"Low-value" glycemic outcomes among older adults with diabetes cared for by primary care nurse practitioners or physicians: A retrospective cohort study.

BACKGROUND: "Low-value" healthcare is care without benefit to patients. Overly intensive glycemic control (i.e., HgbA1C < 7 %) can cause harm to patients at high risk of hypoglycemia, particularly among older adults with co-morbidities. It is unknown whether overly intensive glycemic control differs among patients with diabetes and at high-risk of hypoglycemia cared for by primary care nurse practitioners versus physicians. OBJECTIVE: This study examined patients with diabetes at high risk of hypoglycemia receiving primary care between Jan 2010 and Jan 2012, comparing patients reassigned to nurse practitioners to those reassigned to physicians after their previous physician separated from practice in an integrated United States health system. DESIGN: This was a retrospective cohort study. Study outcomes were collected at two years after reassignment to a new primary care provider. Outcomes were predicted probabilities of HgbA1C < 7 % using two-stage residual inclusion instrumental variable models, controlling for baseline confounders. SETTING: Primary care clinics within the United States Veterans Health Administration. PARTICIPANTS: 38,543 patients with diabetes at increased risk for hypoglycemia (age ≥ 65 years with renal disease, dementia, or cognitive impairment), who had their primary care physician leave the Veterans Health Administration and who were reassigned to a new primary care provider in the following year. RESULTS: Cohort patients were on average 76 years and 99 % men. Of these, 33,700 were reassigned to physicians and 4843 to nurse practitioners. After two years with their new provider, in adjusted models, patients reassigned to nurse practitioners had a -20.4 percentage-point [95 % CI -37.9 to -2.8] lower probability of two-year HgbA1C < 7 %. CONCLUSIONS: Aligned with prior studies on care quality, rates of overly intensive glycemic control may be appropriately lower among older patients with diabetes at high-risk of hypoglycemia, cared for by nurse practitioners than physicians. TWEETABLE ABSTRACT: Primary care nurse practitioners deliver equivalent or better rates of low-value diabetes care for older patients, compared to physicians.

Potential Explanatory Factors for the Concurrent Experience of Dyspnea and Pain in Patients with COPD.

Previous research has identified unexpectedly strong associations between dyspnea and pain, but the reasons remain unclear. Ascertaining the underlying biological and psychological mechanisms might enhance the understanding of the experience of both conditions, and suggest novel treatments. We sought to elucidate whether demographic factors, disease severity, psychological symptoms and biomarkers might account for the association between pain and dyspnea in individuals with COPD. We analyzed data from 301 patients with COPD who were followed in a prospective longitudinal observational study over 2 years. Measures included self-reported dyspnea and pain, pulmonary function tests, serum levels of inflammatory cytokines, measures of physical deconditioning, and scales for depression and anxiety. Analyses involved cross-sectional and longitudinal linear regression models. Pain and dyspnea were strongly correlated cross-sectionally (r = 0.77, 95% CI 0.72-0.82) and simultaneously across time (r = 0.42, 95% CI 0.28-0.56). Accounting for any of the other health factors only slightly mitigated the associations. Symptoms of pain and dyspnea thus may be fundamentally linked in COPD, rather than being mediated by common biological, psychological, or functional factors. From the patient's perspective, pain and dyspnea may be part of the same essential experience. It is possible that treatments for one condition would improve the other.

Factors Associated With Low-Value Cancer Screenings in the Veterans Health Administration.

