In vitro characterization of anti-inflammatory activities of 3RS, 7R, 11R-phytanic acid.

The aim of the research described here was to investigate the in vitro immunomodulatory effects of 3RS, 7R, 11R-phytanic acid (3RS-PHY) from the perspective of efficacy against autoimmune diseases. 3RS-PHY is a milk component with strong agonist activity at the peroxisome proliferator activated receptor (PPAR). As PPAR is a therapeutic target for several human diseases, 3RS-PHY intake may have possible health benefits. Recently, we chemically synthesized a preparation of 3RS-PHY and demonstrated that 3RS-PHY inhibited T-cell production of interferon (IFN)-γ. However, the overall immunomodulatory effects were not evaluated. In this study, mouse splenocytes, purified T-cells and B-cells were stimulated by mitogens and incubated with 3RS-PHY, followed by evaluation of cytokine and antibody production. A macrophage-like cell line J774.1 was also incubated with 3RS-PHY to evaluate nitric oxide production. 3RS-PHY decreased mRNA levels not only of IFN-γ but also of interleukin (IL)-2, IL-10 and IL-17A in splenocytes and similar effects were confirmed at the protein level. In addition, 3RS-PHY had a direct action on T-cells with preferential inhibitory effects on Th1 and Th17 cytokines such as IFN-γ and IL-17A. Furthermore, 3RS-PHY suppressed antibody secretion by B-cells and nitric oxide production by J774.1 almost completely, indicating that 3RS-PHY is a bioactive fatty acid with anti-inflammatory properties. These findings encourage further investigations, including in vivo experiments, to evaluate whether 3RS-PHY actually shows the potential to prevent autoimmune diseases, and provide basic information to produce milk and dairy products with an increased 3RS-PHY concentration.

Dietary phytanic acid-induced changes in tissue fatty acid profiles in mice.

The aims of this research communication were to investigate the in vivo tissue accumulation of phytanic acid (PA) and any changes in the tissue fatty acid profiles in mice. Previous in vitro studies have demonstrated that PA is a milk component with the potential to cause both beneficial effects on lipid and glucose metabolism and detrimental effects on neuronal cells. However, there is limited information about its in vivo actions. In this study, mice were fed diets containing either 0.00 or 0.05% 3RS, 7R, 11R-PA, which is the isomer found in milk and the human body. After 4 weeks, adipose tissue, liver and brain were harvested and their fatty acid profiles were determined by gas chromatographic analysis. The results showed that PA and its metabolite pristanic acid accumulated in the adipose tissue of PA-fed mice, and that dietary PA decreased the hepatic compositions of several saturated fatty acids such as palmitic acid while increasing the compositions of polyunsaturated fatty acids including linoleic acid and docosahexaenoic acid. However, dietary PA neither accumulated nor had a high impact on the fatty acid profile in the brain. These results suggested that dietary PA could exert its biological activities in adipose tissue and liver, although the brain is relatively less affected by dietary PA. These data provide a basis for understanding the in vivo physiological actions of PA.

Changes in expression of the autophagy-related genes microtubule-associated protein 1 light chain 3ß and autophagy related 7 in skeletal muscle of fattening Japanese Black cattle: a pilot study.

OBJECTIVE: Autophagy is a bulk degradation system for intracellular proteins which contributes to skeletal muscle homeostasis, according to previous studies in humans and rodents. However, there is a lack of information on the physiological role of autophagy in the skeletal muscle of meat animals. This study was planned as a pilot study to investigate changes in expression of two major autophagy-related genes, microtubule-associated protein 1 light chain 3ß (MAP1LC3B) and autophagy related 7 (ATG7) in fattening beef cattle, and to compare them with skeletal muscle growth. METHODS: Six castrated Japanese Black cattle (initial body weight: 503±20 kg) were enrolled in this study and fattened for 7 months. Three skeletal muscles, M. longissimus, M. gluteus medius, and M. semimembranosus, were collected by needle biopsy three times during the observation period, and mRNA levels of MAP1LC3B and ATG7 were determined by quantitative reverse-transcription polymerase chain reaction. The expression levels of genes associated with the ubiquitin-proteasome system, another proteolytic mechanism, were also analyzed for comparison with autophagy-related genes. In addition, ultrasonic scanning was repeatedly performed to measure M. longissimus area as an index of muscle growth. RESULTS: Our results showed that both MAP1LC3B and ATG7 expression increased over the observation period in all three skeletal muscles. Interestingly, the increase in expression of these two genes in M. longissimus was highly correlated with ultrasonic M. longissimus area and body weight. On the other hand, the expression of genes associated with the ubiquitin-proteasome system was unchanged during the same period. CONCLUSION: These findings suggest that autophagy plays an important role in the growth of skeletal muscle of fattening beef cattle and imply that autophagic activity affects meat productivity.

