Tumor immunotherapy using adenovirus vaccines in combination with intratumoral doses of CpG ODN.

The combination of viral vaccination with intratumoral (IT) administration of CpG ODNs is yet to be investigated as an immunotherapeutic treatment for solid tumors. Here, we show that such a treatment regime can benefit survival of tumor-challenged mice. C57BL/6 mice bearing ovalbumin (OVA)-expressing EG.7 thymoma tumors were therapeutically vaccinated with adenovirus type 5 encoding OVA (Ad5-OVA), and the tumors subsequently injected with the immunostimulatory TLR9 agonist, CpG-B ODN 1826 (CpG), 4, 7, 10, and 13 days later. This therapeutic combination resulted in enhanced mean survival times that were more than 3.5× longer than naïve mice, and greater than 40% of mice were cured and capable of resisting subsequent tumor challenge. This suggests that an adaptive immune response was generated. Both Ad5-OVA and Ad5-OVA + CpG IT treatments led to significantly increased levels of H-2 K(b)-OVA-specific CD8+ lymphocytes in the peripheral blood and intratumorally. Lymphocyte depletion studies performed in vivo implicated both NK cells and CD8+ lymphocytes as co-contributors to the therapeutic effect. Analysis of tumor infiltrating lymphocytes (TILs) on day 12 post-tumor challenge revealed that mice treated with Ad5-OVA + CpG IT possessed a significantly reduced percentage of regulatory T lymphocytes (Tregs) within the CD4+ lymphocyte population, compared with TILs isolated from mice treated with Ad5-OVA only. In addition, the proportion of CD8+ TILs that were OVA-specific was reproducibly higher in the mice treated with Ad5-OVA + CpG IT compared with other treatment groups. These findings highlight the therapeutic potential of combining intratumoral CpG and vaccination with virus encoding tumor antigen.

Development of an MHC class I L(d)-restricted PSA peptide-loaded tetramer for detection of PSA-specific CD8+ T cells in the mouse.

We set out to develop a PSA peptide-loaded tetramer for enumeration of PSA-specific CD8(+) T cells in the Balb/c mouse model. A candidate major histocompatibility complex (MHC) class I PSA peptide (HPQKVTKFML(188-197)) was selected on the basis of its ability to restimulate PSA-specific CD8(+) T cells to secrete interferon-γ in our assays. Next, H-2L(d)-restricted peptide-loaded and fluorescently labeled tetramers were produced in conjunction with the NIH Tetramer Core Facility, Atlanta, GA, USA. This tetramer was then tested for staining specificity and optimized for detection of PSA-specific CD8(+) T cells induced by our PSA-encoding adenovirus tumor vaccine. The MHC class I PSA peptide demonstrated successful restimulation of CD8(+) T cells isolated from mice previously vaccinated with a PSA-encoding adenovirus tumor vaccine, with no restimulation observed in control-vaccinated mice. The peptide-loaded H-2L(d) tetramer exhibited the desired binding specificity and allowed for detection and frequency determination of PSA-specific CD8(+) T cells by flow cytometry. We have successfully designed and validated a PSA peptide tetramer for use in the Balb/c mouse model that can be used to test PSA-based prostate cancer vaccines. Until now, PSA-specific CD8(+) T cells in the mouse have only been detectable via cytotoxic T-lymphocyte assays or intracellular cytokine staining, which primarily assess antigen-specific functional activity and not their absolute number. This research tool provides laboratories the ability to directly quantitate CD8(+) T cells elicited by PSA-specific immunotherapies and cancer vaccines that are tested in mouse models.

Prostate cancer: genes, environment, immunity and the use of immunotherapy.

Prostate cancer remains the most prevalent noncutaneous cancer, leading to almost 30,000 deaths every year in men in the United States. A large body of knowledge emphasizes a strong influence of epidemiological factors such as lifestyle, environment and diet, on the development of prostate cancer. Although risk reduction of prostate cancer has been somewhat successful, effective prevention is still lacking. Immunotherapeutic approaches, although moderately complicated, remain promising in an effort to control the progression and development of the disease. Taken together, the parameters of epidemiological studies and immunotherapeutic regimens might eventually be the most effective and preventive approach for prostate cancer. This review highlights some of the events associated with the development and prevention of prostate cancer.

