How Does Prior Experience Pay Off in Large-Scale Quality Improvement Initiatives?

INTRODUCTION: To examine the association of prior investment on the effectiveness of organizations delivering large-scale external support to improve primary care. METHODS: Mixed-methods study of 7 EvidenceNOW grantees (henceforth, Cooperatives) and their recruited practices (n = 1720). Independent Variable: Cooperatives's experience level prior to EvidenceNOW, defined as a sustained track record in delivering large-scale quality improvement (QI) to primary care practices (high, medium, or low). Dependent Variables: Implementation of external support, measured as facilitation dose; effectiveness at improving (1) clinical quality, measured as practices' performance on Aspirin, Blood Pressure, Cholesterol, and Smoking (ABCS); and (2) practice capacity, measured using the Adaptive Reserve (AR) score and Change Process Capacity Questionnaire (CPCQ). Data were analyzed using multivariable linear regressions and a qualitative inductive approach. RESULTS: Cooperatives with High (vs low) levels of prior experience with and investment in large-scale QI before EvidenceNOW recruited more geographically dispersed and diverse practices, with lower baseline ABCS performance (differences ranging from 2.8% for blood pressure to 41.5% for smoking), delivered more facilitation (mean=+20.3 hours, P = .04), and made greater improvements in practices' QI capacity (CPCQ: +2.04, P < .001) and smoking performance (+6.43%, P = .003). These Cooperatives had established networks of facilitators at the start of EvidenceNOW and leadership experienced in supporting this workforce, which explained their better recruitment, delivery of facilitation, and improvement in outcomes. DISCUSSION: Long-term investment that establishes regionwide organizations with infrastructure and experience to support primary care practices in QI is associated with more consistent delivery of facilitation support, and greater improvement in practice capacity and some clinical outcomes.

How Does Prior Experience Pay Off in Large-Scale Quality Improvement Initiatives?

INTRODUCTION: To examine the association of prior investment on the effectiveness of organizations delivering large-scale external support to improve primary care. METHODS: Mixed-methods study of 7 EvidenceNOW grantees (henceforth, Cooperatives) and their recruited practices (n = 1720). Independent Variable: Cooperatives's experience level prior to EvidenceNOW, defined as a sustained track record in delivering large-scale quality improvement (QI) to primary care practices (high, medium, or low). Dependent Variables: Implementation of external support, measured as facilitation dose; effectiveness at improving (1) clinical quality, measured as practices' performance on Aspirin, Blood Pressure, Cholesterol, and Smoking (ABCS); and (2) practice capacity, measured using the Adaptive Reserve (AR) score and Change Process Capacity Questionnaire (CPCQ). Data were analyzed using multivariable linear regressions and a qualitative inductive approach. RESULTS: Cooperatives with High (vs low) levels of prior experience with and investment in large-scale QI before EvidenceNOW recruited more geographically dispersed and diverse practices, with lower baseline ABCS performance (differences ranging from 2.8% for blood pressure to 41.5% for smoking), delivered more facilitation (mean=+20.3 hours, P = .04), and made greater improvements in practices' QI capacity (CPCQ: +2.04, P < .001) and smoking performance (+6.43%, P = .003). These Cooperatives had established networks of facilitators at the start of EvidenceNOW and leadership experienced in supporting this workforce, which explained their better recruitment, delivery of facilitation, and improvement in outcomes. DISCUSSION: Long-term investment that establishes regionwide organizations with infrastructure and experience to support primary care practices in QI is associated with more consistent delivery of facilitation support, and greater improvement in practice capacity and some clinical outcomes.

A Novel Mixed Methods Approach Combining Geospatial Mapping and Qualitative Inquiry to Identify Multilevel Policy Targets: The Focused Rapid Assessment Process (fRAP) Applied to Cancer Survivorship.

Multi-level perspectives across communities, medical systems and policy environments are needed, but few methods are available for health services researchers with limited resources. We developed a mixed method health policy approach, the focused Rapid Assessment Process (fRAP), that is designed to uncover multi-level modifiable barriers and facilitators contributing to public health issues. We illustrate with a study applying fRAP to the issue of cancer survivorship care. Through this multi-level investigation we identified two major modifiable areas impacting high-quality cancer survivorship care: 1) the importance of cancer survivorship guidelines/data, 2) the need for improved oncology-primary care relationships. This article contributes to the mixed methods literature by coupling geospatial mapping to qualitative rapid assessment to efficiently identify policy change targets.

