Knockdown of Serum- and Glucocorticoid-Regulated Kinase 1 Enhances Cisplatin Sensitivity of Gastric Cancer Through Suppressing the Nuclear Factor Kappa-B Signaling Pathway.

BACKGROUND: Previous studies have published the promoting effect of serum and glucocorticoid-regulated kinase 1 (SGK1) in various malignant tumors. However, whether SGK1 promotes gastric cancer remains a mystery. AIMS: To clarify the function of SGK1 in gastric cancer and its potential regulatory mechanism. STUDY DESIGN: Cell culture study. METHODS: The SGK1-silenced model was generated in two gastric cancer cell lines and further evaluated their malignant behavior and susceptibility to cisplatin. The interaction between miR-15a-5p and SGK1 was evaluated by the luciferase reporter assay. The knockdown efficiency of SGK1 was confirmed by RT- qPCR and Western blot assays. Cell proliferation rate was assessed with CCK-8 assay, and flow cytometry was used to determine cell cycle progression and apoptosis. RESULTS: Western blot data displayed an elevated level of SGK1 in gastric cancer cell lines. Functionally, SGK1 deficiency suppressed gastric cancer cell proliferation (P < .01) by acting on cell-cycle progression. Moreover, SGK1 deficiency suppressed cell invasion and migration of gastric cancer cells (P < .01). Further, the silencing of SGK1 obviously suppressed cell proliferation and induced apoptosis of the cells after cisplatin treatment (P < .01), indicating that SGK1 deficiency facilitated the chemosensitivity of these 2 gastric cancer cell lines to cisplatin. Mechanically, downregulation of SGK1 repressed the cytoplasm- to-nucleus translocation of NF-κB p65. Interestingly, we found that miR-15a-5p binds to the 3'UTR of SGK1, which was confirmed using luciferase activity assay (P < .05). Moreover, the data suggested that SGK1 reversed the suppression effect of miR-15a-5p on gastric cancer cell migration (P < .01). CONCLUSION: Loss of SGK1 suppresses the malignant behavior of gastric cancer cells and increases cisplatin sensitivity by restraining the NF-κB signaling pathway. Moreover, SGK1 may exert an inhibitory effect in gastric cancer by being targeted by miR-15a-5p. Therefore, SGK1 may be a prospective target for future gastric cancer therapy.

Serum- and Glucocorticoid-induced Kinase Sgk1 Directly Promotes the Differentiation of Colorectal Cancer Cells and Restrains Metastasis.

PURPOSE: The molecular events that determine intestinal cell differentiation are poorly understood and it is unclear whether it is primarily a passive event or an active process. It is clinically important to gain a greater understanding of the process, because in colorectal cancer, the degree of differentiation of a tumor is associated with patient survival. SGK1 has previously been identified as a gene that is principally expressed in differentiated intestinal cells. In colorectal cancer, there is marked downregulation of SGK1 compared with normal tissue.Experimental Design: An inducible SGK1 viral overexpression system was utilized to induce reexpression of SGK1 in colorectal cancer cell lines. Transcriptomic and phenotypic analyses of these colorectal cancer lines was performed and validation in mouse and human cohorts was performed. RESULTS: We demonstrate that SGK1 is upregulated in response to, and an important controller of, intestinal cell differentiation. Reexpression of SGK1 in colorectal cancer cell lines results in features of differentiation, decreased migration rates, and inhibition of metastasis in an orthotopic xenograft model. These effects may be mediated, in part, by SGK1-induced PKP3 expression and increased degradation of MYC. CONCLUSIONS: Our results suggest that SGK1 is an important mediator of differentiation of colorectal cells and may inhibit colorectal cancer metastasis.

Akt3 inhibits adipogenesis and protects from diet-induced obesity via WNK1/SGK1 signaling.

