Applying recovery biomarkers to calibrate self-report measures of sodium and potassium in the hispanic community health study/study of latinos.

This corrects the article DOI: 10.1038/jhh.2016.98.

Clinical Trials in Veterinary Medicine: A New Era Brings New Challenges.

Randomized clinical trials (RCTs) are among the most rigorous ways to determine the causal relationship between an intervention and important clinical outcome. Their use in veterinary medicine has become increasingly common, and as is often the case, with progress comes new challenges. Randomized clinical trials yield important answers, but results from these studies can be unhelpful or even misleading unless the study design and reporting are carried out with care. Herein, we offer some perspective on several emerging challenges associated with RCTs, including use of composite endpoints, the reporting of different forms of risk, analysis in the presence of missing data, and issues of reporting and safety assessment. These topics are explored in the context of previously reported veterinary internal medicine studies as well as through illustrative examples with hypothetical data sets. Moreover, many insights germane to RCTs in veterinary internal medicine can be drawn from the wealth of experience with RCTs in the human medical field. A better understanding of the issues presented here can help improve the design, interpretation, and reporting of veterinary RCTs.

Applying recovery biomarkers to calibrate self-report measures of sodium and potassium in the Hispanic Community Health Study/Study of Latinos.

Measurement error in assessment of sodium and potassium intake obscures associations with health outcomes. The level of this error in a diverse US Hispanic/Latino population is unknown. We investigated the measurement error in self-reported dietary intake of sodium and potassium and examined differences by background (Central American, Cuban, Dominican, Mexican, Puerto Rican and South American). In 2010-2012, we studied 447 participants aged 18-74 years from four communities (Miami, Bronx, Chicago and San Diego), obtaining objective 24-h urinary sodium and potassium excretion measures. Self-report was captured from two interviewer-administered 24-h dietary recalls. Twenty percent of the sample repeated the study. We examined bias in self-reported sodium and potassium from diet and the association of mismeasurement with participant characteristics. Linear regression relating self-report with objective measures was used to develop calibration equations. Self-report underestimated sodium intake by 19.8% and 20.8% and potassium intake by 1.3% and 4.6% in men and women, respectively. Sodium intake underestimation varied by Hispanic/Latino background (P<0.05) and was associated with higher body mass index (BMI). Potassium intake underestimation was associated with higher BMI, lower restaurant score (indicating lower consumption of foods prepared away from home and/or eaten outside the home) and supplement use. The R2 was 19.7% and 25.0% for the sodium and potassium calibration models, respectively, increasing to 59.5 and 61.7% after adjusting for within-person variability in each biomarker. These calibration equations, corrected for subject-specific reporting error, have the potential to reduce bias in diet-disease associations within this largest cohort of Hispanics in the United States.

Therapeutic drug monitoring of posaconazole oral suspension in paediatric patients younger than 13 years of age: a retrospective analysis and literature review.

WHAT IS KNOWN AND OBJECTIVE: Posaconazole is an extended-spectrum triazole antifungal with activity against a variety of clinically significant yeasts and moulds. Posaconazole is not currently approved by the U.S. Food and Drug Administration for use in children younger than 13 years of age. Our primary objective was to describe the dosing and observed trough concentrations with posaconazole oral suspension in paediatric patients at the National Institutes of Health Clinical Center (Bethesda, MD). METHODS: This retrospective single-centre study reviewed paediatric patients younger than 13 years of age initiated on posaconazole oral suspension. Patients were included if they were initiated on posaconazole for prophylaxis or treatment for fungal infections from September 2006 through March 2013 with at least one trough concentration collected after at least 7 days of therapy. RESULTS AND DISCUSSION: A total of 20 male patients were included, of whom 15 (75%) had chronic granulomatous disease. The median age of patients was 6·5 years (range: 2·8-10·7). A total of 79 posaconazole trough concentrations were measured in patients receiving posaconazole as prophylaxis (n = 8) or treatment (n = 12). Posaconazole dose referenced to total body weight ranged from 10·0 to 49·2 mg/kg/day. Posaconazole trough concentrations ranged from undetectable (<50 ng/mL) up to 3620 ng/mL and were ≥500, ≥700 and ≥1250 ng/mL in 95%, 60% and 25% of patients, respectively. WHAT IS NEW AND CONCLUSIONS: Patients younger than 13 years of age had highly variable trough concentrations, and recommendations for the appropriate dosing of posaconazole oral suspension remain challenging. Until studies are conducted to determine the appropriate dosing of posaconazole in this patient population, therapeutic drug monitoring should be considered to ensure adequate posaconazole exposure.

