Targeting Unique Ligand Binding Domain Structural Features Downregulates DKK1 in Y537S ESR1 Mutant Breast Cancer Cells.

Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ERα) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ERα ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ERα breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, in vitro, and in vivo approaches to better T6I-29's activities in breast cancer cells harboring Y537S ERα. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of DKK1, a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.

Targeting Unique Ligand Binding Domain Structural Features Downregulates DKK1 in Y537S ESR1 Mutant Breast Cancer Cells.

Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ERα) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ERα ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ERα breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, in vitro, and in vivo approaches to better T6I-29's activities in breast cancer cells harboring Y537S ERα. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of DKK1, a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.

Mice that lack the interferon-gamma receptor have profoundly altered responses to infection with Bacillus Calmette-Guérin and subsequent challenge with lipopolysaccharide.

Mice with a targeted disruption of the interferon gamma receptor gene (IFN-gamma R0/0 mice) and control wild-type mice were inoculated with the Bacillus Calmette-Guérin (BCG) strain of Mycobacterium bovis. BCG infection was not lethal for wild-type mice whereas all IFN-gamma R0/0 mice died approximately 7-9 wk after inoculation. Histological examination at 2 and 6 wk after BCG inoculation showed that livers of IFN-gamma R0/0 mice had higher numbers of acid-fast bacteria than wild-type mice, especially at 6 wk. In parallel, the livers of IFN-gamma R0/0 mice showed a reduction in the formation of characteristic granulomas at 2 wk after inoculation. Injection of lipopolysaccharide (LPS) 2 wk after BCG inoculation was significantly less lethal for IFN-gamma R0/0 mice than for wild-type mice. Reduced lethality of LPS correlated with a drastically reduced production of tumor necrosis factor alpha (TNF-alpha) in the IFN-gamma R0/0 mice. Interleukin 1 alpha (IL-1 alpha) and IL-6 levels in the serum were also significantly reduced in the IFN-gamma R0/0 mice after BCG infection and LPS challenge. The greatly reduced capacity of BCG-infected IFN-gamma R0/0 mice to produce TNF-alpha may be an important factor in their inability to resist BCG infection. These results show that the presence of a functional IFN-gamma receptor is essential for the recovery of mice from BCG infection, and that IFN-gamma is a key element in the complex process whereby BCG infection leads to the sensitization to endotoxin.

Requirement for transcription factor IRF-1 in NO synthase induction in macrophages.

Production of nitric oxide (NO) by macrophages is important for the killing of intracellular infectious agents. Interferon (IFN)-gamma and lipopolysaccharide stimulate NO production by transcriptionally up-regulating the inducible NO synthase (iNOS). Macrophages from mice with a targeted disruption of the IFN regulatory factor-1 (IRF-1) gene (IRF-1-/- mice) produced little or no NO and synthesized barely detectable iNOS messenger RNA in response to stimulation. Two adjacent IRF-1 response elements were identified in the iNOS promoter. Infection with Mycobacterium bovis (BCG) was more severe in IRF-1-/- mice than in wild-type mice. Thus, IRF-1 is essential for iNOS activation in murine macrophages.

How accurately do routinely reported HIV viral load suppression proportions reflect progress towards the 90-90-90 target in the population on antiretroviral treatment in Khayelitsha, South Africa?