Three Akt isoforms, encoded by 3 separate genes, are expressed in mammals. While the roles of Akt1 and Akt2 in metabolism are well established, it is not yet known whether Akt3 plays a role in metabolic diseases. We now report that Akt3 protects mice from high-fat diet-induced obesity by suppressing an alternative pathway of adipogenesis via with no lysine protein kinase-1 (WNK1) and serum/glucocorticoid-inducible kinase 1 (SGK1). We demonstrate that Akt3 specifically phosphorylates WNK1 at T58 and promotes its degradation via the ubiquitin-proteasome pathway. A lack of Akt3 in adipocytes increases the WNK1 protein level, leading to activation of SGK1. SGK1, in turn, promotes adipogenesis by phosphorylating and inhibiting transcription factor FOXO1 and, subsequently, activating the transcription of PPARγ in adipocytes. Akt3-deficient mice have an increased number of adipocytes and, when fed a high-fat diet, display increased weight gain, white adipose tissue expansion, and impaired glucose homeostasis. Pharmacological blockade of SGK1 in high-fat diet-fed Akt3-deficient mice suppressed adipogenesis, prevented excessive weight gain and adiposity, and ameliorated metabolic parameters. Thus, Akt3/WNK1/SGK1 represents a potentially novel signaling pathway controlling the development of obesity.

Role of serum- and glucocorticoid-inducible kinases in stroke.

Increased expression of serum- and glucocorticoid-inducible kinase 1 (SGK1) can be induced by stress and growth factors in mammals, and plays an important role in cancer, diabetes, and hypertension. A recent work suggested that SGK1 activity restores damage in a stroke model. To further investigate the role of SGKs in ischemic brain injury, we examined how SGK inhibitors influence stroke outcome in vivo and neurotoxicity in vitro. Infarct volumes were compared in adult mice with middle cerebral artery occlusion, followed by 24 h reperfusion, in the absence or presence of SGK inhibitors. Neurotoxicity assay, electrophysiological recording, and fluorescence Ca(2+) imaging were carried out using cultured cortical neurons to evaluate the underlying mechanisms. Contrary to our expectation, infarct volume by stroke decreased significantly when SGK inhibitor, gsk650394, or EMD638683, was administrated 30 min before middle cerebral artery occlusion under normal and diabetic conditions. SGK inhibitors reduced neurotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors, a leading factor responsible for cell death in stroke. SGK inhibitors also ameliorated Ca(2+) increase and peak amplitude of NMDA current in cultured neurons. In addition, SGK inhibitor gsk650394 decreased phosphorylation of Nedd4-2 and inhibited voltage-gated sodium currents. These observations suggest that SGK activity exacerbates stroke damage and that SGK inhibitors may be useful candidates for therapeutic intervention. To investigate the role of serum- and glucocorticoid-inducible kinases (SGKs) in ischemic brain injury, we examined how SGK inhibitors influence stroke outcome. Infarct volumes induced by middle cerebral artery occlusion were decreased significantly by SGK inhibitors. The inhibitors also reduced glutamate toxicity, at least partly, by attenuation of NMDA and voltage-gated sodium currents. Thus, SGK inhibition attenuates stroke damage.

Estrogen receptor ß regulates endometriotic cell survival through serum and glucocorticoid-regulated kinase activation.

OBJECTIVE: To determine the expression and biological roles of serum and glucocorticoid-regulated kinase (SGK1) in tissues and cells from patients with endometriosis and from healthy control subjects. DESIGN: Case-control. SETTING: University research setting. PATIENT(S): Premenopausal women. INTERVENTION(S): Endometriotic tissues were obtained from women with ovarian endometriosis, and normal endometrial tissues were obtained from women undergoing hysterectomy for benign conditions. MAIN OUTCOME MEASURE(S): Expression levels of SGK1, the role of SGK1 in endometriosis pathology, and regulation of SGK1 by estrogen receptor (ER) ß. RESULT(S): Transcript and protein levels of SGK1 were significantly higher in endometriotic tissues and cells compared with normal endometrium. SGK1 mRNA and protein levels were stimulated by E2, by the ERß-selective agonist diarylpropionitrile, and by prostaglandin E2. SGK1 was transcriptionally regulated by ERß based on small interfering RNA knockdown and chromatin immunoprecipitation of ERß followed by quantitative polymerase chain reaction. SGK1 knockdown led to increased cleavage of poly(ADP-ribose) polymerase, and SGK1 activation was correlated with the phosphorylation of FOXO3a, a proapoptotic factor. CONCLUSION(S): ERß leads to SGK1 overexpression in endometriosis, which contributes to the survival of endometriotic lesions through inhibition of apoptosis.

