Lifespan socioeconomic context is associated with cytomegalovirus and late-differentiated CD8+ T and NK cells: Initial results in older adults.

OBJECTIVE: Lower socioeconomic status (SES) can accelerate immune aging; however, it is unknown whether and how lifespan socioeconomic context (SEC) -the relative wealth and quality of the communities an individual lives in across their lifespan- impacts immune aging. We examined the effects of childhood and adulthood SEC on late-differentiated immune cells and investigated the mediating and moderating role of cytomegalovirus (CMV), a key driver of immune aging. METHODS: Adults 60 years and older (N = 109) reported their addresses from birth to age 60, which were coded for county-level employment, education, and income to construct a latent SEC variable, averaged across ages 0-18 (childhood SEC) and 19-60 (adulthood SEC). Blood was drawn semiannually over 5 years for CMV serostatus and flow cytometry estimates of late-differentiated CD8+ T and natural killer (NK) cells. Models were adjusted for chronological age, time, gender, and individual SES (current income and education). RESULTS: Lower childhood SEC was associated with higher percentages of late-differentiated CD8+ T and NK cells via CMV seropositivity (indirect effects ps .015-.028). Additionally, an interaction between CMV serostatus and SEC on CD8+ T cell aging (p = .049) demonstrated that adulthood SEC was negatively associated with immune aging among CMV- but not CMV+ adults. CONCLUSIONS: Beyond current SES, socioeconomic context related to immune aging in distinct patterns by lifespan phase. Lower childhood SEC importantly may influence who acquires CMV, which in turn, predicts higher levels of immune aging, whereas higher adulthood SEC was protective against immune aging among CMV- older adults. These initial results need to be explored in larger samples.

Life stressors and immune aging: Protective effects of cognitive reappraisal.

Stressful life events may accelerate aspects of immune aging, but habitual use of an adaptive emotion regulation strategy, cognitive reappraisal, may attenuate these effects. This study examined whether cognitive reappraisal moderates the associations between life stressor frequency and stressor desirability on aspects of immune aging, including late-differentiated CD8+ T and natural killer (NK) cells and inflammatory markers (IL-6, TNF-α, and CRP), both between and within people in a longitudinal sample of 149 older adults (mean age = 77.8, range: 64-92 years). Participants reported stressful life events, use of cognitive reappraisal, and provided blood semiannually for up to 5 years to assess aspects of immune aging. Multilevel models, adjusted for demographic and health covariates, tested the between-person (stable, trait-like differences) and within-person associations (dynamic fluctuations) among life stressors and reappraisal on immune aging. Experiencing more frequent life stressors than usual was associated with higher levels of late-differentiated NK cells within person, but this effect was accounted for by experiencing health-related stressors. Unexpectedly, experiencing more frequent and less desirable stressors were associated with lower average levels of TNF-α. As expected, reappraisal moderated the associations between life stressors and late-differentiated NK cells between people and IL-6 within people. Specifically, older adults who experienced less desirable stressors but also used more reappraisal had significantly lower proportions of late-differentiated NK cells on average and lower levels of IL-6 within-person. These results suggest cognitive reappraisal may play a protective role in attenuating the effects of stressful life events on aspects of innate immune aging in older adults.

Streptococcus gordonii-Induced miRNAs Regulate CCL20 Responses in Human Oral Epithelial Cells.

The mechanisms through which oral commensal bacteria mitigates uncontrolled inflammatory responses of the oral mucosa remain unknown. Here, we show that representative oral bacterial species normally associated with oral health [S. gordonii (Sg), V. parvula (Vp), A. naeslundii (An), C. sputigena (Cs), and N. mucosa (Nm)] enhanced differential chemokine responses in oral epithelial cells (OECs), with some bacteria (An, Vp, and Nm) inducing higher chemokine levels (CXCL1, CXCL8) than others (Sg, Cs). Although all bacterial species (except Cs) increased CCL20 mRNA levels consistent with protein elevations in cell lysates, only An, Vp, and Nm induced higher CCL20 secretion, similar to the effect of the oral pathogen F. nucleatum (Fn). In contrast, most CCL20 remained associated with OECs exposed to Sg and negligible amounts released into the cell supernatants. Consistently, Sg attenuated An-induced CCL20. MiR-4516 and miR-663a were identified as Sg-specifically induced miRNAs modulating validated targets of chemokine-associated pathways. Cell transfection with miR-4516 and miR-663a decreased An- and Fn-induced CCL20. MiRNA upregulation and attenuation of An-induced CCL20 by Sg were reversed by catalase. Up-regulation of both miRNAs was specifically enhanced by oral streptococci H2O2-producers. These findings suggest that CCL20 levels produced by OECs in response to bacterial challenge are regulated by Sg-induced miR-4516 and miR-663a in a mechanism that involves hydrogen peroxide. This type of molecular mechanism could partly explain the central role of specific oral streptococcal species in balancing inflammatory and antimicrobial responses given the critical role of CCL20 in innate (antimicrobial) and adaptive immunity (modulates Th17 responses).