BACKGROUND: The Joint United Nations Programme on HIV/AIDS (UNAIDS) third 90-90-90 target requires 90% of patients on antiretroviral treatment (ART) to be virally suppressed (<1 000 copies/mL). In Khayelitsha, Cape Town, South Africa viral load (VL) suppression of <400 copies/mL was reported as 89% in 2016, but only 56% of patients had a result recorded in routine data. We conceived a VL 'cascade' to represent the steps required for an expected VL to be reported as complete in routine data and thus contribute to reported VL suppression: among those for whom a VL is 'expected', a sample must be collected and tested ('done'), a result must be 'filed' in the patient folder, 'noted' by a clinician and electronically 'captured'. The low reported completion suggested gaps along the VL cascade and cast doubt on the validity of reported suppression. OBJECTIVES: To assess the validity of routinely reported VL suppression and identify barriers to VL completion. METHODS: A retrospective cohort study between 1 July 2015 and 30 June 2016, which included all Khayelitsha patients receiving ART, with a routine VL expected, was conducted. We obtained data routinely captured on site and VL data from the laboratory system. A sample of 1 035 patient folders was reviewed. VL suppression was calculated using laboratory data, including all tests done, and compared with reported suppression based on on-site captured electronic data. Successful progression through each step on the VL cascade was estimated. We used logistic regression to identify factors associated with laboratory data and reported VL testing. RESULTS: Of 22 991 patients for whom a routine VL test was due, 84% were done, 79% filed, 76% noted and 55% captured. Using all laboratory data, VL suppression was  estimated as 82%, 87%, 89% and 91% at the 50, 200, 400 and 1 000 copies/mL thresholds, respectively, but reported suppression using captured results was 80%, 86%, 88% and 89% at those thresholds. Routine VL testing is more likely to be done in children <15 years old (adjusted odds ratio (aOR) 1.89, 95% confidence interval (CI) 1.45 - 2.48) and pregnant women (aOR 1.90, 95% CI 1.28 - 2.81) than in men, adjusted for facility. CONCLUSIONS: Despite a low reported completion, VL testing completion was high. Reported suppression using captured data was similar to suppression calculated using all laboratory data, which provided an accurate measure of progress towards the 90-90-90 target. More work is needed to reach the 16% of patients missed by routine testing.

Practical aspects of planning, building, and interpreting tissue microarrays: the Cooperative Prostate Cancer Tissue Resource experience.

This is a review of several new approaches developed at or adopted by the Cooperative Prostate Cancer Tissue Resource (CPCTR) to resolve issues involved in tissue microarray (TMA) construction and use. CPCTR developed the first needle biopsy TMA, allowing researchers to obtain 200 or more consecutive cancer sections from a single biopsy core. Using radiographs of original paraffin blocks to measure tissue thickness we developed a method to produce TMAs with a larger number of usable sections. The modular approach to plan TMA construction is also a novel concept wherein TMAs of different types, such as tumor grade TMAs, metastasis TMA and hormone refractory tumors TMA can be combined to form an ensemble of TMAs with expanded research utility, such as support for tumor progression studies. We also implemented an open access TMA Data Exchange Specification that allows TMA data to be organized in a self-describing XML document annotated with well-defined common data elements. It ensures inter-laboratory reproducibility because it offers information describing the preparation of TMA blocks and slides. There are many important aspects that may be missed by both beginners and experienced investigators in areas of TMA experimental design, human subjects protection, population sample size, selection of tumor areas to sample, strategies for saving tissues, choice of antibodies for immunohistochemistry, and TMA data management.

Induction of dorsolateral prostate adenocarcinomas and other accessory sex gland lesions in male Wistar rats by a single administration of N-methyl-N-nitrosourea, 7,12-dimethylbenz(a)anthracene, and 3,2'-dimethyl-4-aminobiphenyl after sequential treatment with cyproterone acetate and testosterone propionate.