Naturally occurring 3RS, 7R, 11R-phytanic acid suppresses in vitro T-cell production of interferon-gamma.

BACKGROUND: Among the eight stereoisomers of phytanic acid (PA), the 3RS, 7R, 11R-isomer is naturally occurring and is present in foods and the human body. PA is considered to have possible health benefits in the immune system. However, it remains undetermined whether these effects are elicited by the 3RS, 7R, 11R-PA isomer, because previous studies used a commercially available PA whose isomer configuration is unknown. In this study, we synthesized a preparation of 3RS, 7R, 11R-PA, and investigated its in vitro immunomodulatory effects, especially the T-cell production of interferon (IFN)-γ, which is associated with various autoimmune diseases. This study also investigated the effects of 3RS, 7R, 11R-PA on NF-κB activity in order to address the mechanism of its immunomodulatory effects. METHODS: Mouse splenocytes and purified T-cells were stimulated with T-cell mitogens and incubated with 3RS, 7R, 11R-PA, followed by evaluation of IFN-γ production. The effect of 3RS, 7R, 11R-PA on NF-κB activity was also investigated using an A549 cell line with stable expression of an NF-κB-dependent luciferase reporter gene. RESULTS: 3RS, 7R, 11R-PA significantly reduced in vitro IFN-γ production at both the protein and mRNA levels, and was accompanied by decreased expression of T-bet, a key regulator of Th1 cell differentiation. The results indicated that NF-κB-mediated transcriptional activity was significantly decreased by 3RS, 7R, 11R-PA and that GW6471, an antagonist of peroxisome proliferator activated receptor α (PPARα), abrogated the inhibitory effect of 3RS, 7R, 11R-PA on NF-κB activity. CONCLUSIONS: The present study suggests that 3RS, 7R, 11R-PA is a functional and bioactive fatty acid, and has a potentially beneficial effect for amelioration of T-cell mediated autoimmune diseases. This study also indicates that interference in the NF-κB pathway via PPARα activation is a potential mechanism of the immunomodulatory effects of 3RS, 7R, 11R-PA.

Deletion of DOCK2, a regulator of the actin cytoskeleton in lymphocytes, suppresses cardiac allograft rejection.

Allograft rejection is induced by graft tissue infiltration of alloreactive T cells that are activated mainly in secondary lymphoid organs of the host. DOCK2 plays a critical role in lymphocyte homing and immunological synapse formation by regulating the actin cytoskeleton, yet its role in the in vivo immune response remains unknown. We show here that DOCK2 deficiency enables long-term survival of cardiac allografts across a complete mismatch of the major histocompatibility complex molecules. In DOCK2-deficient mice, alloreactivity and allocytotoxicity were suppressed significantly even after in vivo priming with alloantigens, which resulted in reduced intragraft expression of effector molecules, such as interferon-gamma, granzyme B, and perforin. This is mediated, at least in part, by preventing potentially alloreactive T cells from recruiting into secondary lymphoid organs. In addition, we found that DOCK2 is critical for CD28-mediated Rac activation and is required for the full activation of alloreactive T cells. Although DOCK2-deficient, alloreactive T cells were activated in vitro in the presence of exogenous interleukin-2, these T cells, when transferred adoptively, failed to infiltrate into the allografts that were transplanted into RAG1-deficient mice. Thus, DOCK2 deficiency attenuates allograft rejection by simultaneously suppressing multiple and key processes. We propose that DOCK2 could be a novel molecular target for controlling transplant rejection.

Effects of dietary phytol on tissue accumulation of phytanic acid and pristanic acid and on the tissue lipid profiles in mice.