Improving Smoking and Blood Pressure Outcomes: The Interplay Between Operational Changes and Local Context.

PURPOSE: We undertook a study to identify conditions and operational changes linked to improvements in smoking and blood pressure (BP) outcomes in primary care. METHODS: We purposively sampled and interviewed practice staff (eg, office managers, clinicians) from a subset of 104 practices participating in EvidenceNOW-a multisite cardiovascular disease prevention initiative. We calculated Clinical Quality Measure improvements, with targets of 10-point or greater absolute improvements in the proportion of patients with smoking screening and, if relevant, counseling and in the proportion of hypertensive patients with adequately controlled BP. We analyzed interview data to identify operational changes, transforming these into numeric data. We used Configurational Comparative Methods to assess the joint effects of multiple factors on outcomes. RESULTS: In clinician-owned practices, implementing a workflow to routinely screen, counsel, and connect patients to smoking cessation resources, or implementing a documentation change or a referral to a resource alone led to an improvement of at least 10 points in the smoking outcome with a moderate level of facilitation support. These patterns did not manifest in health- or hospital system-owned practices or in Federally Qualified Health Centers, however. The BP outcome improved by at least 10 points among solo practices after medical assistants were trained to take an accurate BP. Among larger, clinician-owned practices, BP outcomes improved when practices implemented a second BP measurement when the first was elevated, and when staff learned where to document this information in the electronic health record. With 50 hours or more of facilitation, BP outcomes improved among larger and health- and hospital system-owned practices that implemented these operational changes. CONCLUSIONS: There was no magic bullet for improving smoking or BP outcomes. Multiple combinations of operational changes led to improvements, but only in specific contexts of practice size and ownership, or dose of external facilitation.

Cancer Survivorship Care Roles for Primary Care Physicians.

PURPOSE: Despite a burgeoning population of cancer survivors and pending shortages of oncology services, clear definitions and systematic approaches for engaging primary care in cancer survivorship are lacking. We sought to understand how primary care clinicians perceive their role in delivering care to cancer survivors. METHODS: We conducted digitally recorded interviews with 38 clinicians in 14 primary care practices that had national reputations as workforce innovators. Interviews took place during intense case study data collection and explored clinicians' perspectives regarding their role in cancer survivorship care. We analyzed verbatim transcripts using an inductive and iterative immersion-crystallization process. RESULTS: Divergent views exist regarding primary care's role in cancer survivor care with a lack of coherence about the concept of survivorship. A few clinicians believed any follow-up care after acute cancer treatment was oncology's responsibility; however, most felt cancer survivor care was within their purview. Some primary care clinicians considered cancer survivors as a distinct population; others felt cancer survivors were like any other patient with a chronic disease. In further interpretative analysis, we discovered a deeply ingrained philosophy of whole-person care that creates a professional identity dilemma for primary care clinicians when faced with rapidly changing specialized knowledge. CONCLUSIONS: This study exposes an emerging identity crisis for primary care that goes beyond cancer survivorship care. Facilitated national conversations might help specialists and primary care develop knowledge translation platforms to support the prioritizing, integrating, and personalizing functions of primary care for patients with highly complicated issues requiring specialized knowledge.

Understanding primary care-oncology relationships within a changing healthcare environment.

BACKGROUND: Management of care transitions from primary care into and out of oncology is critical for optimal care of cancer patients and cancer survivors. There is limited understanding of existing primary care-oncology relationships within the context of the changing health care environment. METHODS: Through a comparative case study of 14 innovative primary care practices throughout the United States (U.S.), we examined relationships between primary care and oncology settings to identify attributes contributing to strengthened relationships in diverse settings. Field researchers observed practices for 10-12 days, recording fieldnotes and conducting interviews. We created a reduced dataset of all text related to primary care-oncology relationships, and collaboratively identified patterns to characterize these relationships through an inductive "immersion/crystallization" analysis process. RESULTS: Nine of the 14 practices discussed having either formal or informal primary care-oncology relationships. Nearly all formal primary care-oncology relationships were embedded within healthcare systems. The majority of private, independent practices had more informal relationships between individual primary care physicians and specific oncologists. Practices with formal relationships noted health system infrastructure that facilitates transfer of patient information and timely referrals. Practices with informal relationships described shared commitment, trust, and rapport with specific oncologists. Regardless of relationship type, challenges reported by primary care settings included lack of clarity about roles and responsibilities during cancer treatment and beyond. CONCLUSIONS: With the rapid transformation of U.S. healthcare towards system ownership of primary care practices, efforts are needed to integrate strengths of informal primary care-oncology relationships in addition to formal system driven relationships.