Importance: Most clinical practice guidelines recommend stopping cancer screenings when risks exceed benefits, yet low-value screenings persist. The Veterans Health Administration focuses on improving the value and quality of care, using a patient-centered medical home model that may affect cancer screening behavior. Objective: To understand rates and factors associated with outpatient low-value cancer screenings. Design, Setting, and Participants: This cohort study assessed the receipt of low-value cancer screening and associated factors among 5 993 010 veterans. Four measures of low-value cancer screening defined by validated recommendations of practices to avoid were constructed using administrative data. Patients with cancer screenings in 2017 at Veterans Health Administration primary care clinics were included. Excluded patients had recent symptoms or historic high-risk diagnoses that may affect test appropriateness (eg, melena preceding colonoscopy). Data were analyzed from December 23, 2019, to June 21, 2021. Exposures: Receipt of cancer screening test. Main Outcomes and Measures: Low-value screenings were defined as occurring for average-risk patients outside of guideline-recommended ages or if the 1-year mortality risk estimated using a previously validated score was at least 50%. Factors evaluated in multivariable regression models included patient, clinician, and clinic characteristics and patient-centered medical home domain performance for team-based care, access, and continuity previously developed from administrative and survey data. Results: Of 5 993 010 veterans (mean [SD] age, 63.1 [16.8] years; 5 496 976 men [91.7%]; 1 027 836 non-Hispanic Black [17.2%] and 4 539 341 non-Hispanic White [75.7%] race and ethnicity) enrolled in primary care, 903 612 of 4 647 479 men of average risk (19.4%) underwent prostate cancer screening; 299 765 of 5 770 622 patients of average risk (5.2%) underwent colorectal cancer screening; 21 930 of 469 045 women of average risk (4.7%) underwent breast cancer screening; and 65 511 of 458 086 women of average risk (14.3%) underwent cervical cancer screening. Of patients screened, low-value testing was rare for 3 cancers, with receipt of a low-value test in 633 of 21 930 of women screened for breast cancer (2.9%), 630 of 65 511 of women screened for cervical cancer (1.0%), and 6790 of 299 765 of patients screened for colorectal cancer (2.3%). However, 350 705 of 4 647 479 of screened men (7.5%) received a low-value prostate cancer test. Patient race and ethnicity, sociodemographic factors, and illness burden were significantly associated with likelihood of receipt of low-value tests among screened patients. No single patient-, clinician-, or clinic-level factor explained the receipt of a low-value test across cancer screening cohorts. Conclusions and Relevance: This large cohort study found that low-value breast, cervical, and colorectal cancer screenings were rare in the Veterans Health Administration, but more than one-third of patients screened for prostate cancer were tested outside of clinical practice guidelines. Guideline-discordant care has quality implications and is not consistently explained by associated multilevel factors.

Case report: two cases of mature oocytes found in prepubertal girls during ovarian tissue cryopreservation.

OBJECTIVE: To report two cases of mature oocytes found in prepubertal girls undergoing ovarian tissue cryopreservation (OTC). DESIGN: Case report. SETTING: Large tertiary care children's hospital and a private fertility clinic. PATIENTS: An 8-year-old prepubertal girl with ß-thalassemia and a 2-year-old girl with sickle cell disease who both underwent OTC before bone marrow transplantations. INTERVENTIONS: Laparoscopic right oophorectomy was performed in each patient. The ovarian cortical tissue was processed for slow freezing and long-term storage, and all oocytes were subsequently vitrified. MAIN OUTCOME MEASURES: Oocytes found at the time of OTC processing for fertility preservation. RESULTS: After a complete right oophorectomy, one mature metaphase II oocyte was discovered on tissue processing for OTC in each patient. Neither patient has yet returned for use of tissue or oocytes. CONCLUSIONS: To our knowledge, this is the first report of mature oocytes found during prepubertal OTC processing. These findings may indicate the need for increased research regarding prepubertal oocyte development and suggest that the technique of examining the media for both mature and immature oocytes at the time of OTC should become more widespread and perhaps recommended in prepubertal patients to optimize fertility preservation methods.

Nursing Home Residents Face Severe Functional Limitation or Death After Hospitalization for Pneumonia.

