Time of day bias for biological sampling in studies of mammary cancer.

Despite its demonstrated biological significance, time of day is a broadly overlooked biological variable in preclinical and clinical studies. How time of day affects the influence of peripheral tumors on central (brain) function remains unspecified. Thus, we tested the hypothesis that peripheral mammary cancer tumors alter the transcriptome of immune responses in the brain and that these responses vary based on time of day; we predicted that time of day sampling bias would alter the interpretation of the results. Brain tissues collected at mid dark and mid light from mammary tumor-bearing and vehicle injected mice were analyzed using the Nanostring nCounter immune panel. Peripheral mammary tumors significantly affected expression within the brain of over 100 unique genes of the 770 represented in the panel, and fewer than 25% of these genes were affected similarly across the day. Indeed, between 65 and 75% of GO biological processes represented by the differentially expressed genes were dependent upon time of day of sampling. The implications of time-of-day sampling bias in interpretation of research studies cannot be understated. We encourage considering time of day as a significant biological variable in studies and to appropriately control for it and clearly report time of day in findings.

Chronotherapeutics for Solid Tumors.

Circadian rhythms are internal manifestations of the 24-h solar day that allow for synchronization of biological and behavioral processes to the external solar day. This precise regulation of physiology and behavior improves adaptive function and survival. Chronotherapy takes advantage of circadian rhythms in physiological processes to optimize the timing of drug administration to achieve maximal therapeutic efficacy and minimize negative side effects. Chronotherapy for cancer treatment was first demonstrated to be beneficial more than five decades ago and has favorable effects across diverse cancer types. However, implementation of chronotherapy in clinic remains limited. The present review examines the evidence for chronotherapeutic treatment for solid tumors. Specifically, studies examining chrono-chemotherapy, chrono-radiotherapy, and alternative chronotherapeutics (e.g., hormone therapy, TKIs, antiangiogenic therapy, immunotherapy) are discussed. In addition, we propose areas of needed research and identify challenges in the field that remain to be addressed.

Functions of Steroid Hormones in the Male Reproductive Tract as Revealed by Mouse Models.

Steroid hormones are capable of diffusing through cell membranes to bind with intracellular receptors to regulate numerous physiological processes. Three classes of steroid hormones, namely androgens, estrogens and glucocorticoids, contribute to the development of the reproductive system and the maintenance of fertility. During the past 30 years, mouse models have been produced in which the expression of genes encoding steroid hormone receptors has been enhanced, partially compromised or eliminated. These mouse models have revealed many of the physiological processes regulated by androgens, estrogens and to a more limited extent glucocorticoids in the testis and male accessory organs. In this review, advances provided by mouse models that have facilitated a better understanding of the molecular regulation of testis and reproductive tract processes by steroid hormones are discussed.

Time of day as a critical variable in biology.

BACKGROUND: Circadian rhythms are important for all aspects of biology; virtually every aspect of biological function varies according to time of day. Although this is well known, variation across the day is also often ignored in the design and reporting of research. For this review, we analyzed the top 50 cited papers across 10 major domains of the biological sciences in the calendar year 2015. We repeated this analysis for the year 2019, hypothesizing that the awarding of a Nobel Prize in 2017 for achievements in the field of circadian biology would highlight the importance of circadian rhythms for scientists across many disciplines, and improve time-of-day reporting. RESULTS: Our analyses of these 1000 empirical papers, however, revealed that most failed to include sufficient temporal details when describing experimental methods and that few systematic differences in time-of-day reporting existed between 2015 and 2019. Overall, only 6.1% of reports included time-of-day information about experimental measures and manipulations sufficient to permit replication. CONCLUSIONS: Circadian rhythms are a defining feature of biological systems, and knowing when in the circadian day these systems are evaluated is fundamentally important information. Failing to account for time of day hampers reproducibility across laboratories, complicates interpretation of results, and reduces the value of data based predominantly on nocturnal animals when extrapolating to diurnal humans.

Time-restricted feeding alters the efficiency of mammary tumor growth.