Does Ownership Make a Difference in Primary Care Practice?

PURPOSE: We assessed differences in structural characteristics, quality improvement processes, and cardiovascular preventive care by ownership type among 989 small to medium primary care practices. METHODS: This cross-sectional analysis used electronic health record and survey data collected between September 2015 and April 2017 as part of an evaluation of the EvidenceNOW: Advancing Heart Health in Primary Care Initiative by the Agency for Health Care Research and Quality. We compared physician-owned practices, health system or medical group practices, and Federally Qualified Health Centers (FQHC) by using 15 survey-based practice characteristic measures, 9 survey-based quality improvement process measures, and 4 electronic health record-based cardiovascular disease prevention quality measures, namely, aspirin prescription, blood pressure control, cholesterol management, and smoking cessation support (ABCS). RESULTS: Physician-owned practices were more likely to be solo (45.0% compared with 8.1%, P < .001 for health system practices and 12.8%, P = .009 for FQHCs) and less likely to have experienced a major change (eg, moved to a new location) in the last year (43.1% vs 65.4%, P = .01 and 72.1%, P = .001, respectively). FQHCs reported the highest use of quality improvement processes, followed by health system practices. ABCS performance was similar across ownership type, with the exception of smoking cessation support (51.0% for physician-owned practices vs 67.3%, P = .004 for health system practices and 69.3%, P = .004 for FQHCs). CONCLUSIONS: Primary care practice ownership was associated with differences in quality improvement process measures, with FQHCs reporting the highest use of such quality-improvement strategies. ABCS were mostly unrelated to ownership, suggesting a complex path between quality improvement strategies and outcomes.

Cancer Survivorship Care in Advanced Primary Care Practices: A Qualitative Study of Challenges and Opportunities.

Importance: Despite a decade of effort by national stakeholders to bring cancer survivorship to the forefront of primary care, there is little evidence to suggest that primary care has begun to integrate comprehensive services to manage the care of long-term cancer survivors. Objective: To explain why primary care has not begun to integrate comprehensive cancer survivorship services. Design, Setting, and Participants: Comparative case study of 12 advanced primary care practices in the United States recruited from March 2015 to February 2017. Practices were selected from a national registry of 151 workforce innovators compiled for the Robert Wood Johnson Foundation. Practices were recruited to include diversity in policy context and organizational structure. Researchers conducted 10 to 12 days of ethnographic data collection in each practice, including interviews with practice personnel and patient pathways with cancer survivors. Fieldnotes, transcripts, and practice documents were analyzed within and across cases to identify salient themes. Main Outcomes and Measures: Description of cancer survivorship care delivery in advanced patient-centered medical homes, including identification of barriers and promotional factors related to that care. Results: The 12 practices came from multiple states and policy contexts and had a mix of clinicians trained in family or internal medicine. All but 3 were recognized as National Committee on Quality Assurance level 3 patient-centered medical homes. None of the practices provided any type of comprehensive cancer survivorship services. Three interdependent explanatory factors emerged: the absence of a recognized, distinct clinical category of survivorship in primary care; a lack of actionable information to treat this patient population; and current information systems unable to support survivorship care. Conclusions and Relevance: To increase the potential for primary care transformation efforts to integrate survivorship services into routine care, survivorship must become a recognized clinical category with actionable care plans supported by a functional information system infrastructure.

A national evaluation of a dissemination and implementation initiative to enhance primary care practice capacity and improve cardiovascular disease care: the ESCALATES study protocol.