Groups of 20-25 male Wistar rats (Cpb:WU), nine groups of 4-week-old rats, and nine groups of 8-week-old rats, were given cyproterone acetate (CA) s.c. or by gavage daily for 18 days at a dose of 50 mg/kg/day. Directly following CA treatment, the rats received 3 daily s.c. injections with testosterone propionate (TP) at a dose of 100 mg/kg/day. On the day after the last TP administration, a single dose of one of the following carcinogens was given to 3 groups: N-methyl-N-nitrosourea (MNU), 50 mg/kg i.v.; 7,12-dimethylbenz(a)anthracene, 30 mg/kg i.v.; 3,2'-dimethyl-4-aminobiphenyl, 250 mg/kg s.c. Three other groups received the same carcinogen treatments after 7 days of recovery from the CA administration. The last 3 groups received carcinogen without TP treatment, but immediately after CA pretreatment was stopped. A 25% incidence of invasively growing, metastasizing adenocarcinomas was found in the dorsolateral prostate region of 8-week-old rats that had received MNU after treatment with CA plus TP. In addition, this group had a 5% incidence of carcinoma in situ and a 5% incidence of atypical hyperplasia in the dorsolateral prostate. Lower incidences of adenocarcinoma of the dorsolateral prostate region and of carcinoma in situ and atypical hyperplasia of the dorsolateral prostate were found in other groups that were treated with MNU or 7,12-dimethylbenz(a)anthracene after pretreatment with CA, followed by TP or recovery, but never in rats that had been treated with CA only. In the groups treated with 3,2'-dimethyl-4-aminobiphenyl, which is slowly metabolized, these lesions were also found in groups that were pretreated with only CA. The carcinomas seemed to originate from the dorsolateral prostate and their average latency time was approximately 61 weeks. The 8-week-old rat given a MNU injection after sequential treatment with CA and TP may provide a relevant animal model for human prostatic cancer.

Characterization of adenocarcinomas of the dorsolateral prostate induced in Wistar rats by N-methyl-N-nitrosourea, 7,12-dimethylbenz(a)anthracene, and 3,2'-dimethyl-4-aminobiphenyl, following sequential treatment with cyproterone acetate and testosterone propionate.

Carcinomas of the rat prostate induced by a single injection of N-methyl-N-nitrosourea, 7,12-dimethylbenz(a)anthracene, and 3,2'-dimethyl-4-aminobiphenyl, after sequential treatment with cyproterone acetate and testosterone propionate, were evaluated as potential animal models for prostatic cancer. All ten carcinomas examined were located in the dorsolateral prostate region and did not involve the distal parts of the seminal vesicles and coagulating glands. The incidence of urinary obstruction leading to the animals' death was 6 of 10 rats, and metastases in the lung, abdominal lymph nodes, and/or liver also occurred in 6 of 10 rats. The tumors were invasive adenocarcinomas, showing frequent perineural invasion and a variable degree of differentiation. There were ultrastructural similarities with human prostatic carcinomas, such as intracellular lumina. Plasma acid phosphatase was increased. Enzyme histochemical analysis revealed similarities with the Dunning R3327H and -HI prostatic carcinomas but was not helpful in determining the site of origin of the tumors. The gross and microscopic appearance of the tumors and the observation of preneoplastic lesions exclusively located in the dorsolateral prostate suggest this lobe as site of origin of the carcinomas. Preneoplastic lesions (n = 9) included atypical hyperplasias (n = 5) and lesions with all histological characteristics of carcinoma except for local invasion and metastases, which were classified as carcinoma in situ (n = 4). Although androgen sensitivity could not be assessed, the observed characteristics of the tumors [their long latency time (46-80 weeks), the presence of preneoplastic lesions, and the short duration of the treatment, leaving the animals intact] all indicate that the present approach is a valid animal model for the study of prostatic carcinogenesis.

Interactive effects of dietary wheat bran and lard on N-methyl-N'-nitro-N-nitrosoguanidine-induced colon carcinogenesis in rats.

A 3 x 3 factorial experiment was conducted to examine how dietary fiber (wheat bran) and fat (lard) interactively affect the genesis of N-methyl-N'-nitro-N-nitrosoguanidine-induced colon cancer in rats. Groups of 30 male 4-week-old Wistar rats were fed ad libitum one of nine experimental diets containing either 15 (low), 27.5 (medium), or 40% (high) energy as fat in combination with 0.7 (low), 2.2 (medium), or 3.8 g (high) fiber/100 kcal for a period of 37 weeks. After 4 weeks, each rat received a total of five weekly intrarectal instillations of 6 mg N-methyl-N'-nitro-N-nitrosoguanidine/kg. The highest colon carcinoma incidence and the highest total number of carcinomas of the colon were observed in the animals fed the medium-fat/medium-fiber diet. The highest number of polyps and a relatively high polyp incidence occurred in the animals on the high-fat/low-fiber diet. An enhancing effect of fat on both the tumor incidence and tumor multiplicity was clearly present for the low-fiber diets, whereas fat had no effect when the fiber content of the diet was high. In general, the results showed a nonlinear dose-response relationship for fiber and fat. These results indicate that both dietary fiber and fat affect colon carcinogenesis in a complex, interactive manner.