Incidence and Expression of Circulating Cell Free p53-Related Genes in Acute Myocardial Infarction Patients.

AIM: The circulating RNA levels are predictive markers in several diseases. We determined the levels of circulating p53-related genes in patients with acute ST-segment elevation myocardial infarction (STEMI), indicating major heart muscle damage. METHODS: Plasma RNA was extracted from the patients (n=45) upon their arrival to the hospital (STEMI 0h) and at four hours post-catheterization (STEMI 4h) as well as from controls (n=34). RESULTS: Of 18 circulating p53-related genes, nine genes were detectable. A significantly lower incidence of circulating p21 (p ＜ 0.0001), Notch1 (p=0.042) and BTG2 (p ＜ 0.0001) was observed in the STEMI 0h samples in comparison to the STEMI 4h and control samples. Lower expression levels (2.1-fold) of circulating BNIP3L (p=0.011), p21 (3.4-fold, p=0.005) and BTG2 (6.3-fold, p=0.0001) were observed in the STEMI 0h samples in comparison to the STEMI 4h samples, with a 7.4-fold lower BTG2 expression (p ＜ 0.001) and 2.6-fold lower p21 expression (p=0.034) compared to the control samples. Moreover, the BNIP3L expression (borderline significance, p=0.0655) predicted the level of peak troponin, a marker of myocardial infarction. In addition, the BNIP3L levels on admission (p=0.0025), at post-catheterization (p=0.020) and the change between the groups (p=0.0079) were inversely associated with troponin. The BNIP3L (p=0.0139) and p21 levels (p=0.0447) were also associated with a longer time to catheterization. CONCLUSIONS: Our results suggest that circulating downstream targets of p53 are inhibited during severe AMI and subsequently re-expressed after catheterization, uncovering possible novel death-or-survival decisions regarding the fate of p53 in the heart and the potential use of its target genes as prognostic biomarkers for oxygenation normalization.

Impact of the serum- and glucocorticoid-inducible kinase 1 on platelet dense granule biogenesis and secretion.

BACKGROUND: Platelet secretion is critical to development of acute thrombotic occlusion. Platelet dense granules contain a variety of important hemostatically active substances. Nevertheless, biogenesis of platelet granules is poorly understood. OBJECTIVES: Serum- and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be highly expressed in platelets and megakaryocytes, but its role in the regulation of platelet granule biogenesis and its impact on thrombosis has not been investigated so far. METHODS AND RESULTS: Electron microscopy analysis of the platelet ultrastructure revealed a significant reduction in the number and packing of dense granules in platelets lacking SGK1 (sgk1(-/-) ). In sgk1(-/-) platelets serotonin content was significantly reduced and activation-dependent secretion of ATP, serotonin and CD63 significantly impaired. In vivo adhesion after carotis ligation was significantly decreased in platelets lacking SGK1 and occlusive thrombus formation after FeCl3 -induced vascular injury was significantly diminished in sgk1(-/-) mice. Transcript levels and protein abundance of dense granule biogenesis regulating GTPase Rab27b were significantly reduced in sgk1(-/-) platelets without affecting Rab27b mRNA stability. In MEG-01 cells transfection with constitutively active (S422) (D) SGK1 but not with inactive (K127) (N) SGK1 significantly enhanced Rab27b mRNA levels. Sgk1(-/-) megakaryocytes show significantly reduced expression of Rab27b and serotonin/CD63 levels compared with sgk1(+/+) megakaryocytes. Proteome analysis identified nine further vesicular transport proteins regulated by SGK1, which may have an impact on impaired platelet granule biogenesis in sgk1(-/-) platelets independent of Rab27b. CONCLUSIONS: The present observations identify SGK1 as a novel powerful regulator of platelet dense granule biogenesis, platelet secretion and thrombus formation. SGK1 is at least partially effective because it regulates transcription of Rab27b in megakaryocytes.

Urinary serum- and glucocorticoid-inducible kinase SGK1 reflects renal injury in patients with immunoglobulin A nephropathy.