Disruption of circadian rhythms has detrimental host consequences. Indeed, both clinical and foundational science demonstrate a clear relationship between disruption of circadian rhythms and cancer initiation and progression. Because timing of food intake can act as a zeitgeber (i.e., entrainment signal) for the circadian clock, and most individuals in the developed world have access to food at all times of the day in a "24/7" society, we sought to determine the effects of timing of food intake on mammary tumor growth. We hypothesized that restricting access to food to during the inactive phase would accelerate tumor growth. Adult female Balb/C mice received a unilateral orthotopic injection of murine mammary carcinoma 4T1 cells into the ninth inguinal mammary gland. Beginning on the day of tumor injection and continuing until the end of the experiment, mice were food restricted to their active phase (ZT12 (lights off)- ZT0 (lights on), inactive phase (ZT0 - ZT12), or had ad libitum access to food. Mice that were food restricted to their inactive phase displayed a significant increase in body mass on days 7 and 14 of tumor growth relative to active phase or ad libitum fed mice. Additionally, mice fed during their inactive phase demonstrated a 20% reduction in food consumption relative to mice fed during their active phase and a 17% reduction in food consumption relative to ab libitum fed mice. Tumor volume was not significantly different between groups. However, food restricting mice to their inactive phase increased mammary tumor growth efficiency (i.e., mg of tumor mass per gram of food intake) relative to mice fed during the active phase and approached significance (p = .06) relative to ad libitum fed mice. To determine a potential explanation for the increased tumor growth efficiency, we examined rhythms of activity and body temperature. Mice fed during the inactive phase displayed significantly disrupted daily activity and body temperature rhythms relative to both other feeding regimens. Together, these data demonstrate that improperly timed food intake can have detrimental consequences on mammary tumor growth likely via disrupted circadian rhythms.

Nonclassical androgen and estrogen signaling is essential for normal spermatogenesis.

Signaling by androgens through androgen receptor (AR) is essential to complete spermatogenesis in the testis. Similarly, loss of the main estrogen receptor, estrogen receptor 1 (ESR1; also known as ERα), results in male infertility, due in part to indirect deleterious effects on the seminiferous epithelium and spermatogenesis. Effects of steroid hormones are induced primarily through genomic changes induced by hormone-mediated activation of their intracellular receptors and subsequent effects on nuclear gene transcription. However, androgens and estrogens also signal through rapid nonclassical pathways involving actions initiated at the cell membrane. Here we review the data that nonclassical androgen and estrogen signaling pathways support processes essential for male fertility in the testis and reproductive tract. The recent development of transgenic mice lacking nonclassical AR or ESR1 signaling but retaining genomic nuclear signaling has provided a powerful tool to elucidate the function of nonclassical signaling in the overall response to androgens and estrogens. Results from these mice have emphasized that nonclassical signaling is essential for full responses to these hormones, and absence of either nonclassical or classical AR or ESR1 pathways produces abnormalities in spermatogenesis and the male reproductive tract. Although additional work is required to fully understand how classical and nonclassical receptor signaling synergize to produce full steroid hormone responses, here we summarize the known physiological functions of the classical and nonclassical androgen and estrogen signaling pathways in the testis and reproductive tract.

Circadian Influences on Chemotherapy Efficacy in a Mouse Model of Brain Metastases of Breast Cancer.

Chemotherapy is more effective in the treatment of peripheral tumors than brain metastases, likely reflecting the reduced ability of chemotherapy to cross the blood-brain barrier (BBB) and blood-tumor barrier at efficacious concentrations. Recent studies demonstrate circadian regulation of the BBB. Thus, we predicted that optimally timed chemotherapy would increase anti-tumor efficacy in a model of brain metastases of breast cancer (BMBC). First, we characterized novel daily alterations in BBB permeability to a commonly used chemotherapeutic, 14C-paclitaxel, within BMBC following injections given at four time points across the day. Peak and trough 14C-paclitaxel concentrations within BMBC occurred during the mid-dark phase and at the beginning of the light phase, respectively. Notably, chemotherapy injections during the dark phase increased cell death within BMBC and delayed onset of neurological symptoms relative to injections during the light phase. These data provide strong evidence for the beneficial effects of chrono-chemotherapy for the treatment of BMBC.

Artificial Light at Night Reduces Anxiety-like Behavior in Female Mice with Exacerbated Mammary Tumor Growth.

