) was sufficient for activation but maximal activation was detected after 18 hr. However, the kinetics of the cytotoxic assay were not shortened and 16-20 hr was necessary for optimal cytotoxic activity. Significant synergy was obtained when suboptimal concentrations of OK-432 and IFN-gamma were used. The synergy was noted in CMC, supernatant activity, and TNF concentration. These results demonstrate that OK-432 is a potent activator of monocyte cytotoxicity and also activates secretion of TNF. Also, OK-432 is a much more potent activator than rIFN-gamma. The synergy with OK-432 and IFN-gamma suggests that OK-432-mediated activation of monocytes takes place by a different mechanism than that mediated by rIFN-gamma. Thus, monocytes and products thereof may actively participate in the in vivo anti-tumor effect mediated by OK-432.