BACKGROUND: The Agency for Healthcare Research and Quality (AHRQ) launched the EvidenceNOW Initiative to rapidly disseminate and implement evidence-based cardiovascular disease (CVD) preventive care in smaller primary care practices. AHRQ funded eight grantees (seven regional Cooperatives and one independent national evaluation) to participate in EvidenceNOW. The national evaluation examines quality improvement efforts and outcomes for more than 1500 small primary care practices (restricted to those with fewer than ten physicians per clinic). Examples of external support include practice facilitation, expert consultation, performance feedback, and educational materials and activities. This paper describes the study protocol for the EvidenceNOW national evaluation, which is called Evaluating System Change to Advance Learning and Take Evidence to Scale (ESCALATES). METHODS: This prospective observational study will examine the portfolio of EvidenceNOW Cooperatives using both qualitative and quantitative data. Qualitative data include: online implementation diaries, observation and interviews at Cooperatives and practices, and systematic assessment of context from the perspective of Cooperative team members. Quantitative data include: practice-level performance on clinical quality measures (aspirin prescribing, blood pressure and cholesterol control, and smoking cessation; ABCS) collected by Cooperatives from electronic health records (EHRs); practice and practice member surveys to assess practice capacity and other organizational and structural characteristics; and systematic tracking of intervention delivery. Quantitative, qualitative, and mixed methods analyses will be conducted to examine how Cooperatives organize to provide external support to practices, to compare effectiveness of the dissemination and implementation approaches they implement, and to examine how regional variations and other organization and contextual factors influence implementation and effectiveness. DISCUSSION: ESCALATES is a national evaluation of an ambitious large-scale dissemination and implementation effort focused on transforming smaller primary care practices. Insights will help to inform the design of national health care practice extension systems aimed at supporting practice transformation efforts in the USA. CLINICAL TRIAL REGISTRATION: NCT02560428 (09/21/15).

Fshb-iCre mice are efficient and specific Cre deleters for the gonadotrope lineage.

Genetic analysis of development and function of the gonadotrope cell lineage within mouse anterior pituitary has been greatly facilitated by at least three currently available Cre strains in which Cre was either knocked into the Gnrhr locus or expressed as a transgene from Cga and Lhb promoters. However, in each case there are some limitations including CRE expression in thyrotropes within pituitary or ectopic expression outside of pituitary, for example in some populations of neurons or gonads. Hence, these Cre strains often pose problems with regard to undesirable deletion of alleles in non-gonadotrope cells, fertility and germline transmission of mutant alleles. Here, we describe generation and characterization of a new Fshb-iCre deleter strain using 4.7 kb of ovine Fshb promoter regulatory sequences driving iCre expression exclusively in the gonadotrope lineage within anterior pituitary. Fshb-iCre mice develop normally, display no ectopic CRE expression in gonads and are fertile. When crossed onto a loxP recombination-mediated red to green color switch reporter mouse genetic background, in vivo CRE recombinase activity is detectable in gonadotropes at more than 95% efficiency and the GFP-tagged gonadotropes readily purified by fluorescence activated cell sorting. We demonstrate the applicability of this Fshb-iCre deleter strain in a mouse model in which Dicer is efficiently and selectively deleted in gonadotropes. We further show that loss of DICER-dependent miRNAs in gonadotropes leads to profound suppression of gonadotropins resulting in male and female infertility. Thus, Fshb-iCre mice serve as a new genetic tool to efficiently manipulate gonadotrope-specific gene expression in vivo.

Contact lens care solution killing efficacy against Acanthamoeba castellanii by in vitro testing and live-imaging.

In the past decade there has been an increased incidence of Acanthamoeba keratitis, particularly in contact lens wearers. The aim of this study was to utilize in vitro killing assays and to establish a novel, time-lapse, live-cell imaging methodology to demonstrate the efficacy of contact lens care solutions in eradicating Acanthamoeba castellanii (A. castellanii) trophozoites and cysts. Standard qualitative and quantitative in vitro assays were performed along with novel time-lapse imaging coupled with fluorescent dye staining that signals cell death. Quantitative data obtained demonstrated that 3% non-ophthalmic hydrogen peroxide demonstrated the highest percent killing at 87.4% corresponding to a 4.4 log kill. The other contact lens care solutions which showed a 72.9 to 29.2% killing which was consistent with 4.3-2.8 log reduction in trophozoite viability. Both analytical approaches revealed that polyquaternium/PHMB-based was the least efficacious in terms of trophicidal activity. The cysticidal activity of the solutions was much less than activity against trophozoites and frequently was not detected. Live-imaging provided a novel visual endpoint for characterizing the trophocidal activity of the care solutions. All solutions caused rapid rounding or pseudocyst formation of the trophozoites, reduced motility and the appearance of different morphotypes. Polyquaternium/alexidine-based and peroxide-based lens care system induced the most visible damage indicated by significant accumulation of debris from ruptured cells. Polyquaternium/PHMB-based was the least effective showing rounding of the cells but minimal death. These observations are in keeping with care solution biocides having prominent activity at the plasma membrane of Acanthamoeba.