Artificial light at night (ALAN) is a pervasive phenomenon. Although initially assumed to be innocuous, recent research has demonstrated its deleterious effects on physiology and behavior. Exposure to ALAN is associated with disruptions to sleep/wake cycles, development of mood disorders, metabolic disorders, and cancer. However, the influence of ALAN on affective behavior in tumor-bearing mice has not been investigated. We hypothesize that exposure to ALAN accelerates mammary tumor growth and predict that ALAN exacerbates negative affective behaviors in tumor-bearing mice. Adult (>8 weeks) female C3H mice received a unilateral orthotropic injection of FM3A mouse mammary carcinoma cells (1.0 × 105 in 100 µL) into the fourth inguinal mammary gland. Nineteen days after tumor inoculation, mice were tested for sucrose preference (anhedonia-like behavior). The following day, mice were subjected to an open field test (anxiety-like behavior), followed by forced swim testing (depressive-like behavior). Regardless of tumor status, mice housed in ALAN increased body mass through the first ten days. Tumor-bearing ALAN-housed mice demonstrated reduced latency to tumor onset (day 5) and increased terminal tumor volume (day 21). Exposure to ALAN reduced sucrose preference independent of tumor status. Additionally, tumor-bearing mice housed in dark nights demonstrated significantly increased anxiety-like behavior that was normalized via housing in ALAN. Together, these data reaffirm the negative effects of ALAN on tumorigenesis and demonstrate the potential anxiolytic effect of ALAN in the presence of mammary tumors.

Light Pollution and Cancer.

For many individuals in industrialized nations, the widespread adoption of electric lighting has dramatically affected the circadian organization of physiology and behavior. Although initially assumed to be innocuous, exposure to artificial light at night (ALAN) is associated with several disorders, including increased incidence of cancer, metabolic disorders, and mood disorders. Within this review, we present a brief overview of the molecular circadian clock system and the importance of maintaining fidelity to bright days and dark nights. We describe the interrelation between core clock genes and the cell cycle, as well as the contribution of clock genes to oncogenesis. Next, we review the clinical implications of disrupted circadian rhythms on cancer, followed by a section on the foundational science literature on the effects of light at night and cancer. Finally, we provide some strategies for mitigation of disrupted circadian rhythms to improve health.

Social enrichment attenuates chemotherapy induced pro-inflammatory cytokine production and affective behavior via oxytocin signaling.

Breast cancer survivors receiving chemotherapy often report increased anxiety and depression. However, the mechanism underlying chemotherapy-induced changes in affect remains unknown. We hypothesized that chemotherapy increases cytokine production, in turn altering exploratory and depressive-like behavior. To test this hypothesis, female Balb/C mice received two injections, separated by two weeks, of vehicle (0.9% saline) or a chemotherapeutic cocktail [9 mg/kg doxorubicin (A) and 90 mg/kg cyclophosphamide (C)]. Peripheral and central cytokine concentrations were increased one and seven days, respectively, after AC. Because of the beneficial effects of social enrichment on several diseases with inflammatory components, we examined whether social enrichment could attenuate the increase in peripheral and central cytokine production following chemotherapy administration. Socially isolated mice receiving AC therapy demonstrated increased depressive-like and exploratory behaviors with a concurrent increase in hippocampal IL-6. Whereas, group housing attenuated AC-induced IL-6 and depressive-like behavior. Next, we sought to determine whether central oxytocin may contribute to the protective effects of social housing after AC administration. Intracerebroventricular administration of oxytocin to socially isolated mice recapitulated the protective effects of social enrichment; specifically, oxytocin ameliorated the AC-induced effects on IL-6 and depressive-like behavior. Furthermore, administration of an oxytocin antagonist to group housed mice recapitulated the responses of socially isolated mice; specifically, AC increased depressive-like behavior and central IL-6. These data suggest a possible neuroprotective role for oxytocin following chemotherapy, via modulation of IL-6. This study adds to the growing literature detailing the negative behavioral effects of chemotherapy and provides further evidence that social enrichment may be beneficial to health.

Acute exposure to low-level light at night is sufficient to induce neurological changes and depressive-like behavior.

