Clinical validation of a blood-based classifier for diagnostic evaluation of asymptomatic individuals with pulmonary nodules.

BACKGROUND: The number of pulmonary nodules detected in the US is expected to increase substantially following recent recommendations for nationwide CT-based lung cancer screening. Given the low specificity of CT screening, non-invasive adjuvant methods are needed to differentiate cancerous lesions from benign nodules to help avoid unnecessary invasive procedures in the asymptomatic population. We have constructed a serum-based multi-biomarker panel and assessed its clinical accuracy in a retrospective analysis of samples collected from participants with suspicious radiographic findings in the Prostate, Lung, Chest and Ovarian (PLCO) cancer screening trial. METHODS: Starting with a set of 9 candidate biomarkers, we identified 8 that exhibited limited pre-analytical variability with increasing clotting time, a key pre-analytical variable associated with the collection of serum. These 8 biomarkers were evaluated in a training study consisting of 95 stage I NSCLC patients and 186 smoker controls where a 5-biomarker pulmonary nodule classifier (PNC) was selected. The clinical accuracy of the PNC was determined in a blinded study of asymptomatic individuals comprising 119 confirmed malignant nodule cases and 119 benign nodule controls selected from the PLCO screening trial. RESULTS: A PNC comprising 5 biomarkers: CEA, CYFRA 21-1, OPN, SCC, and TFPI, was selected in the training study. In an independent validation study, the PNC resolved lung cancer cases from benign nodule controls with an AUC of 0.653 (p < 0.0001). CEA and CYFRA 21-1, two of the markers included in the PNC, also accurately distinguished malignant lesions from benign controls. CONCLUSIONS: A 5-biomarker blood test has been developed for the diagnostic evaluation of asymptomatic individuals with solitary pulmonary nodules.

Blood-based lung cancer biomarkers identified through proteomic discovery in cancer tissues, cell lines and conditioned medium.

BACKGROUND: Support for early detection of lung cancer has emerged from the National Lung Screening Trial (NLST), in which low-dose computed tomography (LDCT) screening reduced lung cancer mortality by 20 % relative to chest x-ray. The US Preventive Services Task Force (USPSTF) recently recommended annual screening for the high-risk population, concluding that the benefits (life years gained) outweighed harms (false positive findings, abortive biopsy/surgery, radiation exposure). In making their recommendation, the USPSTF noted that the moderate net benefit of screening was dependent on the resolution of most false-positive results without invasive procedures. Circulating biomarkers may serve as a valuable adjunctive tool to imaging. RESULTS: We developed a broad-based proteomics discovery program, integrating liquid chromatography/mass spectrometry (LC/MS) analyses of freshly resected lung tumor specimens (n = 13), lung cancer cell lines (n = 17), and conditioned media collected from tumor cell lines (n = 7). To enrich for biomarkers likely to be found at elevated levels in the peripheral circulation of lung cancer patients, proteins were prioritized based on predicted subcellular localization (secreted, cell-membrane associated) and differential expression in disease samples. 179 candidate biomarkers were identified. Several markers selected for further validation showed elevated levels in serum collected from subjects with stage I NSCLC (n = 94), relative to healthy smoker controls (n = 189). An 8-marker model was developed (TFPI, MDK, OPN, MMP2, TIMP1, CEA, CYFRA 21-1, SCC) which accurately distinguished subjects with lung cancer (n = 50) from high risk smokers (n = 50) in an independent validation study (AUC = 0.775). CONCLUSIONS: Integrating biomarker discovery from multiple sample types (fresh tissue, cell lines and conditioned medium) has resulted in a diverse repertoire of candidate biomarkers. This unique collection of biomarkers may have clinical utility in lung cancer detection and diagnoses.

Facilitating Mental Health Treatment Through Proactive Screening and Concierge Services in the Workplace.