Gonadotrope-specific expression and regulation of ovine follicle stimulating hormone Beta: transgenic and adenoviral approaches using primary murine gonadotropes.

The beta subunit of follicle stimulating hormone (FSHB) is expressed specifically in pituitary gonadotropes in vertebrates. Transgenic mouse studies have shown that enhancers in the proximal promoter between -172/-1 bp of the ovine FSHB gene are required for gonadotrope expression of ovine FSHB. These enhancers are associated with regulation by activins and gonadotropin releasing hormone (GnRH). Additional distal promoter sequence between -4741/-750 bp is also required for expression. New transgenic studies presented here focus on this distal region and narrow it to 1116 bp between -1866/-750 bp. In addition, adenoviral constructs were produced to identify these critical distal sequences using purified primary mouse gonadotropes as an in vitro model system. The adenoviral constructs contained -2871 bp, -750 bp or -232 bp of the ovine FSHB promoter. They all showed gonadotrope-specific regulation since they were induced only in purified primary gonadotropes by activin A (50 ng/ml) and inhibited by GnRH (100 nM) in the presence of activin (except -232FSHBLuc). However, basal expression of all three viral constructs (in the presence of follistatin to block cellular induction by activin) was relatively high in pituitary non-gonadotropes as well as gonadotropes. Thus, gonadotrope-specific regulation associated with the proximal promoter was observed as expected, but the model was blind to distal promoter elements between -2871/-750 necessary for gonadotrope-specific expression of ovine FSHB in vivo. The new adenoviral-based in vitro technique did detect, however, a novel GnRH response element between -750 bp and -232 bp of the ovine FSHB promoter. We conclude that adenoviral-based studies in primary gonadotropes can adequately recognize regulatory elements on the ovine FSHB promoter associated with gonadotrope-specific regulation/expression, but that more physiologically based techniques, such as transgenic studies, will be needed to identify sequences between -1866/-750 bp of the ovine FSHB promoter that are also required for tissue/cell specific expression in vivo.

Central and peripheral corneal thinning in keratoconus.

PURPOSE: Recent data regarding the mid-peripheral portion of the removed corneal button in transplant surgery indicate histopathologically that keratoconus was present also in the peripheral portion of the button. The intent of this study was therefore to investigate if peripheral changes could also be detected clinically by measuring the central and peripheral corneal thickness of KC patients. METHODS: Corneal pachymetry was measured with the Visante optical coherence tomography and Orbscan II. Of 52 subjects, 26 were KC subjects and 26 were age- and sex-matched control subjects. RESULTS: Corneal thickness, in KC patients, was significantly reduced in all areas compared with corneal thickness among controls with both the Visante optical coherence tomography and Orbscan II. CONCLUSION: The results of the present study have provided clinical evidence that KC is a disease affecting a wider area of the cornea. Although the KC cornea shows the greatest change in thickness in the ectatic region, this attenuation declines toward the periphery. The presence of peripheral thinning indicated that KC may be a "pancorneal" pathology and not limited to the ectatic portion.

Defining a functionally distinct subset of human memory CD4+ T cells that are CD25POS and FOXP3NEG.

Surface expression of the IL-2 receptor α-chain (CD25) has been used to discriminate between CD4(+) CD25(HI) FOXP3(+) regulatory T (Treg) cells and CD4(+) CD25(NEG) FOXP3(-) non-Treg cells. However, this study reports that the majority of resting human memory CD4(+) FOXP3(-) T cells expresses intermediate levels of CD25 and that CD25 expression can be used to delineate a functionally distinct memory subpopulation. The CD25(NEG) memory T-cell population contains the vast majority of late differentiated cells that respond to antigens associated with chronic immune responses and are increased in patients with systemic lupus erythematosus (SLE). In contrast, the CD25(INT) memory T cells respond to antigens associated with recall responses, produce a greater array of cytokines, and are less dependent on costimulation for effector responses due to their expression of CD25. Lastly, compared to the CD25(NEG) and Treg-cell populations, the CD25(INT) memory population is lost to a greater degree from the blood of cancer patients treated with IL-2. Collectively, these results show that in humans, a large proportion of CD4(+) memory T cells express intermediate levels of CD25, and this CD25(INT) FOXP3(-) subset is a functionally distinct memory population that is uniquely affected by IL-2.