Perceived stress, cytomegalovirus titers, and late-differentiated T and NK cells: Between-, within-person associations in a longitudinal study of older adults.

Cytomegalovirus (CMV) and psychological stress are implicated as drivers of immunological aging. It is unknown, however, whether associations among CMV titers, stress, and immune aging are more stable or dynamic over time. The present investigation tested the between-person (stable differences) and within-person (dynamic fluctuations) associations of CMV titers and perceived stress on late-differentiated T and natural killer (NK) peripheral blood cells in a longitudinal study of older adults aged 64-92 years (N = 149). Participants reported stress levels and provided blood biannually for 2.5 years (up to 5 waves per person) to assess CMV IgG titers and composites of late-differentiated CD8 T cells (CD28- and CD57 + subsets) and CD56dim NK cells (CD57+, NKG2C+, and FcεRIγ- subsets). In multilevel models that controlled for demographic variables, higher CMV titers were associated with higher proportions and counts of aged T and NK cells between people and lower counts of aged T cells within people. Perceived stress was associated with higher counts of aged T cells between people, but was not associated with aged NK cells. A significant interaction between stress and CMV titers on T cells between people indicated that older adults with lower stress levels and lower CMV titers had the lowest proportions of late-differentiated T cells, whereas those with higher stress levels had high proportions, regardless of CMV control. Our results provide evidence for longer-term, between-person associations among CMV titers, stress, and immunological aging, rather than dynamic within-person associations. We propose that targeting factors that promote low, stable perceived stress in older adults may retard T cell differentiation and ultimately support healthy aging.

A Potential Role of Phospholipase 2 Group IIA (PLA2-IIA) in P. gingivalis-Induced Oral Dysbiosis.

Porphyromonas gingivalis is an oral pathogen with the ability to induce oral dysbiosis and periodontal disease. Nevertheless, the mechanisms by which P. gingivalis could abrogate the host-microbe symbiotic relationship leading to oral dysbiosis remain unclear. We have recently demonstrated that P. gingivalis specifically increased the antimicrobial properties of oral epithelial cells, through a strong induction of the expression of PLA2-IIA in a mechanism that involves activation of the Notch-1 receptor. Moreover, gingival expression of PLA2-IIA was significantly increased during initiation and progression of periodontal disease in non-human primates and interestingly, those PLA2-IIA expression changes were concurrent with oral dysbiosis. In this chapter, we present an innovative hypothesis of a potential mechanism involved in P. gingivalis-induced oral dysbiosis and inflammation based on our previous observations and a robust body of literature that supports the antimicrobial and proinflammatory properties of PLA2-IIA as well as its role in other chronic inflammatory diseases.

The periodontal war: microbes and immunity.

Maintenance of periodontal health or transition to a periodontal lesion reflects the continuous and ongoing battle between the vast microbial ecology in the oral cavity and the array of resident and emigrating inflammatory/immune cells in the periodontium. This war clearly signifies many 'battlefronts' representing the interface of the mucosal-surface cells with the dynamic biofilms composed of commensal and potential pathogenic species, as well as more recent knowledge demonstrating active invasion of cells and tissues of the periodontium leading to skirmishes in connective tissue, the locality of bone and even in the local vasculature. Research in the discipline has uncovered a concerted effort of the microbiome, using an array of survival strategies, to interact with other bacteria and host cells. These strategies aid in colonization by 'ambushing, infiltrating and outflanking' host cells and molecules, responding to local environmental changes (including booby traps for host biomolecules), communicating within and between genera and species that provide MASINT (Measurement and Signature Intelligence) to enhance sustained survival, sabotage the host inflammatory and immune responses and by potentially adopting a 'Fabian strategy' with a war of attrition and resulting disease manifestations. Additionally, much has been learned regarding the ever-increasing complexity of the host-response armamentarium at both cellular and molecular levels that is addressed in this review. Knowledge regarding how these systems fully interact requires both new laboratory and clinical tools, as well as sophisticated modeling of the networks that help maintain homeostasis and are dysregulated in disease. Finally, the triggers resulting in a 'coup de main' by the microbiome (exacerbation of disease) and the characteristics of susceptible hosts that can result in 'pyrrhic victories' with collateral damage to host tissues, the hallmark of periodontitis, remains unclear. While much has been learned, substantial gaps in our understanding of the 'parameters of this war' remain elusive toward fulfilling the Sun Tzu adage: 'If you know the enemy and know yourself, you need not fear the result of a hundred battles.'