Recent in vitro evidence suggests that the phytol-derived fatty acids, phytanic acid (PA) and pristanic acid (PrA), are components of animal products with the potential to cause both beneficial and harmful effects on human health. In this study, we investigated the in vivo tissue accumulation of PA and PrA and the changes in tissue lipid profiles, using mice fed a phytol-containing diet. After 4 weeks of treatment with a diet containing 1.0% phytol, plasma, adipose tissue, liver, and brain were collected and their lipid profiles were biochemically and gas-chromatographically determined. Dietary phytol caused PA and PrA accumulation in the adipose tissue and liver but not in the brain, and reduced plasma and liver triacylglycerol levels. Phytol intake also decreased the fatty acid concentrations in the adipose tissue, especially polyunsaturated fatty acids such as linoleic acid, but increased the concentrations of these fatty acids in the liver. However, dietary phytol had a low impact on the brain lipid profile. This study suggests that dietary phytol intake caused accumulation of PA and PrA and modified lipid profiles in the adipose tissue and liver, but that the brain is an insusceptible tissue to dietary phytol-induced changes.

Improving obstetric hemorrhage morbidity by a checklist-based management protocol; a quality improvement initiative.

OBJECTIVE: Postpartum hemorrhage is a leading cause of maternal morbidity and mortality worldwide. Institutions are encouraged to have a standardized approach to the management of obstetric hemorrhage. The purpose of this quality improvement project was to investigate postpartum hemorrhage associated morbidity before and after implementing an obstetric hemorrhage checklist-based protocol. STUDY DESIGN: In 2015, a resident-driven initiative for obstetric hemorrhage was initiated at a single institution using a checklist-based protocol for postpartum hemorrhage. The project included development of the obstetric hemorrhage checklist by a multidisciplinary team and implementation using low cost education and training strategies. Following implementation, a pre-and post-protocol retrospective analysis was performed measuring maternal morbidity surrogates and protocol compliance. During the 18 month study period, 422 women were identified for review and 147 met criteria in the pre-protocol group and 150 met criteria in the post-protocol group. RESULTS: There was a significant decrease in severe postpartum hemorrhage rates in the post-protocol group (p = 0.04) and all other surrogates for maternal morbidity decreased in the post-protocol group. Protocol compliance was 62.2% and compliance with screening using an assessment of hemorrhage risk was 75.7%. CONCLUSION: The implementation of a checklist-based management protocol for postpartum hemorrhage has shown a promising trend in improving maternal morbidity, screening, early diagnosis, and healthcare delivery for obstetric hemorrhage at our institution and has been approved for larger scale implementation within our health system.

A blocking anti-CD28-specific antibody induces long-term heart allograft survival by suppression of the PKC theta-JNK signal pathway.

This study investigated the effects of a blocking anti-CD28 antibody (Anti-CD28-PV1-IgG3) in vitro and in vivo. Anti-CD28-PV1-IgG3, a hamster-mouse chimeric antibody against murine CD28, which does not provide CD28-positive signaling during TCR-driven T cell activation, enabled long-term survival of heart allografts across a complete mismatch of the MHC in rats. Among the T cell signaling proteins tested in the spleens from recipients, we found that recipients treated with anti-CD28-PV1-IgG3 exhibited suppression of alloantigen-initiated proximal TCR signaling events, including Lck, Zap70, Vav, and PI3K expression, and their PKC theta- and JNK-regulated expression/activation. This leads to attenuation of intragraft T cell infiltration and expression of T cell effector molecules. These results indicate that targeting the CD28 receptor with a blocking antibody leads to long-term allograft survival by reducing activation of alloantigen-mediated key signaling events in T cells that might be crucial for full T cell activation.

Orally administered conjugated linoleic acid ameliorates allergic dermatitis induced by repeated applications of oxazolone in mice.

Conjugated linoleic acid (CLA) is one of the constituents of animal products with possible health benefits such as anti-carcinogenic and anti-obesity effects. In this study, we investigated the immunomodulatory effects of CLA using a mouse model of allergic dermatitis. Mice were orally administered either a CLA mixture containing equal amounts of 9c, 11 t-CLA and 10 t, 12c-CLA, or high linoleic acid safflower oil, and allergic dermatitis was induced on the ear by repeated topical applications of oxazolone. Oral administration of the CLA mixture but not the high linoleic safflower oil attenuated the symptoms of allergic dermatitis in both ear weights and clinical scores. This effect was associated with decreased levels of ear interleukin-4 (IL-4) and plasma immunoglobulin E. The immunomodulatory effects of the CLA isomers were compared by an in vitro cytokine production assay. The results showed that 9c, 11 t-CLA, the most predominant isomer in animal products, significantly inhibited IL-4 and interferon-γ production from mouse splenocytes with similar potency to 10 t, 12c-CLA. These findings suggest that CLA, a constituent of animal products, has a potentially beneficial effect for amelioration of allergic dermatitis.