Morphology of contact lens-induced conjunctival epithelial flaps: a pilot study.

PURPOSE: Conjunctival epithelial flap (CEF) is a conjunctival condition most commonly seen in silicone hydrogel contact lens wearers. This study utilized impression cytology to investigate the cellular composition and health of CEFs. METHODS: Nine (9) subjects were enrolled - 3 non-lens wearers, 3 contact lens wearers without CEF, and 3 contact lens wearers with CEFs wearing 8.4/13.8 lotrafilcon A lenses. Impression cytology samples were collected from the flap or similar locations, if CEF was absent, using rectangular 5 mm × 2 mm Millipore HAWP02500 membrane filters. The filters were gently pressed onto the conjunctiva, subsequently fixed in 95% alcohol, stained with hematoxylin-eosin and evaluated under an Olympus IX70 microscope. Measurements of the longest cell and nucleus dimensions were measured on 40 cells from each filter by utilizing NIH Image 1.63. RESULTS: CEF consisted of multilayers of epithelial and goblet cells and were devoid of inflammatory cells, basement membrane material and stromal tissue. The cytoplasmic and nuclear dimensions were similar within the groups and the cytoplasm-to-nucleus ratio was not different between the flap group and the non-lens wearing group. CONCLUSION: The CEF appeared to be formed by healthy epithelial and goblet cells that have been dislocated from their normal location along the conjunctival surface by the lens edge. No inflammatory cells were present in this contact lens induced condition, which is reported to be associated most commonly with the silicone hydrogel material.

Activin A induces ovine follicle stimulating hormone beta using -169/-58 bp of its promoter and a simple TATA box.

BACKGROUND: Activin A increases production of follicle stimulating hormone (FSH) by inducing transcription of its beta subunit (FSHB). This induction has been studied here in LbetaT2 gonadotropes using transient expression of ovine FSHBLuc (-4741 bp of ovine FSHB promoter plus exon/intron 1 linked to Luc). Several sequences between -169/-58 bp of the ovine FSHB proximal promoter are necessary for induction by activin A in LbetaT2 cells, but deletions between -4741/-752 bp decrease induction > 70% suggesting the existence of other important 5' sequences. Induction disappears if a minimal T81 thymidine kinase promoter replaces the ovine FSHB TATA box and 3' exon/intron. The study reported here was designed to determine if sequences outside -169/-58 bp are important for induction of ovine FSHB by activin A. METHODS: Progressively longer deletions of ovine FSHBLuc were created between -4741/-195 bp. Deletions internal to this region were created also, but replaced with substitute DNA. The ovine FSHB TATA box region (-40/+3 bp) was replaced by thymidine kinase and rat prolactin minimal promoters, and substitutions were made in 3' intron/exon sequences. All constructs were tested for basal and activin A-induced expression in LbetaT2 cells. RESULTS: Successive 5' deletions progressively lowered fold-induction by activin A from 9.5 to zero, but progressively increased basal expression. Replacing deletions with substitute DNA showed no changes in basal expression or fold-induction. Induction by activin A was supported by the minimal rat prolactin promoter (TATA box) but not the thymidine kinase promoter (no TATA box). Replacement mutations in the 3' region did not decrease induction by activin A. CONCLUSION: The data show that specific ovine FSHB sequences 5' to -175 bp or 3' of the transcription start site are not required for induction by activin A. A minimal TATA box promoter supports induction by activin A, but the sequence between the TATA box and transcription start site seems unimportant.

Expression and regulation of the beta-subunit of ovine follicle-stimulating hormone relies heavily on a promoter sequence likely to bind Smad-associated proteins.