OBJECTIVE: The aim of this study is to determine if a proactive employer-sponsored mental health program closed gaps in detection and treatment of mental health conditions. METHODS: Of n = 56,442 eligible, n = 8170 (14.5%) participated in the optional screening. Participants with mental health risk were offered care concierge services including support, care planning, and connection to care. Difference in behavioral health care utilization, diagnoses, and prescriptions were evaluated postintervention through claims analysis. RESULTS: Compared with controls (n = 2433), those receiving concierge services (n = 369) were more likely to fill mental health prescriptions (adjusted hazards ratio [HR], 1.2; 1.0-1.5; P = 0.042), use professional mental health services (adjusted HR, 1.4; 1.1-1.8; P = 0.02), and use new mental health services (adjusted HR, 1.9; 1.2-2.8; P = 0.004) in the following 6 months. CONCLUSIONS: This proactive mental health program with care concierge services identified risk, connected individuals to mental health care, and facilitated mental health treatment, among program participants.

Assessing the impact of a comprehensive mental health program on frontline health service workers.

Mental health issues are a growing concern in the workplace, linked to negative outcomes including reduced productivity, increased absenteeism, and increased turnover. Employer-sponsored mental health benefits that are accessible and proactive may help address these concerns. The aim of this retrospective cohort study was to evaluate the impact of a digital mental health benefit (Spring Health) on frontline healthcare service workers' clinical and workplace outcomes. The benefit was sponsored by a national health services company from 2021-2022 and included mental health screening, care navigation, psychotherapy and/or medication management. We hypothesized program use would be associated with improvements in depression and anxiety symptoms, and increased productivity and retention. Participants were employees enrolled in the benefit program, had at least moderate anxiety or depression, at least 1 treatment appointment, and at least 2 outcome assessments. Clinical improvement measures were PHQ-9 scale (range, 0-27) for depression and GAD-7 scale (range, 0-21) for anxiety; workplace measures were employee retention and the Sheehan Disability Scale (SDS) for functional impairment. A total of 686 participants were included. Participants using the mental health benefit had a 5.60 point (95% CI, 4.40-6.79, d = 1.28) reduction in depression and a 5.48 point (95% CI, 3.88-7.08, d = 1.64) reduction in anxiety across 6 months. 69.9% (95% CI, 61.8%-78.1%) of participants reliably improved (≥5 point change) and 84.1% (95% CI, 78.2%-90.1%) achieved reliable improvement or recovery (<10 points). Participants reported 0.70 (95% CI, 0.26-1.14) fewer workdays per week impacted by mental health issues, corresponding to $3,491 (95% CI, $1305-$5677) salary savings at approximately federal median wage ($50,000). Furthermore, employees using the benefit were retained at 1.58 (95% CI, 1.4-1.76) times the rate of those who did not. Overall, this evaluation suggests that accessible, proactive, and comprehensive mental health benefits for frontline health services workers can lead to positive clinical and workplace outcomes.

Impact of a Digital Diabetes Prevention Program on Estimated 8-Year Risk of Diabetes in a Workforce Population.

OBJECTIVE: We asked whether the estimated 8-year risk of diabetes could be reduced within the first 2 years of a digital Diabetes Prevention Program (dDPP) in a workforce population. METHODS: Employees and spouses were eligible if they had prediabetes-range fasting glucose or hemoglobin A 1c and body mass index ≥25 kg/m 2 . Diabetes risk was assessed using the Framingham diabetes risk score in the year before and the 2 years after dDPP initiation. RESULTS: Among participants completing at least nine dDPP lessons ( n = 286), diabetes risk decreased 5.3% the year after dDPP initiation, after a 5.4% increase the year before initiation (difference in differences, -10.6%; 95% confidence interval, -13.4% to -7.9%; P < 0.001), with risk maintained at reduced levels after the second year of the program. CONCLUSION: This dDPP reduced the estimated 8-year risk of diabetes over the first 2 years of the program.

Workplace Outreach Program Improves Management of Chronic Kidney Disease.