Influence of N-methyl-N-nitrosourea, testosterone, and N-(4-hydroxyphenyl)-all-trans-retinamide on prostate cancer induction in Wistar-Unilever rats.

The influence of chemical carcinogen, hormonal stimulation, and chronic dietary administration of the synthetic retinoid, N-(4-hydroxyphenyl)-all-trans-retinamide (4-HPR), on the induction of prostate cancer in male Wistar-Unilever rats was determined. Three different tumor induction regimens were used: (a) a single i.v. dose of 50 mg of N-methyl-N-nitrosourea (MNU) per kg body weight, followed by chronic androgen stimulation via s.c. implantation of two silastic capsules containing 40 mg testosterone each; (b) a single i.v. dose of 50 mg of MNU per kg body weight (no testosterone treatment); and (c) chronic androgen stimulation with implanted testosterone capsules (no MNU treatment). In a fourth series of animals, the incidence of spontaneous prostate tumors was determined in groups of rats receiving neither carcinogen nor hormone stimulation. Within each series, parallel groups of animals were fed a control (vehicle-supplemented) diet or control diet supplemented with 4-HPR beginning 1 day after carcinogen administration; retinoid administration was continuous until termination of the study at 450 days. The incidence of accessory sex gland cancer in rats treated sequentially with MNU + testosterone was >60%, in comparison with cancer incidences of <20% in rats receiving MNU only and <5% in rats treated with testosterone only. No spontaneous accessory sex gland tumors were observed in rats receiving no carcinogen and no testosterone. Tumor induction in the accessory sex glands by MNU + testosterone was relatively specific for the prostate: the incidence of carcinoma of the dorsolateral/anterior prostate was more than 5-fold greater than the incidence of cancer present only in the seminal vesicle. 4-HPR conferred no protection against cancer induction in the prostate by any regimen of MNU and/or testosterone. These results demonstrate the importance of both carcinogen exposure and hormone stimulation on the induction of neoplasia in the prostate of Wistar-Unilever rats.

Chemoprevention of rat prostate carcinogenesis by 9-cis-retinoic acid.

A chemoprevention study was conducted to evaluate the activity of 9-cis-retinoic acid (9-cis-RA) as an inhibitor of prostate carcinogenesis in male Wistar-Unilever (HsdCpb:Wu) rats. After pretreatment with a sequential regimen of cyproterone acetate (50 mg/kg/day for 21 days) and testosterone propionate (100 mg/kg/day for 3 days), groups of 40 rats received a single i.v. injection of N-methyl-N-nitrosourea (MNU; 30 mg/kg body weight). Beginning 2 weeks after carcinogen administration, rats received chronic exposure to testosterone administered in s.c. implanted silastic capsules. The study was terminated at 13 months after MNU administration, and prostate cancer incidence was determined by histopathological evaluation of step sections of accessory sex glands. Continuous dietary administration of 9-cis-RA at 100 mg/kg diet or 50 mg/kg diet beginning 1 week before MNU administration reduced cancer incidence in the dorsolateral + anterior prostate from 65% in dietary controls to 18 and 20%, respectively (P < 0.001 for both comparisons). Similarly, these dose levels of 9-cis-RA reduced the incidence of cancer in all accessory sex glands from 79% in dietary controls to 48 and 33% (P < 0.01 for both comparisons), respectively. Chronic dietary administration of 9-cis-RA induced no gross or organ-specific toxicity in any animal and did not suppress group mean body weight gain. The potent anticarcinogenic activity of 9-cis-RA in the rat prostate, when considered with its apparent lack of toxicity in rodents, suggests that this and other ligands for the retinoid X receptor merit consideration for evaluation in clinical prostate cancer chemoprevention trials.