The advent and wide-spread adoption of electric lighting over the past century has profoundly affected the circadian organization of physiology and behavior for many individuals in industrialized nations; electric lighting in homes, work environments, and public areas have extended daytime activities into the evening, thus, increasing night-time exposure to light. Although initially assumed to be innocuous, chronic exposure to light at night (LAN) is now associated with increased incidence of cancer, metabolic disorders, and affective problems in humans. However, little is known about potential acute effects of LAN. To determine whether acute exposure to low-level LAN alters brain function, adult male, and female mice were housed in either light days and dark nights (LD; 14 h of 150 lux:10 h of 0 lux) or light days and low level light at night (LAN; 14 h of 150 lux:10 h of 5 lux). Mice exposed to LAN on three consecutive nights increased depressive-like responses compared to mice housed in dark nights. In addition, female mice exposed to LAN increased central tendency in the open field. LAN was associated with reduced hippocampal vascular endothelial growth factor-A (VEGF-A) in both male and female mice, as well as increased VEGFR1 and interleukin-1ß mRNA expression in females, and reduced brain derived neurotrophic factor mRNA in males. Further, LAN significantly altered circadian rhythms (activity and temperature) and circadian gene expression in female and male mice, respectively. Altogether, this study demonstrates that acute exposure to LAN alters brain physiology and can be detrimental to well-being in otherwise healthy individuals.

Molecular Mechanisms of Cancer-Induced Sleep Disruption.

Sleep is essential for health. Indeed, poor sleep is consistently linked to the development of systemic disease, including depression, metabolic syndrome, and cognitive impairments. Further evidence has accumulated suggesting the role of sleep in cancer initiation and progression (primarily breast cancer). Indeed, patients with cancer and cancer survivors frequently experience poor sleep, manifesting as insomnia, circadian misalignment, hypersomnia, somnolence syndrome, hot flushes, and nightmares. These problems are associated with a reduction in the patients' quality of life and increased mortality. Due to the heterogeneity among cancers, treatment regimens, patient populations and lifestyle factors, the etiology of cancer-induced sleep disruption is largely unknown. Here, we discuss recent advances in understanding the pathways linking cancer and the brain and how this leads to altered sleep patterns. We describe a conceptual framework where tumors disrupt normal homeostatic processes, resulting in aberrant changes in physiology and behavior that are detrimental to health. Finally, we discuss how this knowledge can be leveraged to develop novel therapeutic approaches for cancer-associated sleep disruption, with special emphasis on host-tumor interactions.

Prior exposure to dim light at night impairs dermal wound healing in female C57BL/6 mice.

Artificial light at night (LAN) is a pervasive phenomenon in today's society, and the detrimental consequences of LAN exposure are becoming apparent. LAN is associated with the increased incidence of metabolic disorders, cancers, mood alterations, and immune dysfunction in mammals. Consequently, we examined the effects of dim LAN (DLAN) on wound healing. Female C57BL/6 mice were housed for 3 weeks in DLAN or LD conditions prior to wounding. Following wounding, mice were maintained in either their previous light conditions or switched to the opposite lighting conditions for 3 weeks. DLAN prior to wounding impaired healing; specifically, mice in DLAN/DLAN had significantly larger wounds on day 8. Additionally, mice in DLAN/LD had significantly larger wounds on days 5, 7, 8, and 9, and increased average time to closure. These data demonstrate a potential harmful effect of DLAN on wound healing that should be considered and may represent a target for therapeutic intervention.

Mammary tumor and mastectomy synergistically promote neuroinflammation in a breast cancer survivor model.

Understanding why breast cancer survivors are at an increased risk for cognitive and affective disorders is essential for developing targeted treatment plans and improving quality of life. Microglia priming results in chronic neuroinflammation and can contribute to neuronal degeneration and dysfunction, thereby offering a potential mechanism for altered brain function that persists after tumor removal. This study examined whether mammary tumors alter microglia and augment the inflammatory profile and behavior of mice. To test this, non-metastatic mammary tumor cells (67NR) were injected orthotopically into the mammary glands of BALB/c mice, allowed to grow for 16 days, and then the tumors were removed via mastectomy. Following a 14-day surgical recovery, the mice were challenged with lipopolysaccharide (LPS), and then central and peripheral inflammation, anxiety, and depressive-like behavior were evaluated. Here we show that major central and peripheral inflammatory markers were not altered by tumor growth nor mastectomy surgery alone. However, hippocampal mRNA expression of major proinflammatory cytokines IL-1ß and TNFα was increased in tumor removal animals, persisting past surgical recovery. Nonetheless, the immune and behavioral responses following LPS administration were comparable among groups. In sum, these data demonstrate that the combination of tumor and mastectomy promotes neuroinflammation; however, immune challenge did not elucidate this inflammation as maladaptive for the host.