OBJECTIVE: Assess whether an employee outreach program improved management of chronic kidney disease (CKD). METHODS: Participants with suspected CKD (eGFR <60 mL/min/1.73m 2 ) identified in employee health assessments in 2017 and 2018 were contacted by phone and offered physician consultation. Subsequent nephrologist visits at 11 months of follow up were compared between those who were (outreach group) and were not (control group) successfully contacted. RESULTS: Most CKD risk factors at baseline were similar in outreach and control groups. At the end of the follow-up, outreach participants had more than 2-fold greater incidence of visiting a nephrologist compared with controls (HR = 2.3; 95% CI 1.2-4.2, P = 0.01), after adjusting for potential confounders. Conclusions: Employee outreach program increased utilization of nephrologist care.

SOSTDC1 differentially modulates Smad and beta-catenin activation and is down-regulated in breast cancer.

Sclerostin domain containing 1 (SOSTDC1) protein regulates processes from development to cancer by modulating activity of bone morphogenetic protein (BMP) and wingless/int (Wnt) signaling pathways. As dysregulation of both BMP and Wnt signaling has been observed in breast cancer, we investigated whether disruption of SOSTDC1 signaling occurs in breast cancer. SOSTDC1 mRNA expression levels in breast tissue were examined using a dot blot. Affymetrix microarray data on SOSTDC1 levels were correlated with breast cancer patient survival using Kaplan-Meier plots. Correlations between SOSTDC1 protein levels and clinical parameters were assessed by immunohistochemistry of a breast cancer tissue microarray. SOSTDC1 secretion and BMP and Wnt signaling were investigated using immunoblotting. We found that SOSTDC1 is expressed in normal breast tissue and this expression is reduced in breast cancer. High levels of SOSTDC1 mRNA correlated with increased patient survival; conversely, SOSTDC1 protein levels decreased as tumor size and disease stage increased. Treatment of breast cancer cells with recombinant SOSTDC1 or Wise, a SOSTDC1 orthologue, demonstrated that SOSTDC1 selectively blocks BMP-7-induced Smad phosphorylation without diminishing BMP-2 or Wnt3a-induced signaling. In conclusion, SOSTDC1 mRNA and protein are reduced in breast cancer. High SOSTDC1 mRNA levels correlate with increased distant metastasis-free survival in breast cancer patients. SOSTDC1 differentially affects Wnt3a, BMP-2, and BMP-7 signaling in breast cancer cells. These results identify SOSTDC1 as a clinically important extracellular regulator of multiple signaling pathways in breast cancer.

Decreased survival of B cells of HIV-viremic patients mediated by altered expression of receptors of the TNF superfamily.

Human immunodeficiency virus (HIV) infection leads to numerous perturbations of B cells through mechanisms that remain elusive. We performed DNA microarray, phenotypic, and functional analyses in an effort to elucidate mechanisms of B cell perturbation associated with ongoing HIV replication. 42 genes were up-regulated in B cells of HIV-viremic patients when compared with HIV-aviremic and HIV-negative patients, the majority of which were interferon (IFN)-stimulated or associated with terminal differentiation. Flow cytometry confirmed these increases and indicated that CD21(low) B cells, enhanced in HIV-viremic patients, were largely responsible for the changes. Increased expression of the tumor necrosis factor (TNF) superfamily (TNFSF) receptor CD95 correlated with increased susceptibility to CD95-mediated apoptosis of CD21(low) B cells, which, in turn, correlated with HIV plasma viremia. Increased expression of BCMA, a weak TNFSF receptor for B lymphocyte stimulator (BLyS), on CD21(low) B cells was associated with a concomitant reduction in the expression of the more potent BLyS receptor, BAFF-R, that resulted in reduced BLyS binding and BLyS-mediated survival. These findings demonstrate that altered expression of genes associated with IFN stimulation and terminal differentiation in B cells of HIV-viremic patients lead to an increased propensity to cell death, which may have substantial deleterious effects on B cell responsiveness to antigenic stimulation.