Absence of Activation-induced Cytidine Deaminase, a Regulator of Class Switch Recombination and Hypermutation in B Cells, Suppresses Aorta Allograft Vasculopathy in Mice.

BACKGROUND: Antibody-mediated rejection is caused in part by increasing circulation/production of donor-specific antibody (DSA). Activation-induced cytidine deaminase (AID) is a key regulator of class switch recombination and somatic hypermutation of immunoglobulin in B cells, yet its role in antibody-mediated transplant rejection remains unclear. We show here that AID deficiency in mice enables suppression of allograft vasculopathy (AV) after aorta transplantation, a DSA-mediated process. METHODS: Splenocytes from C57BL/6 J (B6) AID(−/−) mice were used for determining in vitro proliferation responses, alloreactivity, cell surface marker expression, and antibody production. BALB/c mouse aortas were transplanted into B6 AID(−/−) mice with or without FK506 treatment. Blood and aorta grafts were harvested on day 30 after transplantation and were subjected to DSA, histological, and immunohistological analyses. RESULTS: The AID(−/−) splenocytes were comparable to wild type splenocytes in proliferation responses, alloreactivity, and expression of cell surface markers in vitro. However, they completely failed to produce immunoglobulin G, although they were not impaired in immunoglobulin M production relative to controls. Furthermore, BALB/c aorta grafts from B6 AID(−/−) recipient mice on day 30 after transplantation showed reduced signs of AV compared to the grafts from B6 wild type recipient mice which had severe vascular intimal hyperplasia, interstitial fibrosis, and inflammation. Treatment with FK506 produced a synergistic effect in the grafts from AID(−/−) recipients with further reduction of intimal hyperplasia and fibrosis scores. CONCLUSIONS: The AID deficiency inhibits DSA-mediated AV after aorta transplantation in mice. We propose that AID could be a novel molecular target for controlling antibody-mediated rejection in organ transplantation.

Microarray-based gene expression profiles of allograft rejection and immunosuppression in the rat heart transplantation model.

BACKGROUND: Gene expression profiling has the potential to produce new insights into complex biologic systems. To test the value of complement DNA arrays in identifying pathways involved in organ transplant rejection, we examined the gene expression profiles of rat heart allografts from recipients treated with or without immunosuppression to prevent acute allograft rejection. METHODS: Heterotopic heart transplantation was performed using ACI or Lewis donors and Lewis recipients. Recipients were treated with tacrolimus (Tac) or cyclosporine (CsA) at the equivalent effective doses, and graft hearts were harvested on days 3, 5, and 7. A commercial microarray was used to measure gene expression levels of 588 genes in day 5 grafts. Selected genes were analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: The expression levels of 118 genes were perturbed in the untreated allograft in comparison with the isograft control, of which 77 genes were categorized as candidate genes for Tac- or CsA-mediated immunosuppression or both, and 41 as genes associated with other pathways. Among the 77 candidate genes, 55 genes shared the same response to suppression by both drugs, including inducible nitric oxide synthase, interferon-gamma, and interferon regulatory factor 1. Drug-specific effects were observed in 22 genes: Fourteen genes were exclusively reversed by Tac and eight by CsA. CONCLUSIONS: Gene expression profiling reveals a large variety of genes affected during acute rejection, indicating that multiple metabolic pathways, including immune and nonimmune responses, are involved in the local graft rejection events. The differences and similarities of the gene expression profiles relative to the two immunosuppressants may provide more detailed therapeutic approaches for optimal immunosuppression.

Tacrolimus and cyclosporine differ in their capacity to overcome ongoing allograft rejection as a result of their differential abilities to inhibit interleukin-10 production.