Chemoprevention of rat prostate carcinogenesis by early and delayed administration of dehydroepiandrosterone.

Two in vivo bioassays were conducted to evaluate the efficacy of dehydroepiandrosterone (DHEA) as an inhibitor of prostate carcinogenesis in rats. Prostate adenocarcinomas were induced in male Wistar-Unilever rats by a sequential regimen of cyproterone acetate and testosterone propionate, followed by a single i.v. injection of N-methyl-N-nitrosourea (MNU) and chronic androgen stimulation. In the first experiment, DHEA (1000 or 2000 mg/kg diet) was administered continuously to rats beginning 1 week before MNU exposure. In the second experiment, continuous administration of DHEA (2000 mg/kg diet) was begun either 1 week before, 20 weeks after, or 40 weeks after MNU exposure. Controls received basal diet without added DHEA. Studies were terminated at 13 months after MNU administration, and prostate cancer incidence was determined by histopathological evaluation of step sections of accessory sex glands. In the first study, continuous dietary administration of DHEA beginning 1 week before MNU resulted in a dose-related inhibition of prostate cancer induction. In the second experiment, comparable reductions in prostate cancer incidence were observed in groups exposed to DHEA beginning 1 week before, 20 weeks after, and 40 weeks after carcinogen exposure. These data demonstrate that nontoxic doses of DHEA confer significant protection against prostate carcinogenesis in rats. The efficacy of delayed administration of DHEA suggests that the compound confers protection against later stages of prostate cancer induction and can suppress the progression of existing preneoplastic lesions to invasive disease.

The role of steroid hormones in prostate carcinogenesis.

Carcinoma of the prostate is the most frequently diagnosed malignancy and the second leading cause of death as a result of cancer in men in the United States and in many other Western countries. Notwithstanding the importance of this malignancy, little is understood about its causes. The epidemiology of prostate cancer strongly suggests that environmental factors, particularly diet and nutrition, are major determinants of risk for this disease, and evidence is mounting that there are important genetic risk factors for prostate cancer. Human prostate carcinomas are often androgen sensitive and react to hormonal therapy by temporary remission, followed by relapse to an androgen-insensitive state. These well-established features of prostate cancer strongly suggest that steroid hormones, particularly androgens, play a major role in human prostatic carcinogenesis, but the precise mechanisms by which androgens affect this process are unknown. In addition, the possible involvement of estrogenic hormones is not entirely clear. The purpose of this overview is to summarize the literature about steroid hormonal factors, androgens and estrogens, and prostate carcinogenesis. From these literature observations, a multifactorial general hypothesis of prostate carcinogenesis emerges with androgens as strong tumor promoters acting via androgen receptor-mediated mechanisms to enhance the carcinogenic activity of strong endogenous genotoxic carcinogens, such as reactive estrogen metabolites and estrogen- and prostatitis-generated reactive oxygen species and possible weak environmental carcinogens of unknown nature. In this hypothesis, all of these processes are modulated by a variety of environmental factors such as diet and by genetic determinants such as hereditary susceptibility and polymorphic genes that encode for steroid hormone receptors and enzymes involved in the metabolism and action of steroid hormones.

Adenocarcinomas of the prostate induced by N-nitroso-N-methylurea in rats pretreated with cyproterone acetate and testosterone.

Prostatic adenocarcinomas were induced in 5 out of 20 Wistar rats upon a single administration of 50 mg/kg N-nitroso-N-methylurea (NMU). The rats were pretreated with a daily dose of 50 mg/kg cyproterone acetate for 3 weeks followed by 3 daily injections of 100 mg/kg testosterone. All tumours developed in the dorsolateral prostate and were invasively growing. In 2 cases distant metastases were found. Three proliferative lesions classified as carcinomas in situ were also found in the dorsolateral prostate. A total of 7/20 animals (35%) carried an adenocarcinoma and/or a carcinoma in situ. In addition, 6 epithelial hyperplasias were observed in the dorsolateral and 1 in the ventral prostate of non-tumour-bearing rats. The method described may provide a good animal model for cancer of the prostate and lead to a better understanding of prostatic carcinogenesis.