Immunization with type 5 adenovirus recombinant for a tumor antigen in combination with recombinant canarypox virus (ALVAC) cytokine gene delivery induces destruction of established prostate tumors.

Prostate-specific antigen (PSA) is expressed by prostate epithelial cells and has a highly restricted tissue distribution. Prostatic malignancies in 95% of patients continue to express PSA, making this antigen a good candidate for targeted immunotherapy. The goals of our studies are to generate a recombinant PSA adenovirus type 5 (Ad5-PSA) that is safe and effectively activates a PSA-specific T-cell response capable of eliminating prostate cancer cells, and to characterize the immunologic basis for this rejection. Here we show that immunization of mice with Ad5-PSA induced PSA-specific cellular and humoral immunity that was protective against a subcutaneous challenge with RM11 prostate cancer cells expressing PSA (RM11psa), but not mock-transfected RM11 tumor cells (RM11neo). Mice immunized with recombinant adenovirus type 5 encoding beta-galactosidase (Ad5-lacZ) did not generate protective immunity. Antitumor activity was predominantly mediated by CD8(+) T lymphocytes. Although Ad5-PSA immunization prior to RM11psa challenge was protective, Ad5-PSA immunization alone was not able to control the growth of existing RM11psa tumors. In contrast, established RM11psa tumors ranging in size from 500 to 1,000 mm(3) were efficiently eliminated if Ad5-PSA priming was followed 7 days later by intratumoral injection of recombinant canarypox viruses (ALVAC) encoding interleukin-12 (IL-12), IL-2, and tumor necrosis factor-alpha. In this case, antitumor immunity was still dominated by CD8(+) T lymphocytes, but natural killer cells became necessary for a maximal response. These data provide information on the effector cell populations in a protective immune response to prostate cancer and demonstrate the utility of an Ad5-PSA vaccine combined with cytokine gene delivery to eliminate large established tumors that are refractory to other interventional methods.

Pain and immunologic response to root canal treatment and subsequent health outcomes.

OBJECTIVE: This study examined the effects of pain and stress associated with a dental procedure, root canal treatment (RCT), on natural killer cell cytotoxicity (NKCC) and the subsequent development of symptoms of upper respiratory illness during the following month. METHODS: Patients (N = 33) were recruited from those scheduled for RCT appointments. Subjects for a non-RCT comparison group (N = 14) were also recruited from dental clinic patients. Peripheral blood was drawn by use of an indwelling catheter three times: just before RCT, 30 minutes after injection of a local anesthetic, and 30 minutes after RCT (a parallel time course was followed for the comparison group.) Blood was assayed for cortisol and NKCC. Subjects completed a health diary in the month after RCT. RESULTS: Patients showed a significant increase in NKCC between baseline and RCT and a significant decrease from RCT to after RCT, whereas the comparison group did not. The NKCC following the RCT was negatively correlated with the pain level during RCT (r = -0.48, p < .01) and pain levels 2 and 6 hours after RCT (r = -0.43, p < .05; r = -0.44 p < .05, respectively). The patient group reported significantly more illness episodes 2 weeks after RCT than the comparison group (Wilcoxon rank sum = 4.78, p = .03). Discriminant function analysis correctly classified 88% of the subjects into the illness category using predictor variables of post-RCT NKCC, stress, and pain levels during RCT (F(3,21) = 8.23, p < .001). CONCLUSIONS: Transitory changes in NKCC associated with pain and stress may be implicated in the development of infectious disease episodes after an acute stressful event.

Effects of housing relocation on immunocompetence and psychosocial functioning in older adults.