AIM: Serum- and glucocorticoid-inducible kinase SGK1 functions as an important regulator of transepithelial sodium transport by activating epithelial sodium channel in renal tubules. Considerable evidence demonstrated that SGK1 was associated with hypertension and fibrosing diseases, such as diabetic nephropathy and glomerulonephritis. The present study was performed to evaluate the role of SGK1 played in immunoglobulin A (IgA) nephropathy. METHODS: Seventy-six patients of biopsy-proven IgA nephropathy and 33 healthy volunteers were enrolled in this study. All patients and healthy volunteers' urinary and serum samples were tested for SGK1 expression by indirect enzyme-linked immunosorbent assay. Meanwhile all patients' renal tissues were semi-quantified for SGK1 expression by immunohistochemistry assay. The relationships between SGK1 expressions and clinical or pathological parameters were also assessed. RESULTS: SGK1 expression was upregulated in urine and renal tubules in patients of Oxford classification T1 and T2, whereas its expression in serum did not increase significantly. Relationship analysis indicated that urinary and tissue SGK1 expressions were associated with heavy proteinuria and renal insufficiency in patients with IgA nephropathy. On the other hand, RAS blockades would reduce the SGK1 levels both in urine and renal tissues. CONCLUSION: These results suggested that urinary SGK1 should be a good indicator of tubulointerstitial damage in patients of IgA nephropathy. SGK1 expressions in urine and renal tissues were associated with the activity of renin-angiotensin-aldosterone system.

Serum- and glucocorticoid-inducible kinase SGK1 regulates reorganization of actin cytoskeleton in mast cells upon degranulation.

Aggregation of the high-affinity IgE receptor (FcεRI) on mast cells (MCs) causes MC degranulation, a process that involves cortical F-actin disassembly. Actin depolymerization may be triggered by increase of cytosolic Ca(2+). Entry of Ca(2+) through the Ca(2+) release-activated Ca(2+) (CRAC) channels is under powerful regulation by the serum- and glucocorticoid-inducible kinase SGK1. Moreover, FcεRI-dependent degranulation is decreased in SGK1-deficient (sgk1(-/-)) MCs. The present study addressed whether SGK1 is required for actin cytoskeleton rearrangement in MCs and whether modulation of actin architecture could underlie decreased degranulation of sgk1(-/-) MCs. Confirming previous results, release of ß-hexosaminidase reflecting FcεRI-dependent degranulation was impaired in sgk1(-/-) MCs compared with sgk1(+/+) MCs. When CRAC channels were inhibited by 2-aminoethoxydiphenyl borate (2-APB; 50 µM), MC degranulation was strongly decreased in both sgk1(+/+) and sgk1(-/-) MCs and the difference between genotypes was abolished. Moreover, degranulation was impaired by actin-stabilizing (phallacidin) and enhanced by actin-disrupting (cytochalasin B) agents to a similar extent in sgk1(+/+) MCs and sgk1(-/-) MCs, implying a regulatory role of actin reorganization in this event. In line with this, measurements of monomeric (G) and filamentous (F) actin content by FACS analysis and Western blotting of detergent-soluble and -insoluble cell fractions indicated an increase of the G/F-actin ratio in sgk1(+/+) MCs but not in sgk1(-/-) MCs upon FcεRI ligation, an observation reflecting actin depolymerization. In sgk1(+/+) MCs, FcεRI-induced actin depolymerization was abolished by 2-APB. The observed actin reorganization was confirmed by confocal laser microscopic analysis. Our observations uncover SGK1-dependent Ca(2+) entry in mast cells as a novel mechanism regulating actin cytoskeleton.

Low SGK1 expression in human adrenocortical tumors is associated with ACTH-independent glucocorticoid secretion and poor prognosis.