Decreased survival of B cells of HIV-viremic patients mediated by altered expression of receptors of the TNF superfamily.

Human immunodeficiency virus (HIV) infection leads to numerous perturbations of B cells through mechanisms that remain elusive. We performed DNA microarray, phenotypic, and functional analyses in an effort to elucidate mechanisms of B cell perturbation associated with ongoing HIV replication. 42 genes were up-regulated in B cells of HIV-viremic patients when compared with HIV-aviremic and HIV-negative patients, the majority of which were interferon (IFN)-stimulated or associated with terminal differentiation. Flow cytometry confirmed these increases and indicated that CD21(low) B cells, enhanced in HIV-viremic patients, were largely responsible for the changes. Increased expression of the tumor necrosis factor (TNF) superfamily (TNFSF) receptor CD95 correlated with increased susceptibility to CD95-mediated apoptosis of CD21(low) B cells, which, in turn, correlated with HIV plasma viremia. Increased expression of BCMA, a weak TNFSF receptor for B lymphocyte stimulator (BLyS), on CD21(low) B cells was associated with a concomitant reduction in the expression of the more potent BLyS receptor, BAFF-R, that resulted in reduced BLyS binding and BLyS-mediated survival. These findings demonstrate that altered expression of genes associated with IFN stimulation and terminal differentiation in B cells of HIV-viremic patients lead to an increased propensity to cell death, which may have substantial deleterious effects on B cell responsiveness to antigenic stimulation.

Use of an immunoaffinity-mass spectrometry-based approach for the quantification of protein biomarkers from serum samples of lung cancer patients.

It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometry-based approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry-based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated, and digested with trypsin. Peptides were analyzed by LC coupled with multiple reaction monitoring approach, and quantification was achieved by the addition of stable isotope-labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 to 250 ng/ml for carcinoembryonic antigen (CEA), a known tumor biomarker. Moreover we observed elevated levels of CEA in sera samples from lung cancer patients that to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. A linear response was obtained from a multiplex spiking experiment in normal human sera for secretory leukocyte peptidase inhibitor (4-500 ng/ml), tissue factor pathway inhibitor (TFPI) (42-1000 ng/ml), tissue factor pathway inhibitor 2 (TFPI2) (2-250 ng/ml), and metalloproteinase inhibitor 1 (TIMP1) (430-1000 ng/ml). A replicate experiment for a single concentration value yielded a relative coefficient of variation better than 11% for TFPI, secretory leukocyte peptidase inhibitor, and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by an immunoaffinity-multiple reaction monitoring method, and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry-based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples especially for those proteins for which the necessary reagents for ELISA development are unavailable.

Impact of a Digital Diabetes Prevention Program on Risk Factors for Chronic Disease in a Workforce Cohort.

OBJECTIVE: Evaluate the effect of a digital Diabetes Prevention Program (dDPP) on chronic disease risk factors in a workplace population. METHODS: dDPP participants were employees and spouses with BMI ≥ 24 kg/m and prediabetes or diabetes (n = 84). Annual change in risk factors before and after dDPP were assessed in the dDPP group and in a retrospectively identified matched control group drawn from those who participated in a dDPP after the conclusion of this study (n = 252). RESULTS: In the dDPP group, body weight, BMI, fasting glucose, triglycerides, total cholesterol and LDL-cholesterol decreased in the post-dDPP period compared with the pre-dDPP period (P < 0.05). In the control group, no difference between the annual change before and after dDPP was observed (P > 0.37). CONCLUSION: The dDPP was effective in reducing risk factors for chronic disease in a workplace setting.

Population health screenings for the prevention of chronic disease progression.