BACKGROUND: The psychological and physical response to moderate life stressors among older adults has not been well characterized. This research examines effects of voluntary housing relocation on distress and immune function in healthy older adults as a model for studying the effects of moderate life stress. METHODS: Thirty older adults moving to congregate living facilities were assessed 1 month premove, 2 weeks postmove, and 3 months postmove. Twenty-eight nonmoving control subjects were assessed at similar time points. Subjects completed psychosocial questionnaires and had early morning blood draws in their homes. Blood samples were assayed for natural killer cell cytotoxicity (NKCC), interleukin-6 (IL-6), and IgG antibody titers to the Epstein Barr virus (EBV) viral capsid antigen. RESULTS: Movers demonstrated decreased vigor and elevated thought intrusion 1 month premove and 2 weeks postmove. By the 3-month follow-up, vigor increased, and intrusion decreased to levels commensurate with the controls. Averaged across all time points, movers showed lower NKCC than controls; however, post-hoc analyses indicate that by the 3-month follow-up time point, these differences were no longer significant. There were no differences between groups in IL-6 or in EBV antibody titers. Independent of the effects of group, higher levels of vigor were associated with greater NKCC at all assessments and with lower EBV titers at 2 weeks postmove. CONCLUSIONS: Findings suggest that in general, healthy older adults recover well psychologically from moderate. temporary life stressors such as moving. Whereas movers showed generally lower NKCC than controls, IL-6 and EBV antibody titers appeared not to be strongly affected by the stress of moving.

Cutting edge: restoration of the ability to generate CTL in mice immune to adenovirus by delivery of virus in a collagen-based matrix.

Viruses are commonly used for the delivery of genes coding for tumor-associated Ags to elicit tumor-specific immune responses. The success of viral vectors has been limited in preclinical and clinical trials in part because of antiviral immunity. We investigated the ability of a collagen-based matrix (Gelfoam; Pharmacia and Upjohn, Kalamazoo, MI) to improve CTL activation by recombinant adenovirus. The data show that coinjection of Gelfoam with type 5 adenovirus recombinant for prostate-specific Ag (Ad5-PSA) enhanced CTL activation. Ad5-PSA priming in Gelfoam also abrogated the inhibitory effects of adenoviral immunity on CTL activation in mice naive to PSA but immune to adenovirus. Finally, Gelfoam enhanced immunization in a self-Ag model using type 5 adenovirus recombinant for membrane-bound OVA (Ad5-mOVA) in rat insulin promoter (RIP)-mOVA-transgenic mice. Thus, Gelfoam enhances CTL activation by recombinant viral vectors in a setting where preformed Ab to the virus is present and also in a tolerant self-Ag model.

Effects of stress and relaxation on capsaicin-induced pain.

A sizable body of research has been devoted to understanding the relationship between pain sensitivity and the psychological state of the individual. Considerable disagreement as to the direction of the association still exists. This study examines the effects of 2 experimental manipulations, cognitive/emotional stress and relaxation, on capsaicin-induced pain. Subjects were pretrained in relaxation and then randomized to experimental stress produced by a 20-minute Stroop test, relaxation (tape), or a control condition (neutral video), followed by a capsaicin injection in the forearm. Cardiovascular measures were taken at regular intervals, and cortisol, norepinephrine (NE), and self-reports of arousal (relaxation index) were taken immediately before and after the experimental task. The manipulation significantly interacted with sex to predict capsaicin-induced maximum pain. Women in the stress condition reported greater pain than both men in the stress condition and women in the relaxation condition. Pain was correlated negatively with task-induced changes in NE and cortisol and positively with self-reported arousal (decreased relaxation). However, separate analyses showed that some physiologic indexes of heightened arousal (increased blood pressure and NE) predicted lower pain only in men, whereas subjective increases in arousal predicted higher pain only in women. Multiple hierarchical regression analyses confirmed that physiologic and self-reported arousal predicted pain independently and in opposite directions, and a model including both accounted for 56% of the overall variance. These findings suggest that a unidimensional model of arousal may be insufficient to explain the effects of stress on pain and that these effects operate differently in men and women.

Effects of relaxation and stress on the capsaicin-induced local inflammatory response.