FSH is essential for normal gonadal function in mammals. Expression of its beta-subunit (FSHB) controls overall production/secretion of FSH and is induced by activin. Studies with ovine FSHB promoter/reporter constructs in L beta T2 gonadotropes show that induction by activin requires a putative Smad binding element in the ovine FSHB promoter (-162AGAC-159). Similar studies reported here show that another site, juxtaposed to the Smad binding element, was also required for 81% activin induction in L beta T2 cells. This site was similar to several that bind proteins known to partner with Smads. When this site (-171ACTgcgtTT-163) was mutated by changing the nucleotides shown in lowercase letters, the resulting ovine-derived construct (mut-oFSHBLuc) was expressed poorly as a transgene in primary mouse gonadotropes (<0.001 times compared with ovine wild-type transgenes). This decrease in expression demonstrated the importance of this site for activin induction and, perhaps, basal expression, although studies with L beta T2 cells did not suggest this latter possibility. Expression of mut-oFSHBLuc in male mouse gonadotropes in vivo was at least 644 times greater than expression in all but one nongonadotrope tissue tested, indicating that mut-oFSHBLuc retained significant gonadotrope-specific expression. An increase in FSHB expression occurs during estrus in mice and is faithfully reproduced with wild-type ovine FSHBLuc transgenes, but not with mut-oFSHBLuc, indicating that the mutated site is needed for this secondary FSH surge. These data suggest that activin gathers Smads and Smad-associated proteins at the -171/-159 promoter region to regulate expression of the ovine FSHB and overall FSH production.

Chronic gonadotropin-releasing hormone inhibits activin induction of the ovine follicle-stimulating hormone beta-subunit: involvement of 3',5'-cyclic adenosine monophosphate response element binding protein and nitric oxide synthase type I.

FSH is induced by activin, and this expression is modulated by GnRH through FSHB expression. This report focuses on the inhibitory effect of GnRH on activin-induced FSHB expression. Activin-treated primary murine pituitary cultures robustly express mutant ovine FSHBLuc-DeltaAP1, a luciferase transgene driven by 4.7 kb of ovine FSHB promoter. This promoter lacks two GnRH-inducible activator protein-1 sites, making it easier to observe GnRH-mediated inhibition. Luciferase expression from this transgene was decreased 94% by 100 nM GnRH with a half-time of approximately 4 h in pituitary cultures, and this inhibition was independent of follistatin. Activators of cAMP and protein kinase C like forskolin and phorbol 12-myristate 3-acetate (PMA), respectively, mimicked GnRH action. Kinetic studies of wild-type ovine FSHBLuc in LbetaT2 cells showed continuous induction by activin (4-fold) over 20 h. Most of this induction (78%) was blocked, beginning at 6 h. cAMP response element binding protein (CREB) was implicated in this inhibition because overexpression of its constitutively active mutant mimicked GnRH, and its inhibitor (inducible cAMP early repressor isoform II) reversed the inhibition caused by GnRH, forskolin, or PMA. In addition, GnRH, forskolin, or PMA increased the expression of a CREB-responsive reporter gene, 6xCRE-37PRL-Luc. Inhibition of nitric oxide type I (NOSI) by 7-nitroindazole also reversed GnRH-mediated inhibition by 60%. It is known that GnRH and CREB induce production of NOSI in gonadotropes and neuronal cells, respectively. These data support the concept that chronic GnRH inhibits activin-induced ovine FSHB expression by sequential activation of CREB and NOSI through the cAMP and/or protein kinase C pathways.

Conjunctival cytokine expression in symptomatic moderate dry eye subjects.