Randomised controlled trial of alternative messages to increase enrolment in a healthy food programme among individuals with diabetes.

OBJECTIVES: We compared the effectiveness of diabetes-focused messaging strategies at increasing enrolment in a healthy food programme among adults with diabetes. METHODS: Vitality is a multifaceted wellness benefit available to members of Discovery Health, a South Africa-based health insurer. One of the largest Vitality programmes is HealthyFood (HF), an incentive-based programme designed to encourage healthier diets by providing up to 25% cashback on healthy food purchases. We randomised adults with type 2 diabetes to 1 of 5 arms: (1) control, (2) a diabetes-specific message, (3) a message with a recommendation of HF written from the perspective of a HF member with diabetes, (4) a message containing a physician's recommendation of HF, or (5) the diabetes-specific message from arm 2 paired with an 'enhanced active choice'(EAC). In an EAC, readers are asked to make an immediate choice (in this case, to enrol or not enrol); the pros and cons associated with the preferred and non-preferred options are highlighted. HF enrolment was assessed 1 month following the first emailed message. RESULTS: We randomised 3906 members. After excluding those who enrolled in HF or departed from the Vitality programme before the first intervention email, 3665 (94%) were included in a modified intent-to-treat analysis. All 4 experimental arms had significantly higher HF enrolment rates compared with control (p<0.0001 for all comparisons). When comparing experimental arms, the diabetes-specific message with the EAC had a significantly higher enrolment rate (12.6%) than the diabetes-specific message alone (7.6%, p=0.0016). CONCLUSIONS: Messages focused on diabetes were effective at increasing enrolment in a healthy food programme. The addition of a framed active choice to a message significantly raised enrolment rates in this population. These findings suggest that simple, low-cost interventions can enhance enrolment in health promoting programmes and also be pragmatically tested within those programmes. TRIAL REGISTRATION NUMBER: NCT02462057.

Biomarker-predicted sugars intake compared with self-reported measures in US Hispanics/Latinos: results from the HCHS/SOL SOLNAS study.

OBJECTIVE: Measurement error in self-reported total sugars intake may obscure associations between sugars consumption and health outcomes, and the sum of 24 h urinary sucrose and fructose may serve as a predictive biomarker of total sugars intake. DESIGN: The Study of Latinos: Nutrition & Physical Activity Assessment Study (SOLNAS) was an ancillary study to the Hispanic Community Health Study/Study of Latinos (HCHS/SOL) cohort. Doubly labelled water and 24 h urinary sucrose and fructose were used as biomarkers of energy and sugars intake, respectively. Participants' diets were assessed by up to three 24 h recalls (88 % had two or more recalls). Procedures were repeated approximately 6 months after the initial visit among a subset of ninety-six participants. SETTING: Four centres (Bronx, NY; Chicago, IL; Miami, FL; San Diego, CA) across the USA. SUBJECTS: Men and women (n 477) aged 18-74 years. RESULTS: The geometric mean of total sugars was 167·5 (95 % CI 154·4, 181·7) g/d for the biomarker-predicted and 90·6 (95 % CI 87·6, 93·6) g/d for the self-reported total sugars intake. Self-reported total sugars intake was not correlated with biomarker-predicted sugars intake (r=-0·06, P=0·20, n 450). Among the reliability sample (n 90), the reproducibility coefficient was 0·59 for biomarker-predicted and 0·20 for self-reported total sugars intake. CONCLUSIONS: Possible explanations for the lack of association between biomarker-predicted and self-reported sugars intake include measurement error in self-reported diet, high intra-individual variability in sugars intake, and/or urinary sucrose and fructose may not be a suitable proxy for total sugars intake in this study population.

Mortality among patients with pulmonary non-tuberculous mycobacteria disease.

SETTING: Tertiary referral center, National Institutes of Health (NIH), USA. OBJECTIVE: To estimate the mortality rate and its correlates among persons with pulmonary non-tuberculous mycobacteria (PNTM) disease. DESIGN: A retrospective review of 106 patients who were treated at the NIH Clinical Center and met American Thoracic Society/Infectious Diseases Society of America criteria for PNTM. Eligible patients were aged ⩾18 years and did not have cystic fibrosis or human immunodeficiency virus (HIV) infection. RESULTS: Of 106 patients followed for a median of 4.9 years, 27 (25%) died during follow-up, for a mortality rate of 4.2 per 100 person-years. The population was predominantly female (88%) and White (88%), with infrequent comorbidities. Fibrocavitary disease (adjusted hazard ratio [aHR] 3.3, 95% confidence interval [CI] 1.3-8.3) and pulmonary hypertension (aHR 2.1, 95%CI 0.9-5.1) were associated with a significantly elevated risk of mortality in survival analysis. CONCLUSIONS: PNTM remains a serious public health concern, with a consistently elevated mortality rate across multiple populations. Significant risk factors for death include fibrocavitary disease and pulmonary hypertension. Further research is needed to more specifically identify clinical and microbiologic factors that jointly influence disease outcome.