OBJECTIVES: Early detection of disease enables prompt treatment that can prevent disease progression and costly health outcomes. We report incidence of previously unrecognized disease and investigate the expected effect of early detection and care on health outcomes. STUDY DESIGN: Population health study based on laboratory evidence. METHODS: Laboratory evidence of prediabetes, diabetes, chronic kidney disease, and colorectal cancer was evaluated in an employee and spouse population (65% women; mean [SD] age = 46 [12] years). Expected disease progression was assessed. RESULTS: Annual screening found laboratory evidence for 1185 previously unrecognized cases of prediabetes, 287 cases of diabetes, 73 cases of chronic kidney disease, and 669 positive colorectal screens per 10,000 people. CONCLUSIONS: Early identification and appropriate medical care may delay 34 cases of end-stage kidney disease and prevent diabetes-related complications, 210 cases of diabetes, and 3 cases of late-stage colorectal cancer over 5 years per 1000 cases identified.

An integrated functional genomics screening program reveals a role for BMP-9 in glucose homeostasis.

A coordinated functional genomics program was implemented to identify secreted polypeptides with therapeutic applications in the treatment of diabetes. Secreted factors were predicted from a diverse expressed-sequence tags (EST) database, representing >1,000 cDNA libraries, using a combination of bioinformatic algorithms. Subsequently, approximately 8,000 human proteins were screened in high-throughput cell-based assays designed to monitor key physiological transitions known to be centrally involved in the physiology of type 2 diabetes. Bone morphogenetic protein-9 (BMP-9) gave a positive response in two independent assays: reducing phosphoenolpyruvate carboxykinase (PEPCK) expression in hepatocytes and activating Akt kinase in differentiated myotubes. Purified recombinant BMP-9 potently inhibited hepatic glucose production and activated expression of key enzymes of lipid metabolism. In freely fed diabetic mice, a single subcutaneous injection of BMP-9 reduced glycemia to near-normal levels, with maximal reduction observed 30 hours after treatment. BMP-9 represents the first hepatic factor shown to regulate blood glucose concentration. Using a combination of bioinformatic and high-throughput functional analyses, we have identified a factor that may be exploited for the treatment of diabetes.

Osteostat/tumor necrosis factor superfamily 18 inhibits osteoclastogenesis and is selectively expressed by vascular endothelial cells.

Vascular endothelial cells (EC) participate in the process of bone formation through the production of factors regulating osteoclast differentiation and function. In this study, we report the selective expression in primary human microvascular EC of Osteostat/TNF superfamily 18, a ligand of the TNF superfamily. Osteostat protein is detectable in human microvascular EC and is highly up-regulated by IFN-alpha and IFN-beta. Moreover, an anti-Osteostat antibody strongly binds to the vascular endothelium in human tissues, demonstrating that the protein is present in the EC layers surrounding blood vessels. Functional in vitro assays were used to define Osteostat involvement in osteoclastogenesis. Both recombinant and membrane-bound Osteostat inhibit differentiation of osteoclasts from monocytic precursor cells. Osteostat suppresses the early stage of osteoclastogenesis via inhibition of macrophage colony-stimulating factor-induced receptor activator of NF-kappaB (RANK) expression in the osteoclast precursor cells. This effect appears to be specific for the differentiation pathway of the osteoclast lineage, because Osteostat does not inhibit lipopolysaccharide-induced RANK expression in monocytes and dendritic cells, or activation-induced RANK expression in T cells. These findings demonstrate that Osteostat is a novel regulator of osteoclast generation and substantiate the major role played by the endothelium in bone physiology.

Development of a long-acting insulin analog using albumin fusion technology.