BACKGROUND: Accumulated evidence from clinical transplantation has suggested that tacrolimus-based treatment can reverse ongoing allograft rejection in patients treated with cyclosporine (CsA)-based immunosuppression, even when a high dose of antirejection rescue therapy has failed. This evidence prompted us to investigate whether these two compounds, which share an in vitro mechanism, would differ in their abilities to regulate in situ cellular and molecular events during ongoing allograft rejection. METHODS: The equivalent effective doses of tacrolimus (3.2 mg/kg/day) and CsA (10 mg/kg/day), when administered orally to Lewis rats for 10 days (day 0-9), were predetermined and defined as the ability of the drug to induce a similar survival of Brown Norway rat heart allografts with an equal suppression of intragraft interleukin (IL)-2 mRNA expression. To investigate the ability of each drug to rescue ongoing allograft rejection, Lewis recipients of Brown Norway rat heart grafts were left untreated for the first 5 days after transplantation. Tacrolimus or CsA was then administered at the equivalent effective dose for 10 days (days 5-14). Heart grafts and blood samples, harvested on days 3, 5, 7, and 10, were analyzed by reverse transcriptase-polymerase chain reaction, real-time quantitative polymerase chain reaction, ELISA, and immunohistology. RESULTS: Ongoing allograft rejection was found to be rescued by tacrolimus but not by CsA at the equivalent dose (median survival time: untreated, 6 days; tacrolimus, 18 days; and CsA, 7 days). A significant suppression of local intragraft IL-10 mRNA expression and serum protein production along with a dramatic down-regulation of functional CD8+ T and NKR-P1a+ natural killer cell local infiltration by means of decreased of cytotoxic factor release, including granzyme B and perforin 1, was found to be associated with tacrolimus but not CsA treatment. However, both drugs inhibited other immune cells (CD4+ T cell, ED2+ macrophage) and cytokines (IL-1beta, IL-2, IL-4, IL-6, IL-12, interferon-gamma, transforming growth factor-beta, and tumor necrosis factor-alpha) at almost the same levels. The inability of CsA to overcome ongoing allograft rejection could be rescued by cotreating recipients with neutralizing anti-IL-10 antibody on day 5 and day 6 after transplantation: anti-IL-10 antibody alone did not show such an effect. CONCLUSIONS: Inhibition of IL-10 production is a critical factor in the ability of tacrolimus to reverse ongoing allograft rejection.

FK778, a powerful new immunosuppressant, effectively reduces functional and histologic changes of chronic rejection in rat renal allografts.

BACKGROUND: FK778 is a new derivative of the active leflunomide metabolite A77 1726. It effectively prevented acute allograft rejection in several experimental transplant models, and it is currently in phase II trials in human transplant recipients. In this study, we examined the effects of FK778 in a well-established model of chronic renal allograft rejection in the rat. METHODS: Kidneys of Lewis (LEW) and F344 rats were orthotopically transplanted into bilaterally nephrectomized LEW recipients as the isograft and allograft control, respectively. Allograft recipients were orally administered FK778 at doses of 3 mg/kg per day, 10 mg/kg per day, and 20 mg/kg per day for 10 days. Blood and 24-hr urine samples were collected once a week after grafting for plasma creatinine, allo-specific antibodies, and proteinuria determination. Kidney grafts were harvested on the 90th day after transplantation and subjected to histologic, immunohistologic, and reverse transcriptase-polymerase chain reaction analysis. Histologic sections were semiquantitatively scored using criteria adapted from the Banff' classification for transplant pathologic conditions. RESULTS: Recipients treated with FK778 for 10 days exhibited a dose-dependent decrease in proteinuria and plasma creatinine for the entire 90-day period after transplantation when compared with the allograft control. FK778, at doses of 10 mg/kg per day and 20 mg/kg per day, remarkably reduced chronic histologic changes, including tubular atrophy, glomerulosclerosis, fibrointimal hyperplasia, and transplant glomerulopathy. In addition, FK778 treatment was associated with decreased intragraft mononuclear cell infiltration, serum allo-specific immunoglobulin (Ig)M and IgG antibody production, and intragraft transforming growth factor beta messenger RNA expression in those recipients surviving 90 days after transplantation when compared with the allograft control. CONCLUSION: FK778 effectively reduces functional and histologic chronic kidney allograft rejection in the rat.

Unique gene expression profiles of heart allograft rejection in the interferon regulatory factor-1-deficient mouse.