OBJECTIVE: Although stress is known to modulate the inflammatory response, there has been little experimental examination of the effects of stress and stress reduction on inflammation in humans. In particular, the effects of stress and relaxation on neurogenic inflammation have been minimally studied. This study examines the effects of three experimental manipulations: mental stress, relaxation, and control on the local inflammatory response evoked by the intradermal injection of capsaicin, the active ingredient in chili peppers. METHODS: Fifty subjects (28 men and 22 women) were pretrained in relaxation using an imagery-based relaxation tape and then randomized to experimental condition. Subjects participated in an evening reactivity session including 20 minutes of a stress (Stroop test), relaxation (tape), or control (video) manipulation, followed by a capsaicin injection in the forearm. Digitized flare measurements were taken for 1 hour postcapsaicin, and measurements of cardiovascular variables, cortisol, adrenocorticotrophic hormone, and norepinephrine were taken at regular intervals. RESULTS: The size of the maximum capsaicin-induced flare was significantly smaller in the relaxation condition than in the stress or control conditions, which did not differ from each other. Increases in norepinephrine, heart rate, and systolic blood pressure during the experimental task, but not after capsaicin, significantly predicted size of maximum flare and total area under the curve of flare measurements. CONCLUSIONS: These findings suggest that stress reduction may affect local inflammatory processes. Results are consistent with sympathetic modulation of the effects of relaxation on the flare response.

Macropinocytosis as a mechanism of entry into primary human urethral epithelial cells by Neisseria gonorrhoeae.

Gonococcal entry into primary human urethral epithelial cells (HUEC) can occur by macropinocytosis. Scanning and transmission electron microscopy revealed lamellipodia surrounding gonococci, and confocal laser scanning microscopy analysis showed organisms colocalized with M(r) 70,000 fluorescein isothiocyanate-labeled dextran within the cells. Phosphoinositide 3-kinase inhibitors and an actin polymerization inhibitor prevented macropinocytic entry of gonococci into HUEC.

Life stress, mood disturbance, and elevated interleukin-6 in healthy older women.

BACKGROUND: Although adverse effects of severe chronic stress on immunocompetence and physical well-being in older adults have been reported, the immune response to less severe life stress among healthy older adults, particularly among women, is not well understood. Interleukin-6 (IL-6) has been considered a good overall indicator of immune functioning in older adults because of its contribution to the pathogenesis of several age-related conditions such as osteoporosis. Regulation of IL-6 is impaired in elderly adults, and levels of IL-6 increase with stress and depression. This research cross-sectionally examined levels of IL-6 in three groups of healthy older women with varying levels of life stress and mood disturbance and a healthy group of young women. METHODS: Subjects included 18 caregivers of Alzheimer's patients, 17 older women assessed one month before relocation of their residence, 15 nonmoving and noncaregiving older women, and 20 younger women. Subjects completed the Profile of Mood States (POMS) and had early morning blood draws. RESULTS: Alzheimer's caregivers reported significantly greater distress than women of all other groups. IL-6 levels in caregivers were significantly higher than those of all other women. The older women had significantly higher IL-6 than young controls, but there were no significant differences in IL-6 between movers and older controls. Among all women, greater depression and distress were related to higher levels of IL-6. CONCLUSIONS: These findings suggest that in older women, chronic stressors are associated with significant elevations in IL-6 over and above the elevations associated with normal aging, but that moderate stressors may not be related to appreciable elevations in IL-6.

Suppression of prostate cancer invasive potential and matrix metalloproteinase activity by E-cadherin transfection.

Our previous studies have demonstrated the heterogeneous expression of E-cadherin in a Dunning rat prostate tumor model. From this model, cloned E-cadherin-negative cells exhibited enhanced invasive and metastatic potential when compared with E-cadherin-positive cells. In this report, we examined the invasion suppressor function of E-cadherin in these prostate tumor cell clones. The E-cadherin gene was stably transfected into E-cadherin-negative Dunning clones. E-cadherin transfection resulted in the up-regulation of the three major catenins (alpha-, beta-, and gamma-catenin) and enhanced Ca2+-dependent cellular cohesiveness. Morphological analyses of E-cadherin transfectants revealed a reversion from a fibroblastic, motile phenotype to a more stationary epithelial phenotype. Matrix metalloproteinase 2, an important marker associated with invasive and metastatic potential, was reduced in all six stable transfected lines. A concomitant decrease in cellular invasiveness was observed, as assessed in vitro by the ability of the transfected cells to invade biological matrices. These results lend further support to the hypothesis that in this experimental system, E-cadherin plays a central role in reducing the cellular invasiveness of prostatic adenocarcinoma, due in part to the down-regulation of matrix metalloproteinase 2 activity. Moreover, the data shed additional light on the possible mechanisms involved in E-cadherin-dependent modulation of invasion.