Loss of LKB1 and p53 synergizes to alter fallopian tube epithelial phenotype and high-grade serous tumorigenesis.

Liver kinase B1 (LKB1) is a tumor suppressor ubiquitously expressed serine/threonine protein kinase involved in energy metabolism and cellular polarity. In microarray experiments that compared normal tubal epithelium with high-grade serous carcinoma (HGSC), we observed a decrease in LKB1 mRNA expression in HGSC. In this study, we demonstrate that loss of cytoplasmic and nuclear LKB1 protein expression is frequently observed in tubal cancer precursor lesions as well as in both sporadic and hereditary HGSCs compared with other ovarian cancer histotypes. Bi-allelic genomic loss of LKB1 in HGSC did not account for the majority of cases with a decrease in protein expression. In vitro, shLKB1-fallopian tube epithelial (FTE) cells underwent premature cellular arrest and in ex vivo FTE culture, LKB1 loss and p53 mutant synergized to disrupt apical to basal polarity and decrease the number of ciliated cells. Overexpression of cyclin E1 allowed for bypass of LKB1-induced cellular arrest, and increased both proliferation and anchorage-independent growth of transformed FTE cells. These data suggest that LKB1 loss early in ovarian serous tumorigenesis has an integral role in tumor promotion by disrupting apical to basal polarity in the presence of mutated p53 in fallopian tube cells.

Frequency of adverse reactions to first- and second-line anti-tuberculosis chemotherapy in a Korean cohort.

OBJECTIVE: To determine the frequency of and risk factors for major adverse drug reactions (MADRs) associated with anti-tuberculosis treatment at a tuberculosis (TB) referral hospital in the Republic of Korea. METHODS: Data from an ongoing natural history cohort study were analyzed for permanent regimen changes due to adverse drug reactions and confirmed by chart review. RESULTS: Among 655 subjects, there were 132 MADRs in 112 (17%) subjects. The most common MADRs were gastrointestinal (n = 53), musculoskeletal (n = 22), psychiatric (n = 10), visual (n = 9) and peripheral neuropathic (n = 8). MADRs were more frequent in subjects being treated with second-line regimens (16%) compared to first-line regimens (2.5%). Drugs frequently associated with MADRs were amikacin (3/10, 30%), linezolid (8/29, 28%), para-aminosalicylic acid (47/192, 24%), pyrazinamide (31/528, 5.8%), macrolides (2/44, 4.5%) and cycloserine (12/272, 4.4%). Fluoroquinolones accounted for a single MADR (1/377, 0.003%), despite widespread usage. In multivariate analysis, infection with multi- or extensively drug-resistant disease and previous history of anti-tuberculosis treatment were risk factors for MADR, with adjusted hazard ratios of respectively 2.2 (P = 0.02) and 1.6 (P = 0.04). CONCLUSION: MADRs are common during anti-tuberculosis chemotherapy in this population, occurring in more than one in six subjects. New and less toxic agents to treat drug-resistant TB are urgently needed.

Differentially androgen-modulated genes in ovarian epithelial cells from BRCA mutation carriers and control patients predict ovarian cancer survival and disease progression.