PURPOSE: To compare ocular surface cytokine expression in healthy controls and subjects with moderate dry eye and to study the ability of interleukin (IL)-1beta to modulate cytokine expression in cultured human conjunctival epithelial cells (CECs). METHODS: Subjective (symptom questionnaire) and objective (tear osmolality, fluorescein tear break-up time [TBUT]) measures of dry eye were determined in five healthy controls and five subjects with moderate dry eye. Tear clearance rates were measured with a fluorophotometer. Enzyme immunoassay and a cytokine bead assay were used to quantify IL-1beta in tear fluid. RT-PCR was performed to detect expression of IL-1beta, IL-6, IL-8, growth-related oncogene (GRO)-beta, intercellular adhesion molecule (ICAM)-1, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and ephrin A5 in conjunctival impression cytology (CIC) samples and in CECs (IOBA-NHC cell line, n=3; primary cultured CEC, n=3) exposed to 10 ng/mL IL-1beta for 6 hours. RESULTS: Subjects with moderate dry eye had significantly higher symptom scores, higher tear osmolality, and shorter TBUT than healthy controls. Subjects with dry eye demonstrated slightly slower tear clearance (13.1% per minute) than healthy controls (15.4% per minute). Very low levels of IL-1beta protein were detected in the tear fluid of both groups. TRAIL was constitutively expressed in CIC samples, whereas IL-1beta, IL-6, and GRO-beta were absent. Weak expression of IL-8 (two healthy, four dry eye), ICAM-1 (four healthy, four dry eye), and ephrin A5 (one healthy, two dry eye) was observed. IL-1beta upregulated its own expression and that of IL-6, IL-8, GRO-beta, and ICAM-1 in cultured CECs but not that of ephrin A5 or TRAIL. CONCLUSIONS: The lack of major differences in ocular surface cytokine expression between the two groups of subjects implies other inflammatory pathways or etiologies are involved in moderate dry eye. Although IL-1beta modulated the expression of various cytokines in cultured CECs, its absence in tear fluid and CIC samples suggests that IL-1beta does not play a modulatory role in moderate dry eye.

Differential regulation of follicle stimulating hormone by activin A and TGFB1 in murine gonadotropes.

BACKGROUND: Activins stimulate the synthesis of follicle stimulating hormone (FSH) in pituitary gonadotropes, at least in part, by inducing transcription of its beta subunit (Fshb). Evidence from several laboratories studying transformed murine LbetaT2 gonadotropes indicates that activins signal through Smad-dependent and/or Smad-independent pathways, similar to those used by transforming growth factor beta-1 (TGFB1) in other cell types. Therefore, given common intracellular signaling mechanisms of these two ligands, we examined whether TGFBs can also induce transcription of Fshb in LbetaT2 cells as well as in purified primary murine gonadotropes. METHODS: Murine Fshb promoter-reporter (-1990/+1 mFshb-luc) activity was measured in LbetaT2 cells treated with activin A or TGFB1, and in cells transfected with either activin or TGFB receptors. The ability of the ligands to stimulate phosphorylation of Smads 2 and 3 in LbetaT2 cells was measured by western blot analysis, and expression of TGFB type I and II receptors was assessed by reverse transcriptase polymerase chain reaction in both LbetaT2 cells and primary gonadotropes purified from male mice of different ages. Finally, regulation of endogenous murine Fshb mRNA levels by activin A and TGFB1 in purified gonadotropes and whole pituitary cultures was measured using quantitative RT-PCR. RESULTS: Activin A dose-dependently stimulated -1990/+1 mFshb-luc activity in LbetaT2 cells, but TGFB1 had no effect at doses up to 5 nM. Similarly, activin A, but not TGFB1, stimulated Smad 2 and 3 phosphorylation in these cells. Constitutively active forms of the activin (Acvr1b-T206D) and TGFB (TGFBR1-T204D) type I receptors strongly stimulated -1990/+1 mFshb-luc activity, showing that mechanisms down stream of Tgfbr1 seem to be intact in LbetaT2 cells. RT-PCR analysis of LbetaT2 cells and whole adult murine pituitaries indicated that both expressed Tgfbr1 mRNA, but that Tgfbr2 was not detected in LbetaT2 cells. When cells were transfected with a human TGFBR2 expression construct, TGFB1 acquired the ability to significantly stimulate -1990/+1 mFshb-luc activity. In contrast to LbetaT2 cells, primary murine gonadotropes from young mice (8-10 weeks) contained low, but detectable levels of Tgfbr2 mRNA and these levels increased in older mice (1 yr). A second surprise was the finding that treatment of purified primary gonadotropes with TGFB1 decreased murine Fshb mRNA expression by 95% whereas activin A stimulated expression by 31-fold. CONCLUSION: These data indicate that TGFB1-insensitivity in LbetaT2 cells results from a deficiency in Tgfbr2 expression. In primary gonadotropes, however, expression of Tgfbr2 does occur, and its presence permits TGFB1 to inhibit Fshb transcription, whereas activin A stimulates it. These divergent actions of activin A and TGFB1 were unexpected and show that the two ligands may act through distinct pathways to cause opposing biological effects in primary murine gonadotropes.