The testicular transcriptome associated with spermatogonia differentiation initiated by gonadotrophin stimulation in the juvenile rhesus monkey (Macaca mulatta).

STUDY QUESTION: What is the genetic landscape within the testis of the juvenile rhesus monkey (Macaca mulatta) that underlies the decision of undifferentiated spermatogonia to commit to a pathway of differentiation when puberty is induced prematurely by exogenous LH and FSH stimulation? SUMMARY ANSWER: Forty-eight hours of gonadotrophin stimulation of the juvenile monkey testis resulted in the appearance of differentiating B spermatogonia and the emergence of 1362 up-regulated and 225 down-regulated testicular mRNAs encoding a complex network of proteins ranging from enzymes regulating Leydig cell steroidogenesis to membrane receptors, and from juxtacrine and paracrine factors to transcriptional factors governing spermatogonial stem cell fate. WHAT IS KNOWN ALREADY: Our understanding of the cell and molecular biology underlying the fate of undifferentiated spermatogonia is based largely on studies of rodents, particularly of mice, but in the case of primates very little is known. The present study represents the first attempt to comprehensively address this question in a highly evolved primate. STUDY DESIGN, SIZE, DURATION: Global gene expression in the testis from juvenile rhesus monkeys that had been stimulated with recombinant monkey LH and FSH for 48 h (N = 3) or 96 h (N = 4) was compared to that from vehicle treated animals (N = 3). Testicular cell types and testosterone secretion were also monitored. PARTICIPANTS/MATERIALS, SETTING, METHODS: Precocious testicular puberty was initiated in juvenile rhesus monkeys, 14-24 months of age, using a physiologic mode of intermittent stimulation with i.v. recombinant monkey LH and FSH that within 48 h produced 'adult' levels of circulating LH, FSH and testosterone. Mitotic activity was monitored by immunohistochemical assays of 5-bromo-2'-deoxyuridine and 5-ethynyl-2'-deoxyuridine incorporation. Animals were bilaterally castrated and RNA was extracted from the right testis. Global gene expression was determined using RNA-Seq. Differentially expressed genes (DEGs) were identified and evaluated by pathway analysis. mRNAs of particular interest were also quantitated using quantitative RT-PCR. Fractions of the left testis were used for histochemistry or immunoflouresence. MAIN RESULTS AND THE ROLE OF CHANCE: Differentiating type B spematogonia were observed after both 48 and 96 h of gonadotrophin stimulation. Pathway analysis identified five super categories of over-represented DEGs. Repression of GFRA1 (glial cell line-derived neurotrophic factor family receptor alpha 1) and NANOS2 (nanos C2HC-type zinc finger 2) that favor spermatogonial stem cell renewal was noted after 48 and 96 h of LH and FSH stimulation. Additionally, changes in expression of numerous genes involved in regulating the Notch pathway, cell adhesion, structural plasticity and modulating the immune system were observed. Induction of genes associated with the differentiation of spermatogonia stem cells (SOHLH1(spermatogenesis- and oogenesis-specific basic helix-loop-helix 1), SOHLH2 and KIT (V-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog)) was not observed. Expression of the gene encoding STRA8 (stimulated by retinoic acid 8), a protein generally considered to mark activation of retinoic acid signaling, was below our limit of detection. LARGE SCALE DATA: The entire mRNA data set for vehicle and gonadotrophin treated animals (N = 10) has been deposited in the GEO-NCBI repository (GSE97786). LIMITATIONS REASONS FOR CAUTION: The limited number of monkeys per group and the dilution of low abundance germ cell transcripts by mRNAs contributed from somatic cells likely resulted in an underestimation of the number of differentially expressed germ cell genes. WIDER IMPLICATIONS OF THE FINDINGS: The findings that expression of GDNF (a major promoter of spermatogonial stem cell renewal) was not detected in the control juvenile testes, expression of SOHLH1, SOHLH2 and KIT, promoters of spermatogonial differentiation in mice, were not up-regulated in association with the gonadotrophin-induced generation of differentiating spermatogonia, and that robust activation of the retinoic acid signaling pathway was not observed, could not have been predicted. These unexpected results underline the importance of non-human primate models in translating data derived from animal research to the human situation. STUDY FUNDING/COMPETING INTEREST(S): The work described was funded by NIH grant R01 HD072189 to T.M.P. P.A. was supported by an Endocrine Society Summer Research Fellowship Award and CONICET (Argentine Research Council), S.N. by a grant from Vali-e-Asr Reproductive Health Research Center of Tehran University of Medical Sciences (grant #24335-39-92) to Dr Batool Hosseini Rashidi, and M.P.H. by grants from the National Health and Medical Research Council of Australia, and the Victorian State Government's Operational Infrastructure Support Program. The authors have nothing to disclose.