CONTEXT: Using single-nucleotide polymorphism analysis, we observed allelic loss of the gene for serum glucocorticoid (GC) kinase 1 (SGK1), a GC-responsive kinase involved in multiple cellular functions, in a subset of cortisol-secreting adenomas. OBJECTIVE: Our objective was to analyze SGK1 expression in adrenocortical tumors and to further characterize its role in ACTH-independent cortisol secretion, tumor progression, and prognosis. DESIGN AND SETTING: Gene expression levels of SGK1, SGK3, and CTNNB1 (coding for ß-catenin) and protein expression levels of SGK1, nuclear ß-catenin, and phosphorylated AKT were determined in adrenocortical tumors and normal adrenal glands. PATIENTS: A total of 227 adrenocortical tumors (40 adenomas and 187 carcinomas) and 25 normal adrenal tissues were included. Among them, 62 frozen tumor samples were used for mRNA analysis and 203 tumors were investigated on tissue microarrays or full standard slides by immunohistochemistry. MAIN OUTCOME MEASURES: We evaluated the relationship between SGK1 mRNA and/or protein levels and clinical parameters. RESULTS: SGK1 mRNA levels were lower in cortisol-secreting than in nonsecreting tumors (P < 0.005). Nonsecreting neoplasias showed a significant correlation between SGK1 and CTNNB1 mRNA levels (P < 0.001; r = 0.57). Low SGK1 protein levels, but not nuclear ß-catenin and phosphorylated AKT, were associated with poor overall survival in patients with adrenocortical carcinoma (P < 0.005; hazard ratio = 2.0; 95% confidence interval = 1.24-3.24), independent of tumor stage and GC secretion. CONCLUSION: Low SGK1 expression is related to ACTH-independent cortisol secretion in adrenocortical tumors and is a new prognostic factor in adrenocortical carcinoma.

Lack of prompt expansion of cytomegalovirus pp65 and IE-1-specific IFNgamma CD8+ and CD4+ T cells is associated with rising levels of pp65 antigenemia and DNAemia during pre-emptive therapy in allogeneic hematopoietic stem cell transplant recipients.

Rising levels of cytomegalovirus (CMV) DNAemia and/or pp65 antigenemia have been observed during pre-emptive ganciclovir therapy in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-SCT). We assessed the incidence of this event in our series, and investigated whether its occurrence was associated with an impairment in the CMV-specific T-cell response. A total of 36 allo-SCT recipients experienced one or more episodes of active CMV infection (n=68) that were pre-emptively treated with val(ganciclovir). Rising levels of antigenemia and DNAemia, and an isolated increase in antigenemia, were observed in 39.7 and 2.9% of all episodes, respectively. Receipt of corticosteroids was associated with rising levels of antigenemia and DNAemia. Median increases of 12- and 6.8-fold of IFNgamma CD8(+) T and IFNgamma CD4(+) T cells, respectively, were observed at a median of 16.5 days after initiation of therapy in episodes with decreasing levels in antigenemia and DNAemia. In contrast, the numbers of both T-cell subsets at a median of 13.5 days after initiation of therapy did not differ significantly from those of pre-treatment samples in episodes with rising levels of antigenemia and DNAemia. Lack of prompt expansion of CMV pp65 and IE-1-specific IFNgamma CD8(+) and CD4(+) T cells is associated with rising levels in antigenemia and DNAemia during pre-emptive therapy.

[Evaluation of detection of Epstein-Barr virus Rta/IgG in nasopharyngeal carcinoma].

OBJECTIVE: This study was aimed to investigate the clinical value of Epstein-Barr virus (EBV) Rta/IgG in the diagnosis of nasopharyngeal carcinoma (NPC). METHODS: Serum samples derived from 211 untreated patients with NPC, 413 subjects including 203 non-NPC ENT patients and 210 healthy volunteers as control were examined for the presence of antibodies directed against Rta/IgG by using enzyme-linked immnunosorbent assay (ELISA). Receiver operating characteristic (ROC) curve was applied to perform methodical evaluation of this tumor marker. RESULTS: The rA value median of Rta/IgG in NPC group was significantly higher than one in control group (P < 0.001). The area under ROC was 0.933. The sensitivity and specificity of this marker were 90.5% and 90.1%, respectively, when the best cutoff value was defined. CONCLUSION: Rta/IgG detected with ELISA method is a new target of EBV, and may be one of important marker for NPC diagnosis.

Plasma proteome profiling of a mouse model of breast cancer identifies a set of up-regulated proteins in common with human breast cancer cells.