Human natural killer cell microRNA: differential expression of MIR181A1B1 and MIR181A2B2 genes encoding identical mature microRNAs.

Natural killer (NK) and T lymphocytes share many properties, yet only NK cells respond rapidly to infection and cancer without pre-activation. We found that few microRNAs (miRNAs) differed significantly between human NK and T cells. Among those miRNAs, miR-181a and miR-181b levels rose during NK cell differentiation. Prior studies indicate that miR-181a and miR-181b are critical for human NK cell development and are co-transcribed from genes on chromosome 1 (MIR181A1B1) and on chromosome 9 (MIR181A2B2). We mapped human MIR181A1B1 and MIR181A2B2 transcription start sites to 78.3 kb and 34.0 kb upstream of the mature miRNAs, generating predominantly unspliced transcripts of 80-127 kb and ~60 kb, respectively. Unlike mouse thymocytes, human T cells expressed both MIR181A1B1 and MIR181A2B2. We tested the hypothesis that NK cells differentially transcribe the two genes during development and in response to immune regulatory cytokines. During NK-cell differentiation, MIR181A2B2 expression rose markedly and exceeded that of MIR181A1B1. TGF-ß treatment increased NK-cell MIR181A2B2 transcription, whereas IL-2, IL-15 and IL-12/IL-18 treatments upregulated MIR181A1B1. The MIR181A2B2 promoter was strongly transactivated by SMAD3 and SMAD4 transcription factors, suggesting that TGF-ß signaling upregulates MIR181A2B2 expression, at least in part, through SMAD-dependent promoter activation.

Differential transcription factor use by the KIR2DL4 promoter under constitutive and IL-2/15-treated conditions.

KIR2DL4 is unique among human KIR genes in expression, cellular localization, structure, and function, yet the transcription factors required for its expression have not been identified. Using mutagenesis, EMSA, and cotransfection assays, we identified two redundant Runx binding sites in the 2DL4 promoter as essential for constitutive 2DL4 transcription, with contributions by a cyclic AMP response element (CRE) and initiator elements. IL-2- and IL-15-stimulated human NK cell lines increased 2DL4 promoter activity, which required functional Runx, CRE, and Ets sites. Chromatin immunoprecipitation experiments show that Runx3 and Ets1 bind the 2DL4 promoter in situ. 2DL4 promoter activity had similar transcription factor requirements in T cells. Runx, CRE, and Ets binding motifs are present in 2DL4 promoters from across primate species, but other postulated transcription factor binding sites are not preserved. Differences between 2DL4 and clonally restricted KIR promoters suggest a model that explains the unique 2DL4 expression pattern in human NK cells.

Human NK cells proliferate and die in vivo more rapidly than T cells in healthy young and elderly adults.

NK cells are essential for health, yet little is known about human NK turnover in vivo. In both young and elderly women, all NK subsets proliferated and died more rapidly than T cells. CD56(bright) NK cells proliferated rapidly but died relatively slowly, suggesting that proliferating CD56(bright) cells differentiate into CD56(dim) NK cells in vivo. The relationship between CD56(dim) and CD56(bright) proliferating cells indicates that proliferating CD56(dim) cells both self-renew and are derived from proliferating CD56(bright) NK cells. Our data suggest that some dying CD56(dim) cells become CD16(+)CD56(-) NK cells and that CD16(-)CD56(low) NK cells respond rapidly to cellular and cytokine stimulation. We propose a model in which all NK cell subsets are in dynamic flux. About half of CD56(dim) NK cells expressed CD57, which was weakly associated with low proliferation. Surprisingly, CD57 expression was associated with higher proliferation rates in both CD8(+) and CD8(-) T cells. Therefore, CD57 is not a reliable marker of senescent, nonproliferative T cells in vivo. NKG2A expression declined with age on both NK cells and T cells. Killer cell Ig-like receptor expression increased with age on T cells but not on NK cells. Although the percentage of CD56(bright) NK cells declined with age and the percentage of CD56(dim) NK cells increased with age, there were no significant age-related proliferation or apoptosis differences for these two populations or for total NK cells. In vivo human NK cell turnover is rapid in both young and elderly adults.