Mouse Spermatogenesis Requires Classical and Nonclassical Testosterone Signaling.

Testosterone acts though the androgen receptor in Sertoli cells to support germ cell development (spermatogenesis) and male fertility, but the molecular and cellular mechanisms by which testosterone acts are not well understood. Previously, we found that in addition to acting through androgen receptor to directly regulate gene expression (classical testosterone signaling pathway), testosterone acts through a nonclassical pathway via the androgen receptor to rapidly activate kinases that are known to regulate spermatogenesis. In this study, we provide the first evidence that nonclassical testosterone signaling occurs in vivo as the MAP kinase cascade is rapidly activated in Sertoli cells within the testis by increasing testosterone levels in the rat. We find that either classical or nonclassical signaling regulates testosterone-mediated Rhox5 gene expression in Sertoli cells within testis explants. The selective activation of classical or nonclassical signaling pathways in Sertoli cells within testis explants also resulted in the differential activation of the Zbtb16 and c-Kit genes in adjacent spermatogonia germ cells. Delivery of an inhibitor of either pathway to Sertoli cells of mouse testes disrupted the blood-testis barrier that is essential for spermatogenesis. Furthermore, an inhibitor of nonclassical testosterone signaling blocked meiosis in pubertal mice and caused the loss of meiotic and postmeiotic germ cells in adult mouse testes. An inhibitor of the classical pathway caused the premature release of immature germ cells. Collectively, these observations indicate that classical and nonclassical testosterone signaling regulate overlapping and distinct functions that are required for the maintenance of spermatogenesis and male fertility.

Risk of ESRD and all cause mortality in type 2 diabetes according to circulating levels of FGF-23 and TNFR1.

INTRODUCTION: Recent studies demonstrated that circulating fibroblast growth factor (FGF)-23 was associated with risk of end stage renal disease (ESRD) and mortality. This study aims to examine whether the predictive effect of FGF-23 is independent from circulating levels of tumor necrosis factor receptor 1 (TNFR1), a strong predictor of ESRD in Type 2 diabetes (T2D). METHODS: We studied 380 patients with T2D who were followed for 8-12 years and were used previously to examine the effect of TNFR1. Baseline plasma FGF-23 was measured by immunoassay. RESULTS: During follow-up, 48 patients (13%) developed ESRD and 83 patients (22%) died without ESRD. In a univariate analysis, baseline circulating levels of FGF-23 and TNFR1 were significantly higher in subjects who subsequently developed ESRD or died without ESRD than in those who remained alive. In a Cox proportional hazard model, baseline concentration of FGF-23 was associated with increased risk of ESRD, however its effect was no longer significant after controlling for TNFR1 and other clinical characteristics (HR 1.3, p = 0.15). The strong effect of circulating level of TNFR1 on risk of ESRD was not changed by including circulating levels of FGF-23 (HR 8.7, p<0.001). In the Cox multivariate model, circulating levels of FGF-23 remained a significant independent predictor of all-cause mortality unrelated to ESRD (HR 1.5, p<0.001). CONCLUSIONS: We demonstrated that the effect of circulating levels of FGF-23 on the risk of ESRD is accounted for by circulating levels of TNFR1. We confirmed that circulating levels of FGF-23 have an independent effect on all-cause mortality in T2D.

Circulating TNF receptors 1 and 2 predict ESRD in type 2 diabetes.