We have applied an in-depth quantitative proteomic approach, combining isotopic labeling extensive intact protein separation and mass spectrometry, for high confidence identification of protein changes in plasmas from a mouse model of breast cancer. We hypothesized that a wide spectrum of proteins may be up-regulated in plasma with tumor development and that comparisons with proteins expressed in human breast cancer cell lines may identify a subset of up-regulated proteins in common with proteins expressed in breast cancer cell lines that may represent candidate biomarkers for breast cancer. Plasma from PyMT transgenic tumor-bearing mice and matched controls were obtained at two time points during tumor growth. A total of 133 proteins were found to be increased by 1.5-fold or greater at one or both time points. A comparison of this set of proteins with published findings from proteomic analysis of human breast cancer cell lines yielded 49 proteins with increased levels in mouse plasma that were identified in breast cancer cell lines. Pathway analysis comparing the subset of up-regulated proteins known to be expressed in breast cancer cell lines with other up-regulated proteins indicated a cancer related function for the former and a host-response function for the latter. We conclude that integration of proteomic findings from mouse models of breast cancer and from human breast cancer cell lines may help identify a subset of proteins released by breast cancer cells into the circulation and that occur at increased levels in breast cancer.

Connective tissue growth factor is an indicator of bone involvement in multiple myeloma, but matrix metalloproteinase-9 is not.

Bone disease (BD) in multiple myeloma (MM) is because of the activation of osteoclasts and impairment of osteoblast differentiation. Connective tissue growth factor (CTGF) is known to participate in the differentiation of mesenchymal stem cells to committed osteoprogenitor cells. We analysed the concentration of circulating CTGF in 35 MM patients and 22 malignant lymphoma (ML) patients and 14 normal individuals. CTGF is protease-sensitive and thus is found as both an N-terminal half fragment (N-half CTGF) and whole (W-CTGF). Serum levels of W-CTGF and N-half CTGF + W-CTGF were determined by separate sandwich enzyme-linked immunosorbent assays. The level of W-CTGF was significantly lower (P < 0.005) in MM patients compared with ML patients and normal individuals, while N-half + W-CTGF was similar in all groups. Furthermore, W-CTGF was significantly lower in MM patients with BD compared with those without BD (P < 0.005) and this was independent of previous treatment. Matrix metalloproteinase (MMP)-9 is produced by myeloma cells and is thought to be related to BD in MM. However, MMP-9 does not cleave CTGF and serum MMP-9 level was not related to BD in MM. Thus, CTGF is an indicator of BD in MM; its metabolism and function in MM should be clarified.

Vaccine properties of a novel marker gene-free recombinant modified vaccinia Ankara expressing immunodominant CMV antigens pp65 and IE1.

CMV tegument protein pp65 and CMV immediate early gene product IE1 are both considered immunodominant targets of cell-mediated immunity (CMI) and potentially capable of controlling CMV infection. To better assess their role in host defense, we have constructed a novel MVA transfer vector named pZWIIA and generated a recombinant MVA (rMVA) expressing both full-length pp65 and exon4 of IE1 (pp65-IE1-MVA) at high levels, followed by the genetic removal of the bacterial marker gene used to distinguish recombinant forms. Immunogenicity evaluation indicates that pp65-IE1-MVA not only can induce robust primary CMI to both antigens in HLA A2.1 Tg mice, but also can stimulate vigorous expansion of memory T lymphocyte responses to pp65 and IE1 in PBMC of CMV-positive donors. These properties make the MVA-based vaccine ideal for the dual role of priming and boosting CMV-specific T cell immunity as a means to control CMV disease in recipients of hematopoietic cell or solid organ transplantation (HCT or SOT). pZWIIA alone or in combination with other MVA transfer vectors can be used to generate MVA based multiple-antigen vaccine which have application in vaccine development for a wide spectrum of infectious diseases and cancer.

The circulating proangiogenic factors CYR61 (CCN1) and NOV (CCN3) are significantly decreased in placentae and sera of preeclamptic patients.

It is thought that preeclampsia results from a shallow invasion of the extravillous trophoblast into the decidua and maternal vessels, which in turn leads to hypoxia and uteroplacental insufficiency. Here, the authors focus on the expression of the proangiogenic secreted molecules CYR61 (CCN1) and NOV (CCN3) in human placentae during normal pregnancy compared with preeclamptic placentae. CYR61 and NOV are strongly expressed in endothelial cells as well as in the extravillous trophoblast, with increasing levels during placental development. Interestingly, the authors found significantly decreased levels in early preeclamptic placentae compared with matched controls. Whereas both CYR61 and NOV proteins are present at constant high levels in the sera of nonpregnant and pregnant women, in the sera of patients with early-onset preeclampsia, levels were significantly reduced. The authors suggest that the reduction of both CCN molecules in preeclampsia could be 1 reason underlying the failure of uterine vascular remodeling. Moreover, their low maternal serum levels could serve as biomarkers for early diagnosis of this disease.