Epidemiological studies have implicated androgens in the etiology and progression of epithelial ovarian cancer. We previously reported that some androgen responses were dysregulated in malignant ovarian epithelial cells relative to control, non-malignant ovarian surface epithelial (OSE) cells. Moreover, dysregulated androgen responses were observed in OSE cells derived from patients with germline BRCA-1 or -2 mutations (OSEb), which account for the majority of familial ovarian cancer predisposition, and such altered responses may be involved in ovarian carcinogenesis or progression. In the present study, gene expression profiling using cDNA microarrays identified 17 genes differentially expressed in response to continuous androgen exposure in OSEb cells and ovarian cancer cells as compared to OSE cells derived from control patients. A subset of these differentially affected genes was selected and verified by quantitative real-time reverse transcription-polymerase chain reaction. Six of the gene products mapped to the OPHID protein-protein interaction database, and five were networked within two interacting partners. Basic leucine zipper transcription factor 2 (BACH2) and acetylcholinesterase (ACHE), which were upregulated by androgen in OSEb cells relative to OSE cells, were further investigated using an ovarian cancer tissue microarray from a separate set of 149 clinical samples. Both cytoplasmic ACHE and BACH2 immunostaining were significantly increased in ovarian cancer relative to benign cases. High levels of cytoplasmic ACHE staining correlated with decreased survival, whereas nuclear BACH2 staining correlated with decreased time to disease recurrence. The finding that products of genes differentially responsive to androgen in OSEb cells may predict survival and disease progression supports a role for altered androgen effects in ovarian cancer. In addition to BACH2 and ACHE, this study highlights a set of potentially functionally related genes for further investigation in ovarian cancer.

Long-term follow-up of patients following negative colposcopy: a new gold standard and its implications for cervical screening.

From 1189 colposcopy referrals in 1997 at a single cervical screening centre, 88 women who had no biopsy taken at colposcopy (negative colposcopy) were identified. We followed up these women for a maximum of 4 years and calculated the positive predictive value (PPV) of a single smear before and after follow-up. Using slide review we attempted to correlate the grade of smear leading to colposcopy referral with final outcome. Our results showed that long-term follow-up alters the PPV of cervical cytology. Analysis showed a strong correlation between the review grade of the referring smear and the final outcome after follow-up. From these results we suggest an evidence-based protocol for cervical screening follow-up after negative colposcopy.

An alternate model of thyroid cytology: practical not perfect.

A sample of 384 thyroid cytology specimens prepared by cytospin over a 2.5-year period was classified by original report into inadequate, non-neoplastic and suspicious of neoplasia or worse. This was then compared with subsequent histology. The resulting data showed an inadequacy rate of 33%, a sensitivity of 55%, a specificity of 59%, a positive predictive value of 64% and a negative predictive value of 93%. On review of the cytology, in knowledge of the subsequent histology, the maximum achievable results were determined to have a positive predictive value of 79% and a negative predictive value of 97%. No clinically significant adverse event was detected.

Histopathologic features of genetically determined ovarian cancer.

Inheritance of germline mutations of BRCA1 or BRCA2 genes account for approximately 10% of ovarian carcinomas, but the characterization of these genetically determined cancers is incomplete. The objective of our study was to characterize the histologic features of ovarian carcinomas associated with germline mutations of BRCA1 and BRCA2. Thirty-two ovarian carcinomas associated with germline BRCA1 or BRCA2 mutations and 40 ovarian carcinomas from patients screened as negative for germline mutations were obtained from three centers. A gynecologic pathologist, blinded to mutation status, reviewed each case, with documentation of the histologic type, Gynecologic Oncology Group (GOG) grade, architectural and nuclear grade, Silverberg grade, and mitotic activity. All BRCA1 and BRCA2 mutation-associated cases were invasive serous carcinomas, and of these 50% were GOG grade 3, 41% had an architectural grade of 3 (predominant solid architecture), 84% a nuclear grade of 3, 72% a mitotic score of 3 (>25 mitoses per 10 HPF), and 75% a Silverberg grade of 3. The differences in histologic type (p = 0.001) and Silverberg grade (p = 0.002) between these tumors and the control group were statistically significant and remained so when comparisons between BRCA carriers and noncarriers were restricted to carcinomas of serous histology alone. Ovarian carcinomas associated with germline mutations of BRCA1/BRCA2 are, in this study, invasive serous carcinomas, with a statistically significant higher histologic grade than ovarian carcinomas without BRCA mutations when using the recently proposed Silverberg grading system.

Activation of androgen receptor-associated protein 70 (ARA70) mRNA expression in ovarian cancer.