Cutting edge: microRNA-181 promotes human NK cell development by regulating Notch signaling.

MicroRNAs (miRs) have recently been identified as important regulators of gene expression at the posttranscriptional level. Although it has clearly been established that miRs influence the ontogeny of several immune cell lineages, the role of individual miRs during NK cell development has not been described. In this study, we show that miR-181 expression levels have a profound impact on the development of human NK cells from CD34(+) hematopoietic progenitor cells and IFN-γ production in primary CD56(+) NK cells. We also demonstrate that nemo-like kinase (NLK), an inhibitor of Notch signaling, is a target of miR-181 in NK cells, and knockdown of NLK mirrors the developmental effect of miR-181 overexpression. We conclude that miR-181 promotes NK cell development, at least in part, through the suppression of NLK, providing an important link between miRs and Notch signaling.

Resources and lymphocyte terminal maturity among older adults.

OBJECTIVE: Women's financial resources were associated with more terminal maturity in natural killer lymphocytes, generally associated with loss of proliferative potential, during the "Great Recession". This preregistered analysis expanded on that finding in a longitudinal design including both genders and examining the role of cytomegalovirus (CMV) serostatus. METHOD: Older adults (N = 138, 57% women) were assessed longitudinally during 2012-2017; including self-reported psychological, social, financial, and status-skill resources, CMV antibody titers and serostatus, and assessment of T and natural killer lymphocyte terminal maturity (LTM). RESULTS: Neither total nor financial resources were associated with LTM. Adjusting only for age, more psychological resources (e.g., meaning, hope, humor) were associated with lower T LTM (percent: γ = -1.11 [-1.78, -.44]; number: γ = -.99 [-1.70, -.27]). There were no significant interactions with age, gender, or CMV serostatus; however, additionally adjusting for serostatus reduced the effect of psychological resources (percent: γ = -.41 [-93, .12]; number: (γ = -.40 [-.94, .13]). CONCLUSIONS: Outside the context of the "Great Recession", psychological resources but not financial resources were associated with terminal maturity in T cells, a relationship related to CMV serostatus. Further studies in different and more diverse samples, and in different eras, are needed to understand what resources are most protective against immunological aging, when, and for whom. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Automation in flow cytometry: Guidelines and review of systems.

Automation in flow cytometry has recently advanced from the partial laboratory automation and robotics islets, to more fully integrated systems. This article reviews three manufacturers' newest sample preparation systems: the Beckman CellMek, the Sysmex PS-10, and the BD FACSDuet. These three instruments are capable of performing many of the manual steps in flow cytometry sample processing (pipetting, staining, lysing, washing, fixing). General description, capabilities, advantages, and disadvantages of each system are compared. Overall, these systems have the potential to become mainstay items in today's busy clinical flow cytometry laboratories, and save a significant amount of hands-on time for laboratory staff.

Differential Inhibition of Anaplerotic Pyruvate Carboxylation and Glutaminolysis-Fueled Anabolism Underlies Distinct Toxicity of Selenium Agents in Human Lung Cancer.

Past chemopreventive human trials on dietary selenium supplements produced controversial outcomes. They largely employed selenomethionine (SeM)-based diets. SeM was less toxic than selenite or methylseleninic acid (MSeA) to lung cancer cells. We thus investigated the toxic action of these Se agents in two non-small cell lung cancer (NSCLC) cell lines and ex vivo organotypic cultures (OTC) of NSCLC patient lung tissues. Stable isotope-resolved metabolomics (SIRM) using 13C6-glucose and 13C5,15N2-glutamine tracers with gene knockdowns were employed to examine metabolic dysregulations associated with cell type- and treatment-dependent phenotypic changes. Inhibition of key anaplerotic processes, pyruvate carboxylation (PyC) and glutaminolysis were elicited by exposure to MSeA and selenite but not by SeM. They were accompanied by distinct anabolic dysregulation and reflected cell type-dependent changes in proliferation/death/cell cycle arrest. NSCLC OTC showed similar responses of PyC and/or glutaminolysis to the three agents, which correlated with tissue damages. Altogether, we found differential perturbations in anaplerosis-fueled anabolic pathways to underlie the distinct anti-cancer actions of the three Se agents, which could also explain the failure of SeM-based chemoprevention trials.