Onset and duration of cytomegalovirus immediate early 1 mRNA expression in the blood of renal transplant recipients.

Human cytomegalovirus (CMV) messenger (m) RNA expression in circulating leukocytes reflects directly viral activity in the human host. In this study, sixty-nine patients were monitored prospectively for CMV infection and mRNA expression during the first year after renal transplantation. Of the 69 recipients, 58 (84%) recipients were positive for CMV immediate early 1 (IE1) mRNA as detected by nucleic acid sequence-based amplification. The median onset of IE1 expression started at day 22 after transplantation and continued for a median duration of 82 days. IE1 mRNA expression started significantly earlier in recipients who developed an active CMV infection (P = 0.001) and in mycophenolate mofetil (MMF) treated recipients (P = 0.002). The duration of IE1 mRNA expression was significantly longer in recipients that had previously an early onset of IE1 mRNA expression (P = 0.001) and in recipients with active CMV infection (P = 0.007). Remarkably, longer prednisolone intake was correlated with a significantly (P = 0.02) shorter duration of IE1 expression compared to a longer duration of IE1 expression in recipients with only a short prednisolone intake. In recipients infected with glycoprotein B (gB) type 1 CMV strains, the duration of IE1 expression was significantly (P = 0.04) shorter compared to recipients infected with non-gB type 1 CMV strains (64 days vs. 150 days). The study indicates that multiple factors play a role in the onset and/or duration of CMV IE1 mRNA expression, for example, MMF treatment, prednisolone intake, and gB type of the specific CMV strain. The clinical significance of these correlations remains to be studied in more detail.

Characterization of human NOV in biological fluids: an enzyme immunoassay for the quantification of human NOV in sera from patients with diseases of the adrenal gland and of the nervous system.

Immunochromatography has shown that human NOV (NOVH), a member of the CCN (CTGF/CYR61/NOV) family, forms a physiological complex with fibulin-1 in blood. We developed an enzyme immunoassay specific for NOVH and showed for the first time that the concentration of NOVH differs in each of these biological fluids. The normal concentration of NOVH circulating in the blood is 350-400 ng/ml, but this concentration varies with age. By using sera from patients with adrenal gland diseases we found that in vivo ACTH or glucocorticoids are not responsible for the high concentration of NOVH in this endocrine gland. However, the NOVH concentration was significantly modified in malignant adrenocortical tumors, but not in benign adrenocortical tumors. The concentration of NOVH was significantly decreased in patients suffering from astrocytomas or multiple sclerosis, two diseases of the nervous system. Thus, NOVH is a potentially useful marker for the diagnosis of these diseases.

Monitoring cytomegalovirus infection by antigenemia assay and two distinct plasma real-time PCR methods after hematopoietic stem cell transplantation.

We compared a CMV virus load determined by real-time PCR with an antigenemia value to analyze the correlation between these two methods. We also compared the values for virus load determined by the two distinct real-time PCR methods, which amplify the US17 region and immediate-early (IE) gene of CMV, respectively, to evaluate the reliability of these methods. Two hundred and sixty-five samples were obtained weekly from 29 patients, who had engraftment after unrelated bone marrow transplantation or HLA-mismatched related blood stem cell transplantation. CMV infection was detected in 115 samples from 22 patients by US17-PCR and 69 samples from 20 patients by the antigenemia assay. Fifty-eight samples were positive for both assays, but 57 and 11 samples were positive only for US17-PCR and antigenemia, respectively. A good correlation of the results of US17-PCR and antigenemia was demonstrated (r = 0.61). All antigenemia-positive samples and randomly selected antigenemia-negative samples were subjected to IE-PCR. The results of IE-PCR showed a good correlation with those of antigenemia (r = 0.64). Furthermore, the best correlation was observed between US17-PCR and IE-PCR (r = 0.83). In conclusion, both real-time PCR methods showed a good correlation with the antigenemia assay, and could be used to monitor CMV infection after hematopoietic stem cell transplantation.