OBJECTIVE: Androgens have been implicated in ovarian cancer and androgen receptor expression has been reported in 70-95% of ovarian adenocarcinomas, implying a role in ovarian cancer cell biology. Androgen receptor-associated protein 70 (ARA70) is a reported androgen receptor coactivator that enhances the transactivational potential of the androgen receptor up to 10-fold. Because ARA70 expression could amplify androgen action in ovarian cancer cells, we examined patient samples of ovarian cancer for ARA70 expression. METHODS: Twenty invasive ovarian carcinomas and four nonmalignant ovaries were tested for ARA70 mRNA expression by in situ hybridization using a 35S-labeled riboprobe. RESULTS: The probe was first assessed using a sample of human benign prostatic hyperplasia. Expression was restricted to cells within the epithelial glands, which are known to express the highest levels of androgen receptor. In the nonmalignant ovary, ARA70 mRNA was expressed in moderate levels in thecal cells associated with antral follicles, with less labeling observed in granulosa cells and stroma. The surface epithelium was negative for ARA70 transcripts, with only low levels observed in occasional cells. In contrast, a high level of ARA70 expression was observed in 17 of the 20 ovarian carcinomas of various histological types. Labeling was associated with the tumor cells while little if any ARA70 mRNA was observed in stromal cells associated with the carcinoma. CONCLUSION: These observations indicate that ARA70 expression is activated in invasive ovarian cancer tumor cells, and suggest that amplification of androgen action by ARA70 may be involved in the etiology/progression of this disease.

Sympathetic and parasympathetic regulation of cystatin S gene expression.

The autonomic nervous system plays a regulatory role in the differentiation and growth of salivary glands, and in the expression of salivary specific genes. Cystatin S, a member of the evolutionarily conserved family 2 of the cysteine proteinase inhibitor superfamily, expressed in submandibular and parotid glands of rats during development, can be induced in adults by the beta-adrenergic agonist isopreterenol (IPR). It was shown previously that unilateral sympathectomy or bilateral parasympathectomy reduces IPR-induced cystatin S expression. The present experiments demonstrate that IPR-induced cystatin S gene expression in submandibular glands is reduced as early as 3 days post bilateral denervation of both branches of the autonomic nervous system. The reduction is nearly equal to that of either sympathectomy or parasympathectomy alone, suggesting that factor(s) in both sympathetic and parasympathetic fibers are simultaneously required for IPR-induced cystatin S gene expression.

Autonomic regulation of cystatin S gene expression in rat submandibular glands.

Innervation of rat submandibular and parotid glands by the autonomic nervous system regulates saliva volume, its rate of secretion and its composition. The autonomic nervous system also plays a regulatory role in the differentiation and growth of salivary glands, and in the expression of specific sets of genes. Rat cystatin S, a member of family 2 of the cysteine proteinase inhibitor superfamily, is expressed in submandibular and parotid glands of human and rat. In the rat, cystatin S gene expression is tissue- and cell type-specific, is temporally regulated during postnatal development, and not observed in adult animals. The beta-adrenergic agonist isoproterenol (IPR) induces hypertrophic and hyperplastic enlargements of rat salivary glands and the expression of a number of genes including cystatin S. Sympathectomy reduces, but does not completely block, IPR-induced expression of the cystatin S gene in submandibular glands of adult female rats, indicating the participation of sympathetic factor(s) in its regulation. Bilateral parasympathectomy also reduces IPR-induced cystatin S gene expression, suggesting a role of the parasympathetic nervous system in its regulation. Experiments described in this paper suggest that similar factor(s) arising from both the sympathetic and parasympathetic branches of the autonomic nervous system simultaneously participate in IPR-induced cystatin S gene expression in submandibular glands.

Cervical disease in women referred to colposcopy following inadequate smears.

The aim of this audit was to determine if inadequate cervical smears are associated with significant cervical pathology. Case records for 52 women with three consecutive inadequate smears referred for colposcopy to the Leicester Royal Infirmary (LRI) were retrieved. Sixteen women underwent large loop excision of the transformation zone (LLETZ) and cervical intraepithelial neoplasia (CIN) was identified in six cases. There were no cases of inadequate smears initiating the diagnosis in 100 consecutive women with invasive cervical cancer. Inadequate smears are associated with high rates of treatment for a low yield of CIN. To reduce morbidity associated with colposcopy it may be acceptable to repeat an inadequate smear after 6 months rather than arranging immediate recall.

Accumulation of p53 protein is frequent in ovarian cancers associated with BRCA1 and BRCA2 germline mutations.