Rat liver DNA damage by the non-hepatocarcinogen 3,2'-dimethyl-4-aminobiphenyl.

The intestinal carcinogen 3,2'-dimethyl-4-aminobiphenyl (DMAB) and the hepatocarcinogen N-2-acetylaminofluorene (AAF) were administered intraperitoneally to male F344 rats to determine if both would produce DNA damage in the liver. Hepatocyte primary cultures (HPC) were prepared and DNA-repair was assessed in vitro by autoradiography. Hepatocytes from both DMAB- and AAF-exposed rats displayed DNA repair synthesis. These results confirm other observations that non-hepatocarcinogens can produce DNA damage in the liver, and that they can be detected in both in vitro and in vivo HPC/DNA repair assays.

The role of stromal cells in prostate cancer development and progression.

Prostate cancer is one of the most common malignancies in males, and tumor progression critically determines its clinical significance. Prostatic stromal cells may be critically involved in growth and progression of prostate cancer. There is substantial evidence that the stromal component of the embryological tissue of origin, the urogenital sinus, is essential in directing outgrowth and prostatic differentiation of the epithelial anlage of the prostate. The presence of a stromal androgen receptor is required for this effect, and humoral factors, such as keratinocyte growth factor, have been shown to be able to mediate it in a paracrine fashion. The adult prostate is also under control of multiple steroid hormone and paracrine peptide factors, and there is evidence that the prostatic stroma plays a major role in mediation of androgen effects on prostatic epithelium. Normal seminal vesicle mesenchyme can cause differentiation of the Dunning R3327H prostate carcinoma. Normal rat prostatic fibroblasts influence the in vivo and soft agar growth of epithelial cells derived from chemically/hormonally induced rat prostate carcinomas, as do fibroblasts that are isolated from these tumors. Both growth-enhancing and growth-inhibiting effects were observed, apparently depending on the stage of progression of both cell types as well as on whether fibroblasts were derived from the same or a different tumor than the epithelial cells. These findings indicate that stromal cells critically influence epithelial prostate cancer growth, and they suggest that these effects can significantly vary in different tumors as well as in different stages of tumor progression.

Biological functions of IFN-gamma and IFN-alpha/beta: lessons from studies in gene knockout mice.

Mice with a targeted disruption in the IFN-gamma receptor gene (IFN-gamma R0/0) provided a useful model to ask to what extent other cytokines could replace IFN-gamma in macrophage activation. In thioglycollate-elicited peritoneal macrophages from wild-typy (WT) mice, TNF enhanced nitric oxide (NO) release in the presence of IFN-gamma, though TNF alone was not effective. In macrophages from IFN-gamma R0/0 mice, which are not responsive to IFN-gamma, TNF completely failed to stimulate NO release. The NO inducing effects of IFN-alpha/beta were indistinguishable in IFN-gamma R0/0 and WT macrophages. The important role of IFN-gamma in the regulation of the induced expression of MHC class II antigen (Ia) was confirmed by showing that after systemic infection with the BCG strain of Mycobacterium bovis, peritoneal macrophages from IFN-gamma R0/0 mice had a lower level of Ia expression than macrophages from WT mice. BCG infection was not lethal for WT mice whereas all IFN-gamma R0/0 mice died 7-9 weeks after infection. It is well known that BCG infection greatly sensitizes mice to lethal action of LPS. Injection of LPS 2 weeks after BCG inoculation was significantly less lethal for IFN-gamma R0/0 mice than for WT mice. Reduced lethality of LPS correlated with a drastically reduced TNF-alpha production in the IFN-gamma R0/0 mice after BCG infection and LPS challenge. The greatly reduced ability of BCG-infected IFN-gamma R0/0 mice to produce TNF-alpha may be an important factor in their inability to resist BCG infection.(ABSTRACT TRUNCATED AT 250 WORDS)