Levels of proinflammatory cytokines associate with risk for developing type 2 diabetes but whether chronic inflammation contributes to the development of diabetic complications, such as ESRD, is unknown. In the 1990s, we recruited 410 patients with type 2 diabetes for studies of diabetic nephropathy and recorded their characteristics at enrollment. During 12 years of follow-up, 59 patients developed ESRD (17 per 1000 patient-years) and 84 patients died without ESRD (24 per 1000 patient-years). Plasma markers of systemic inflammation, endothelial dysfunction, and the TNF pathway were measured in the study entry samples. Of the examined markers, only TNF receptors 1 and 2 (TNFR1 and TNFR2) associated with risk for ESRD. These two markers were highly correlated, but ESRD associated more strongly with TNFR1. The cumulative incidence of ESRD for patients in the highest TNFR1 quartile was 54% after 12 years but only 3% for the other quartiles (P<0.001). In Cox proportional hazard analyses, TNFR1 predicted risk for ESRD even after adjustment for clinical covariates such as urinary albumin excretion. Plasma concentration of TNFR1 outperformed all tested clinical variables with regard to predicting ESRD. Concentrations of TNFRs moderately associated with death unrelated to ESRD. In conclusion, elevated concentrations of circulating TNFRs in patients with type 2 diabetes at baseline are very strong predictors of the subsequent progression to ESRD in subjects with and without proteinuria.

Circulating TNF receptors 1 and 2 predict stage 3 CKD in type 1 diabetes.

Elevated plasma concentrations of TNF receptors 1 and 2 (TNFR1 and TNFR2) predict development of ESRD in patients with type 2 diabetes without proteinuria, suggesting these markers may contribute to the pathogenesis of renal decline. We investigated whether circulating markers of the TNF pathway determine GFR loss among patients with type 1 diabetes. We followed two cohorts comprising 628 patients with type 1 diabetes, normal renal function, and no proteinuria. Over 12 years, 69 patients developed estimated GFR less than 60 mL/min per 1.73 m(2) (16 per 1000 person-years). Concentrations of TNFR1 and TNFR2 were strongly associated with risk for early renal decline. Renal decline was associated only modestly with total TNFα concentration and appeared unrelated to free TNFα. The cumulative incidence of estimated GFR less than 60 mL/min per 1.73 m(2) for patients in the highest TNFR2 quartile was 60% after 12 years compared with 5%-19% in the remaining quartiles. In Cox proportional hazards analysis, patients with TNFR2 values in the highest quartile were threefold more likely to experience renal decline than patients in the other quartiles (hazard ratio, 3.0; 95% confidence interval, 1.7-5.5). The risk associated with high TNFR1 values was slightly less than that associated with high TNFR2 values. TNFR levels were unrelated to baseline free TNFα level and remained stable over long periods within an individual. In conclusion, early GFR loss in patients with type 1 diabetes without proteinuria is strongly associated with circulating TNF receptor levels but not TNFα levels (free or total).

Chemokine receptor 7 (CCR7) gene expression is regulated by NF-κB and activator protein 1 (AP1) in metastatic squamous cell carcinoma of head and neck (SCCHN).

The chemokine receptor CCR7 is a seven-transmembrane domain G-protein-coupled receptor that facilitates leukocyte migration to regional lymph nodes. Aberrant CCR7 expression in a number of human malignancies has been linked to pro-survival, -invasive, and -metastatic pathways. We demonstrate here that up-regulation of CCR7 in squamous cell carcinoma of the head and neck (SCCHN) patient tumors correlates with lower survival because of metastatic disease. Because of this important oncogenic phenotype, we investigated the mechanisms that regulate CCR7 expression in these tumors. Interestingly, the inflammatory transcription factor NF-κB has been associated with a more aggressive SCCHN phenotype. Immunohistochemical staining of a SCCHN tumor cohort (n = 47) strongly linked NF-κB staining and CCR7 expression in SCCHN. Thus, we investigated whether NF-κB contributes to metastatic disease by promoting CCR7 expression in SCCHN tumor cells. We characterized four novel, potential NF-κB binding sites in the 1000-bp promoter region upstream of the CCR7 gene, using luciferase, ChIP, and EMSA. However, NF-κB inhibition only resulted in partial reduction in CCR7 expression, prompting consideration of other co-regulators of CCR7. Indeed, cooperation between NF-κB and AP1 transcription factors, which are often co-activated, is crucial to the regulation of CCR7 mRNA expression in metastatic SCCHN cells. Thus, our findings support an important biological role for inflammatory NF-κB and AP1 in the regulation of CCR7 expression in metastatic SCCHN. As such, CCR7, NF-κB, and AP1 could be potentially useful therapeutic targets in controlling the progression and metastasis of SCCHN tumors.