The primary therapeutic goal for the treatment of diabetes is maintenance of a long-term, near-normoglycemic condition and prevention of the onset or progression of the complications associated with the disease. Although several analogs of human insulin have been developed, the currently prescribed long-acting insulin analogs do not provide a stable basal glycemia for more than a few hours. Here, we report the development of Albulin, a long-acting insulin analog obtained by direct gene fusion of a single-chain human insulin to human serum albumin. Albulin showed an elimination t(1/2) of approximately 7 h in normoglycemic mice. In vitro pharmacodynamic profiles for Albulin characterized by receptor binding, inhibition of gluconeogenesis, induction of glucose uptake, and global regulation of gene expression in relevant cell types showed that Albulin produced similar activity profiles compared with that of recombinant human insulin. A single Albulin administration in vivo normalized blood glucose level in diabetic mice in a relatively peakless and sustained (24-h) fashion. A further reduction in glucose levels was achieved by administering a recombinant human insulin a few hours after Albulin injection in mice, indicating the potential for Albulin therapy in combination with available fast-acting insulin derivatives. In summary, Albulin displays characteristics of a potent long-acting insulin analog that can be evaluated for use as a novel insulin therapy for patients with insulin-dependent diabetes.

Global transcriptional effects of PEG-IFN-alpha and ribavirin on peripheral blood cells obtained from patients with chronic hepatitis C.

The global transcriptional profile during the first 4 weeks of treatment with pegylated interferon alfa (PEG-IFN-alpha) therapy for chronic hepatitis C (CHC) was evaluated. cDNA array technology was used to assess expression of 10,918 human genes in peripheral blood cells obtained from 17 CHC patients at days 0, 7, and 28 following treatment with PEG-IFN-alpha and ribavirin. Hierarchical average linkage clustering identified seven temporal profiles of differential expression comprising 148 genes. Gene expression profiles were comparable between the PEG-IFN-alpha-2a and PEG-IFN-alpha-2b therapy. Genes representing a broad range of molecular functions were differentially regulated with distinct temporal patterns of expression. The initial global response to interferon treatment appears to be a net up-regulation of genes, consistent with gene responses identified previously in vitro, though by 4 weeks an overall down-regulation of genes was observed. Novel transcription factors potentially involved in secondary gene regulation cascades, a potential dsRNA receptor and members of the ubiquitin signaling, including a novel predicted deubiquitinating peptidase were all identified as being up-regulated upon treatment with IFN. The overall findings provide new light on possible physiological effects of IFN-alpha and open new lines of investigations on the mode of action of PEG-IFN-alpha combination therapy.

An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates.

The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-beta (IFN-beta ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-beta induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 microg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 microg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-beta, and the terminal half-life was 36-40 h compared with 8 h for IFN-beta. Importantly, Albuferon beta induced sustained increases in serum neopterin levels and 2',5' mRNA expression. At a molar dose equivalent to one-half the dose of IFN-beta, Albuferon beta elicited comparable neopterin responses and significantly higher 2',5'-OAS mRNA levels in rhesus monkeys. The enhanced in vivo pharmacologic properties of IFN-beta when fused to serum albumin suggest a clinical opportunity for improved IFN-beta therapy.

LCN6, a novel human epididymal lipocalin.

BACKGROUND: The lipocalin (LCN) family of structurally conserved hydrophobic ligand binding proteins is represented in all major taxonomic groups from prokaryotes to primates. The importance of lipocalins in reproduction and the similarity to known epididymal lipocalins prompted us to characterize the novel human epididymal LCN6. METHODS AND RESULTS: LCN6 cDNA was identified by database analysis in a comprehensive human library sequencing program. Macaca mulatta (rhesus monkey) cDNA was obtained from an epididymis cDNA library and is 93% homologous to the human. The gene is located on chromosome 9q34 adjacent LCN8 and LCN5. LCN6 amino acid sequence is most closely related to LCN5, but the LCN6 beta-barrel structure is best modeled on mouse major urinary protein 1, a pheromone binding protein. Northern blot analysis of RNAs isolated from 25 human tissues revealed predominant expression of a 1.0 kb mRNA in the epididymis. No other transcript was detected except for weak expression of a larger hybridizing mRNA in urinary bladder. Northern hybridization analysis of LCN6 mRNA expression in sham-operated, castrated and testosterone replaced rhesus monkeys suggests mRNA levels are little affected 6 days after castration. Immunohistochemical staining revealed that LCN6 protein is abundant in the caput epithelium and lumen. Immunofluorescent staining of human spermatozoa shows LCN6 located on the head and tail of spermatozoa with the highest concentration of LCN6 on the post-acrosomal region of the head, where it appeared aggregated into large patches. CONCLUSIONS: LCN6 is a novel lipocalin closely related to Lcn5 and Lcn8 and these three genes are likely products of gene duplication events that predate rodent-primate divergence. Predominant expression in the epididymis and location on sperm surface are consistent with a role for LCN6 in male fertility.