The prognostic and predictive power of redox protein expression for anthracycline-based chemotherapy response in locally advanced breast cancer.

Neoadjuvant chemotherapy has become the standard of care for locally advanced primary breast cancer. Anthracycline-based regimens have proven to be one of the most effective treatments in this setting. As certain cytotoxic antineoplastic agents, such as anthracyclines, generate reactive oxygen species as a by-product of their mechanism of action, we examined whether redox protein expression was involved in the response to anthracycline-based chemotherapy and with clinical outcome. Pre-treatment needle core biopsy and post-anthracycline treatment tumour sections were analysed from 98 cases. In all, 32 individuals had a complete clinical response and 17 had a complete pathological response. Immunohistochemical staining was performed for eight redox proteins: thioredoxin, thioredoxin reductase, thioredoxin interacting protein (TxNIP), glutathione S-transferase (GST) π, θ and α, catalase and manganese superoxide dismutase. GST π (P=0.05) and catalase (P=0.045) were associated with pathological complete response in pre-chemotherapy samples. TxNIP (P=0.017) and thioredoxin reductase (P=0.022) were independent prognostic factors for distant metastasis-free survival and TxNIP for overall survival (P=0.014). In oestrogen receptor negative patients that are known to have a poor overall survival, a considerably worse prognosis was seen in cases that exhibited low expression of TxNIP (P=0.000003), stratifying patients into more defined groups. This study indicates the importance of redox regulation in determining breast cancer response to anthracycline-based chemotherapy and provides ways of further stratifying pre-chemotherapy patients to potentially allow more tailored treatments.

The role of late-differentiated T cells, a proxy for IFN-γ-production, in older adults' social networks.

Interferon-γ (IFN-γ), an inflammatory biomarker that promotes antiviral immunity, may be a prerequisite for sociability. IFN-γ production in older adulthood is driven by late-differentiated CD8+ T cells, particularly CD28-and CD57+ subsets, which increase with age, reduce immune response, and increase chronic disease risk. The present study investigated the relationship between late-differentiated T cells (LDTC) and sociability in a longitudinal study of healthy aging. 139 older adults (Mage = 77.95, range 65-93; 58% female, 57% college educated, and 94% Caucasian) provided data at up to 10 occasions (M = 7). Social network size and diversity and cytomegalovirus (CMV) status were collected at every wave. Percentage of LDTC was measured at up to 4 waves and averaged for each participant. There were no significant main effects of LDTC or interactions between LDTC and time on social network size or diversity. Adjustment for baseline age, gender, and sensitivity analyses including CMV and imputed data did not change results. IFN-γ may not play a role in dictating social behavior in older adults. Alternately, LDTC may not have accurately represented circulating levels of IFN-γ. Future work should continue exploring IFN-γ and social behavior, particularly as it relates to age-related changes. The role of IFN-γ-producing, late-differentiated T cells in older adults' social networks.

Mechanisms underlying the effects of caloric restriction on hypertension.

Hypertension is a major risk factor for cardiovascular disease (CVD) as well as a major contributor to all-cause mortality and disability worldwide. The pathophysiology of hypertension is highly attributed to a dysfunctional endothelium and vascular remodeling. Despite the wide use of pharmacological therapies that modulate these pathways, a large percentage of patients continue to have uncontrolled hypertension, and the use of non-pharmacological interventions is increasingly investigated. Among these, caloric restriction (CR) appears to be a promising nutritional intervention for the management of hypertension. However, the mechanisms behind this effect are not yet fully understood, although an evolving view supports a significant impact of CR on vascular structure and function, specifically at the level of vascular endothelial cells, vascular smooth muscle cells along with their extracellular matrix (ECM). Accumulating evidence suggests that CR promotes endothelium-dependent vasodilation through activating eNOS and increasing nitric oxide (NO) levels through multiple cascades involving modulation of oxidative stress, autophagy, and inflammation. Indeed, CR diminishes phenotypic shift, and suppresses proliferation and migration of VSMCs via pathways involving NO and mTOR. By regulating transforming growth factor-ß and matrix metalloproteinases, CR appears to reduce ECM and collagen deposition in vascular walls. Here, we offer a detailed discussion of how these mechanisms contribute to CR's influence on reducing blood pressure. Such mechanisms could then provide a valuable foundation on which to base new therapeutic interventions for hypertension.

The chromosome 3p21.3-encoded gene, LIMD1, is a critical tumor suppressor involved in human lung cancer development.