BACKGROUND: Mutations in the BRCA1 or BRCA2 genes are responsible for up to 95% of hereditary ovarian cancer cases. Both genes function as tumour suppressor genes, and development of a cancer is thought to require an accumulation of somatic genetic events in addition to the inherited germline predisposition. It is unknown whether these somatic events in BRCA associated ovarian cancer are similar to or distinct from those in sporadic cases. The most frequent somatic genetic event in ovarian cancer is a mutation of the p53 gene. AIM: To study the role of p53 in hereditary ovarian cancer, by analysing accumulation of the p53 protein in ovarian cancers which occurred in BRCA1 or BRCA2 germline mutation carriers and comparing the results with a panel of ovarian cancers from patients who tested negative for both BRCA1 and BRCA2. METHODS: The study group consisted of 39 ovarian cancer patients in whom a BRCA mutation had been confirmed previously. p53 Immunohistochemistry was performed on archival tissue using a standard microwave antigen retrieval technique. The rate of p53 accumulation was compared with 40 ovarian cancer cases who tested negative for BRCA1 and BRCA2 germline mutations. RESULTS: P53 Accumulation was similar in BRCA related ovarian cancers and BRCA negative controls. Overall 27 of 39 BRCA1 or BRCA2 positive cases (69%) had evidence of p53 accumulation, compared with 24 of 40 invasive ovarian cancer cases (60%) which tested negative for BRCA1 and BRCA2 germline mutations. BRCA1 related ovarian cancers showed p53 accumulation in 22 of 30 cases (73%); p53 accumulation was present in five of nine BRCA2 related ovarian cancers. CONCLUSIONS: In addition to germline BRCA1 and BRCA2 mutations, somatic p53 alterations leading to p53 accumulation are an important event in hereditary ovarian cancer and are as frequent as in non-BRCA-related ovarian cancer.

Activation of SPARC expression in reactive stroma associated with human epithelial ovarian cancer.

OBJECTIVE: SPARC (secreted protein, acidic, rich in cysteine) is a calcium-binding counteradhesive glycoprotein that has the potential to play an important role in promoting tumor progression and invasiveness. SPARC has been reported to be markedly down-regulated in ovarian carcinomas relative to the normal surface epithelium and has been suggested to act as a tumor suppressor in ovarian cancer. To more precisely define potential changes in SPARC expression associated with malignant transformation of the ovary, we compared the distribution of SPARC mRNA and protein expression in patient specimens of malignant and nonmalignant ovaries. METHOD: SPARC mRNA and protein expression was examined in 24 human invasive ovarian cancers, 5 tumors of low malignant potential (LMP), and 8 nonmalignant ovaries by in situ hybridization and immunohistochemistry. RESULTS: In nonmalignant ovaries, SPARC mRNA expression was restricted to thecal and granulosa cells of vessiculated follicles. Cytoplasmic SPARC immunoreactivity was observed in these compartments, whereas variable SPARC immunostaining was observed in normal surface epithelial cells. In contrast, high-level expression of SPARC mRNA and protein was detected in stroma of ovaries containing malignant tumor cells, particularly at the tumor-stromal interface of the invading tumors. Lower levels and a more diffuse pattern of SPARC mRNA expression were associated with LMP specimens. SPARC mRNA was not expressed by ovarian adenocarcinoma or by surface epithelial cells. Consistent with the in situ hybridization data, SPARC immunoreactivity was found throughout the reactive stroma of specimens containing ovarian carcinoma. However, despite the lack of detectable SPARC mRNA, SPARC immunoreactivity was consistently observed within the cytoplasm of cancer cells. CONCLUSION: The pattern of SPARC expression shown in this study indicates that SPARC is up-regulated in reactive stroma associated with invasive ovarian cancer. Moreover, these results raise the possibility that SPARC secreted from the stroma is internalized by ovarian cancer cells and may exert important intracellular effects upon these cells.

The 5'-flanking sequence and regulatory elements of the cystatin S gene.

The gene encoding rat cystatin S (Cys S), a salivary gland-specific secretory protein, has CAAT and TATA boxes upstream of the inititation codon (Cox and Shaw, 1992), and contains regions that resemble those of other hormonally responsive eukaryotic genes. The 5'-flanking sequence of the rat Cys S gene has a potential CREB/AP-1 binding site (Rupp et al., 1990; Trejo et al., 1992), two potential glucocorticoid responsive elements (GREs, Drouin et al., 1989), and a possible GR/PR (glucocorticoid/progesterone) responsive element (Forman and Samuels, 1990). One of these potential GREs is adjacent to a potential AP-2 binding site, and another is typical of the glucocorticoid and progesterone receptor binding site. In this report, we have identified three regions in the 5'-flanking region of the Cys S gene that are found in salivary gland-specific genes (Ting et al., 1992) with a GT-rich region located between conserved elements II and III. Transfection experiments described in this paper suggest that a 281-bp DNA fragment from the Cys S gene promoter region with conserved elements II and III, the GT-rich region, and a possible GR/PR responsive element contains a negative regulatory element. In addition, our experiments suggest that the GT-rich region by itself is acting as a positive regulatory element.