A novel immunological model for the study of prostate cancer.

The Dunning R-3327 rat prostatic adenocarcinoma is a widely accepted model for in vivo experimental studies of prostate cancer. We have previously derived phenotypically distinct cell lines from a s.c. tumor resulting from the inoculation of the R-3327-5 subclone into Copenhagen rats. In this study, we report studies using a gelatin sponge model for the delivery of tumor cells and the retrieval of tumor-specific leukocytes responsive to different prostatic cell lines. S.c. preimplanted sponges were inoculated with tumor cells previously selected for differential properties of tumor formation and metastasis and examined for leukocyte content at time points of 1, 3, and 5 weeks after tumor cell inoculation. Cytospin and flow cytometric analyses revealed fewer tumor-associated leukocytes present in sponges inoculated with tumorigenic R-3327-5' and R-3327-5'B lines, with lesser sponge degradation, than in experiments with the nontumorigenic R-3327-5'A line, suggestive of a tumor cell-induced immunomodulatory mechanism. Morphological studies indicate an intermittent tumor growth pattern that gradually disappears in sponges inoculated with the nontumorigenic R-3327-5'A cells but a robust growth pattern in sponges inoculated with the tumorigenic cell lines. Cytokine analyses show the secretion of higher levels of active transforming growth factor-beta by the more invasive and metastatic lines. Total transforming growth factor-beta levels are higher in the epithelial, tumorigenic R-3327-5'B line. Additionally, the more tumorigenic lines secrete interleukin 10, a potent immunosuppressive molecule. In this report, we demonstrate the ability to retrieve viable leukocyte populations from a prostate tumor line bearing sponges, which offers an important model for further in vitro and in vivo manipulations and holds promise for testing adoptive immunotherapeutic strategies.

Sense of coherence moderates the relationship between life stress and natural killer cell activity in healthy older adults.

A sense of coherence (SOC) has been found to be a strong predictor of health outcomes and life satisfaction in older adults. This study investigated mood and immune effects of anticipated voluntary housing relocation in 30 healthy older adults and 28 age-matched controls and examined whether SOC would buffer effects of relocation on natural killer (NK) cell activity. Movers completed assessments and had blood drawn 1 month before relocation to congregate living facilities; controls were assessed concurrently. Compared with the control group, movers showed decreased positive mood and NK activity and elevated thought intrusion. Positive mood mediated the relationship of moving with NK activity, whereas SOC moderated this relationship. Low SOC movers had the poorest NK activity; that of high SOC movers was less compromised. These findings are consistent with possible salutogenic contributions of SOC and positive mood to immune function in older adults facing stressful life transitions.

Neisseria gonorrhoeae induces focal polymerization of actin in primary human urethral epithelium.

The pathogenic Neisseria species induce cytoskeletal reorganization in immortalized cell lines. In Chang conjunctival epithelium and T84 intestinal epithelium, focal cytoskeletal rearrangements in which bacteria contacted the epithelial surface were observed. We show that actin footprints are induced in gonococcus-challenged primary urethral epithelium. Moreover, the microbes induced microvillus extension from the epithelial cell surface. Our results indicate that formation of actin footprints is not an artifact of commonly used immortalized cell lines.

Alterations in constituent urinary proteins in response to bladder outlet obstruction in rats.