Interferon regulatory factor-1 (IRF1) is a transcription factor for many genes involved in innate and adaptive immune responses. By using DNA array technology, we have previously demonstrated that IRF1 is significantly upregulated during acute rejection in rat heart allografts and is restored to isograft levels when recipients are treated with the immunosuppressants tacrolimus or cyclosporin A (CsA). To understand the precise role of IRF1 in transplant rejection, we investigated the rejection responses of mice completely deficient of IRF1 protein. Heterotopic heart transplantations were performed using C57BL/6J wild-type (WT B6) and IRF1-deficient (IRF1-/-) mice as recipients, and C3H mice as donors. Graft survival was determined by abdominal palpation and rejection was confirmed by histology. On day 6 after transplantation, isografts and allografts were harvested and subjected to gene expression analysis by a commercial nylon array and by real-time RT-PCR. Median survival time of heart allografts was 8 days in the WT B6 mice and 10 days in the IRF1-/- mice. The gene expression profiles of allografts from the WT B6 and IRF1-/- recipients were nearly identical to each other and very different from the profile of the isograft control. Both WT B6 and IRF1-/- profiles showed 13 genes upregulated (IFN-gamma, MCP-2, MIP-1alpha, MIP-1beta, CCR5, MIG, IP-10 and others) and one gene downregulated (SDF2) among the 76 genes detectable on the array. In more detailed analyses, distinct cytokine and chemokine gene expression profiles were identified in the allografts from the WT B6 and IRF1-/- recipients. Whereas IL-4, IL-6, IL-13, MCP-1, MCP-3, and MPIF-2 were upregulated, RANTES, IL-2Rgamma and gp130 were downregulated in allografts from the IRF1-/- recipients when compared to the WT B6 control. Although the inactivation of the IRF1 gene did not sufficiently prevent acute allograft rejection in this model, a unique cytokine and chemokine gene expression profile was found in the absence of IRF1.

Microarray gene expression profiling of chronic allograft nephropathy in the rat kidney transplant model.

Whole genome gene expression profiles were correlated with renal function and histology in a well-established animal model of chronic allograft nephropathy (CAN). Kidneys of F344 rats were transplanted into LEW recipients treated with a brief dose of FK506 (BFK). Blood and urine samples were collected weekly. Kidney grafts were harvested at an early (day 6) or late (days 30-90) phase after transplantation. BFK kidney grafts showed remarkable changes in function, histology, and gene expression profiles when compared to the isograft controls. In the early phase, renal function and histology were barely affected, yet the expression levels of 225 genes were significantly changed, reflecting both immune and non-immune pathways. In the late phase, however, 826 genes were affected in the BFK kidney grafts, including genes in the pathways of extracellular matrix and cell adhesion. Of these genes, 214 appear to be key factors for development of CAN, since they were affected at both early and late phases, including genes involved in the immune response, the inflammatory response, apoptosis, and metabolism. Kinetic studies with gene expression profiling can identify genes involved in the progressive development of chronic allograft rejection, leading to more detailed therapeutic approaches or useful biomarkers in clinical transplantation.

Common variations in noncoding regions of the human natriuretic peptide receptor A gene have quantitative effects.

Genetic susceptibility to common conditions, such as essential hypertension and cardiac hypertrophy, is probably determined by various combinations of small quantitative changes in the expression of many genes. NPR1, coding for natriuretic peptide receptor A (NPRA), is a potential candidate, because NPRA mediates natriuretic, diuretic, and vasorelaxing actions of the nariuretic peptides, and because genetically determined quantitative changes in the expression of this gene affect blood pressure and heart weight in a dose-dependent manner in mice. To determine whether there are common quantitative variants in human NPR1, we have sequenced the entire human NPR1 gene and identified 10 polymorphic sites in its non-coding sequence by using DNA from 34 unrelated human individuals. Five of the sites are single nucleotide polymorphisms; the remaining five are length polymorphisms, including a highly variable complex dinucleotide repeat in intron 19. There are three common haplotypes 5' to this dinucleotide repeat and three 3' to it, but the 5' haplotypes and 3' haplotypes appear to be randomly associated. Transient expression analysis in cultured cells of reporter plasmids with the proximal promoter sequences of NPR1 and its 3' untranslated regions showed that these polymorphisms have functional effects. We conclude that common NPR1 alleles can alter expression of the gene as much as two-fold and could therefore significantly affect genetic risks for essential hypertension and cardiac hypertrophy in humans.