A human bone morphogenetic protein antagonist is down-regulated in renal cancer.

We analyzed expression of candidate genes encoding cell surface or secreted proteins in normal kidney and kidney cancer. This screen identified a bone morphogenetic protein (BMP) antagonist, SOSTDC1 (sclerostin domain-containing-1) as down-regulated in kidney tumors. To confirm screening results, we probed cDNA dot blots with SOSTDC1. The SOSTDC1 message was decreased in 20/20 kidney tumors compared with normal kidney tissue. Immunohistochemistry confirmed significant decrease of SOSTDC1 protein in clear cell renal carcinomas relative to normal proximal renal tubule cells (p < 0.001). Expression of SOSTDC1 was not decreased in papillary and chromophobe kidney tumors. SOSTDC1 was abundantly expressed in podocytes, distal tubules, and transitional epithelia of the normal kidney. Transfection experiments demonstrated that SOSTDC1 is secreted and binds to neighboring cells and/or the extracellular matrix. SOSTDC1 suppresses both BMP-7-induced phosphorylation of R-Smads-1, -5, and -8 and Wnt-3a signaling. Restoration of SOSTDC1 in renal clear carcinoma cells profoundly suppresses proliferation. Collectively, these results demonstrate that SOSTDC1 is expressed in the human kidney and decreased in renal clear cell carcinoma. Because SOSTDC1 suppresses proliferation of renal carcinoma cells, restoration of SOSTDC1 signaling may represent a novel target in treatment of renal clear cell carcinoma.

Antibacterial properties of the sperm-binding proteins and peptides of human epididymis 2 (HE2) family; salt sensitivity, structural dependence and their interaction with outer and cytoplasmic membranes of Escherichia coli.

During passage through the epididymis, sperm interact with secreted epididymal proteins that promote maturation, including the acquisition of motility and fertilization competence. Viewed previously as distinct from sperm maturation, host defence capabilities are now recognized functions of the human epididymis 2 (HE2) family of sperm-binding proteins. We analysed the potent dose and time-dependent bactericidal activity of recombinant HE2alpha, HE2beta1 and HE2beta2 and found that the full-length proteins (10 microg/ml or approximately 1 microM) caused more than a 50% decrease in Escherichia coli colony forming units within 15 min. By contrast, human beta-defensin-1, at a similar concentration, required more than 90 min to exhibit similar antibacterial activity. The epididymis-specific lipocalin, LCN6, failed to kill bacteria. Higher concentrations (25-100 microg/ml) of HE2 proteins and a longer duration of treatment resulted in near total inhibition of bacterial growth. The C-terminal peptides of HE2alpha, HEbeta1 and HEbeta2 proteins exhibited antibacterial activity similar to their full-length counterparts, indicating that the antibacterial activity of HE2 proteins resides in these C-terminal regions. Antibacterial activities of HE2 proteins and peptides were slightly inhibited by NaCl concentrations of up to 150 mM, while human beta-defensin-1 activity was nearly eliminated. Reduction and alkylation of disulphide bonds in HE2 proteins and their C-terminal peptides abolished their antibacterial activity. Consistent with the ability to kill bacteria, full-length HE2 proteins and C-terminal peptides caused rapid dose-dependent permeabilization of outer and cytoplasmic E. coli membranes. A much longer exposure time was required for human beta-defensin-1-mediated permeabilization of membranes, suggesting a possible difference in mode of action compared with the HE2 antibacterial peptides.