PURPOSE: Benign prostatic hyperplasia, resulting in bladder outflow obstruction, induces well recognized clinical symptoms and morphologic bladder changes. Despite these phenomenon, relatively little is known with regard to the precise molecular events occurring in the bladder as a consequence of obstruction. In an effort to screen for alterations in bladder gene expression induced by obstruction, and/or alterations in uroepithelial integrity, this study compared pre- and post-obstructive constituent urinary proteins in an animal model. MATERIALS AND METHODS: Outlet obstruction was created using a previously established model system. Experimental animals were surgically obstructed for either 2 or 7 days, at which time the urine was aspirated and the bladders removed and weighed. Urinary proteins were separated using 2-D PAGE. Following comparison of sham versus experimental animals, microsequencing was performed on proteins that were down regulated. RESULTS: Duplicate experiments confirmed the presence of outflow obstruction. Statistically significant increases (p <0.01) in bladder weights were seen at 2 and 7 days in the obstructed groups as compared with both sham and control groups. 2-D PAGE demonstrated a down regulation of three urinary proteins post-obstruction. Microsequencing identified these proteins as prostatic steroid-binding protein C3 precursor (pI=5.5, MW=15000), glandular kallikrein 9 (S3) precursor (pI=6.2, MW=19000), and glandular kallikrein 8 (P1) precursor (pI=6.2, MW=33000). CONCLUSIONS: Bladder outflow obstruction alters constituent urinary protein composition in an animal model system. The precise etiology of these alterations remains to be defined.

Fine structural studies of induced tumors arising within the prostatic complex of Lobund-Wistar rats.

BACKGROUND: Morris Pollard, Phyllis Luckert, and colleagues have reported the occurrence of spontaneously arising tumors of the prostatic complex in aged Lobund-Wistar (L-W) rats, and have also shown that the genesis of such tumors may be accelerated by means of intravenous administration of methylnitrosourea, followed by androgen supplementation. METHODS: Light and electron microscopic investigations of the tumors arising under this regime were conducted, with the objective of documenting morphological changes attending the transformation process; 10 tumor samples were used for the electron microscopic studies. RESULTS: All tumors studied were adenocarcinomas arising within the prostatic complex of induced animals. These tumors varied in size, degree of differentiation, and invasiveness. Foci of prostatic intraepithelial neoplasia were noted in light microscopic studies as well. Consistent fine structural features exhibited by cells of the induced adenocarcinomas included a large nuclear to cytoplasmic ratio; large irregular nuclei with heavily marginated chromatin; conspicuous nucleoli; abundant ribosomes and polysomes and a paucity of rough endoplasmic reticulum; and numerous cytoplasmic vesicles and lipid inclusions. Numerous, short microvilli extended from the cell surface into a copious surrounding extracellular matrix. CONCLUSIONS: Thus, these tumors shared many of the fine structural features characteristic of the Dunning (rat) and human prostatic adenocarcinomas.

Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae.

In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected.

Association of smoking, body mass, and physical activity with risk of prostate cancer in the Iowa 65+ Rural Health Study (United States).

Smoking, obesity, alcohol, and physical activity can modulate the endocrine system, and therefore have been hypothesized to play a role in the etiology of prostate cancer. At baseline in 1982, 80 percent (n = 3,673) of the noninstitutionalized persons age 65+ in two rural Iowa (United States) counties were enrolled into the Iowa 65+ Rural Health Study. Follow-up for mortality was complete through 1993, and cancer experience was determined by linkage to the State Health Registry of Iowa cancer database for the years 1973-93. We analyzed data on 1,050 men aged 65 to 101 years (mean age 73.5) with a full interview in 1982 and with no documented cancer in the 10 years prior to baseline. Through 1993 (8,474 person-years of follow-up), there were 71 incident cases of prostate cancer. In a multivariate model, age, cigarette smoking (relative risk [RR] = 2.9 for currently smoking 20 or more cigarettes per day compared with never smoking; P trend = 0.009), greater body mass index (BMI) (wt/ht2) (RR = 1.7 for BMI > 27.8 kg/m2 compared with < 23.6; P trend = 0.1), and greater level of physical activity (RR = 1.9 for high activity level cf inactive; P trend = 0.05) were independent predictors of prostate cancer, and these associations were stronger for regional or disseminated disease at diagnosis. Percent change in BMI from age 50 to baseline was associated positively with risk (P trend = 0.01), and this association appeared to be stronger in heavier men. There were no data on diet. These findings suggest that smoking, overweight, and weight gain in later life are risk factors for prostate cancer and support a hormonal etiology; the positive association for physical activity confirms some previous reports, but remains without a credible biologic mechanism.