Loss of heterozygosity (LOH) and homozygous deletions at chromosome 3p21.3 are common in both small and nonsmall cell lung cancers, indicating the likely presence of tumor suppressor genes (TSGs). Although genetic and epigenetic changes within this region have been identified, the functional significance of these changes has not been explored. Concurrent protein expression and genetic analyses of human lung tumors coupled with functional studies have not been done. Here, we show that expression of the 3p21.3 gene, LIMD1, is frequently down-regulated in human lung tumors. Loss of LIMD1 expression occurs through a combination of gene deletion, LOH, and epigenetic silencing of transcription without evidence for coding region mutations. Experimentally, LIMD1 is a bona fide TSG. Limd1(-/-) mice are predisposed to chemical-induced lung adenocarcinoma and genetic inactivation of Limd1 in mice heterozygous for oncogenic K-Ras(G12D) markedly increased tumor initiation, promotion, and mortality. Thus, we conclude that LIMD1 is a validated chromosome 3p21.3 tumor-suppressor gene involved in human lung cancer development. LIMD1 is a LIM domain containing adapter protein that localizes to E-cadherin cell-cell adhesive junctions, yet also translocates to the nucleus where it has been shown to function as an RB corepressor. As such, LIMD1 has the potential to communicate cell extrinsic or environmental cues with nuclear responses.

Intratumoral T cell infiltration, MHC class I and STAT1 as biomarkers of good prognosis in colorectal cancer.

OBJECTIVE: To evaluate immunosurveillance/editing in colorectal cancer. DESIGN: Transformation stimulates the production of interferon gamma (IFNgamma) which signals via the IFNgamma receptor (IFNGR1) on tumours. This results in stimulation of nuclear STAT1 (nSTAT1), inhibition of tumour growth and upregulation of major histocompatibility complex (MHC) while promoting T cell extravasation. In contrast, downregulation of MHC class I by allele loss results in loss of T cell recognition. A tissue microarray of 462 colorectal tumours with mean follow-up of 42 months (range 1-116) was stained by immunohistochemistry for markers which predict immunosurveillance/editing. RESULTS: The presence of a high level of intratumoral T cells (ITTC) correlated with improved survival compared with a low level of ITTC, with a mean difference in survival of 16.3 months (p=0.006). There was a direct correlation between nSTAT1 expression and ITTC (p<0.001). Patients whose tumours had a high level of ITTC and nSTAT1 survived 20 months longer than those whose tumours had a low level of ITTC and no nSTAT1. A strong correlation was seen between ITTC and MHC class I expression (p=0.0002). A mean survival advantage of 26.1 months was seen in patients whose tumours had strong MHC I expression and high levels of ITTC over those who had weak MHC I and low levels of ITTC (log-rank test=12.023, p=0.034). Both MHC I and ITTC are independent predictors of good survival. CONCLUSIONS: ITTC, nSTAT1 and strong MHC class I expression on tumours identify patients with improved survival and an intact tumour immune system that may benefit from immunotherapy. Conversely, loss of these markers identifies patients whose tumours have escaped immunosurveillance and are unlikely to benefit from immunotherapy.

Cytoplasmic p27 expression is an independent prognostic factor in ovarian cancer.

Dysregulation of the cell cycle is an important prerequisite for cancer development. p27 has an established role in cell cycle control and hence may be disrupted during carcinogenesis. The influence of p27 expression, including its subcellular location, on tumor behavior in ovarian cancer has been controversial. The purpose of this study was to evaluate the expression of p27 in a large population of patients with ovarian cancer and correlate this to clinicopathologic variables including overall survival. Using a tissue microarray of 339 primary ovarian cancers, the expression of p27 was assessed immunohistochemically. Coupled to a comprehensive database of clinicopathologic variables, its effect on these factors and survival was studied. Cytoplasmic p27 showed a progressively negative impact on overall survival (P=0.004). Tumors displaying nuclear p27 also had poorer prognosis (P=0.014). Factors shown to predict prognosis independently of each other were age, stage, and the absence of macroscopic disease after surgery. Cytoplasmic p27 expression, but not nuclear, was independently predictive of prognosis on multivariate analysis (P=0.042). Both subcellular locations of p27 expression were more frequently observed in serous compared with mucinous subtypes. Cytoplasmic p27 independently predicts poorer prognosis in ovarian carcinoma. These results seem counterintuitive, when considering the antiproliferative role of p27, but may reflect a more complex function of p27 within cell cycle regulation. These data support a novel role for p27 within the cytoplasm, possibly through effects on apoptosis, cellular motility, and drug resistance.