OBJECTIVES: Pneumonia is a common cause of hospitalization for nursing home residents and has increased as a cause for hospitalization during the COVID-19 pandemic. Risks of hospitalization, including significant functional decline, are important considerations when deciding whether to treat a resident in the nursing home or transfer to a hospital. Little is known about postdischarge functional status, relative to baseline, of nursing home residents hospitalized for pneumonia. We sought to determine the risk of severe functional limitation or death for nursing home residents following hospitalization for treatment of pneumonia. DESIGN: Retrospective cohort study. SETTING AND PARTICIPANTS: Participants included Medicare enrollees aged ≥65 years, hospitalized from a nursing home in the United States between 2013 and 2014 for pneumonia. METHODS: Activities of daily living (ADL), patient sociodemographics, and comorbidities were obtained from the Minimum Data Set (MDS), an assessment tool completed for all nursing home residents. MDS assessments from prior to and following hospitalization were compared to assess for functional decline. Following hospital discharge, all patients were evaluated for a composite outcome of severe disability (≥4 ADL limitations) following hospitalization or death prior to completion of a postdischarge MDS. RESULTS: In 2013 and 2014, a total of 241,804 nursing home residents were hospitalized for pneumonia, of whom 89.9% (192,736) experienced the composite outcome of severe disability or death following hospitalization for pneumonia. Although we found that prehospitalization functional and cognitive status were associated with developing the composite outcome, 53% of residents with no prehospitalization ADL limitation, and 82% with no cognitive limitation experienced the outcome. CONCLUSIONS AND IMPLICATIONS: Hospitalization for treatment of pneumonia is associated with significant risk of functional decline and death among nursing home residents, even those with minimal deficits prior to hospitalization. Nursing homes need to prepare for these outcomes in both advance care planning and in rehabilitation efforts.

Provider anticipation and experience of patient reaction when deprescribing guideline discordant inhaled corticosteroids.

INTRODUCTION: Despite evidence of possible patient harm and substantial costs, medication overuse is persistent. Patient reaction is one potential barrier to deprescribing, but little research has assessed this in specific instances of medication discontinuation. We sought to understand Veteran and provider experience when de-implementing guideline-discordant use of inhaled corticosteroids (ICS) in those with mild-to-moderate chronic obstructive pulmonary disease (COPD). METHODS: We conducted a mixed-methods analysis in a provider-randomized quality improvement project testing a proactive electronic-consultation from pulmonologists recommending ICS discontinuation when appropriate. PCPs at two Veterans Health Administration healthcare systems were included. We completed interviews with 16 unexposed providers and 6 intervention-exposed providers. We interviewed 9 patients within 3 months after their PCP proposed ICS discontinuation. We conducted inductive and deductive content analysis of qualitative data to explore an emergent theme of patient reaction. Forty-eight PCPs returned surveys (24 exposed and 24 unexposed, response rate: 35%). RESULTS: The unexposed providers anticipated their patients might resist ICS discontinuation because it seems counterintuitive to stop something that is working, patient's fear of worsening symptoms, or if the prescription was initiated by another provider. Intervention-exposed providers reported similar experiences in post-intervention interviews. Unexposed providers anticipated that patients may accept ICS discontinuation, citing tactical use of patient-centered care strategies. This was echoed by intervention-exposed providers who had successfully discontinued an ICS. Veterans reported acceding to their providers out of trust or deference to their advanced training, even after describing an ICS as a 'security blanket'. Our survey findings supported the subthemes from our interviews. Among providers who proposed discontinuation of an ICS, 76% reported that they were able to discontinue it or switch to another more appropriate medication. CONCLUSIONS: While PCPs anticipated that patients would resist discontinuing an ICS, interviews with patient and intervention-exposed PCPs along with surveys suggest that patients were receptive to this change.

Demonstration of reciprocal diurnal variation in human serum T3 and rT3 concentration demonstrated by mass spectrometric analysis and establishment of thyroid hormone reference intervals.

BACKGROUND: There has been a wide range of reference intervals proposed in previous literature for thyroid hormones due to large between-assay variability of immunoassays, as well as lack of correction for collection time. We provided the diurnal reference intervals for five thyroid hormones, namely total thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4), free triiodothyronine (FT3), and reverse T3 (rT3), measured in serum samples of healthy participants using a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method. METHODS: Couplet serum samples (a.m. and p.m.) were collected from 110 healthy females and 49 healthy males. Healthy volunteers were recruited from four participating centers between 2016 and 2018. Measurements of thyroid hormones were obtained by LC-MS/MS analysis. RESULTS: Our study revealed significant uptrend in AM to PM FT4 (p < 0.0001) samples, downtrend in AM to PM TT3 (p = 0.0004) and FT3 samples (p < 0.0001), and AM to PM uptrend in rT3 samples (p < 0.0001). No difference was observed for TT4 between AM and PM. No significant sex differences were seen for any of the five thyroid hormones. CONCLUSION: When diagnosing thyroid disorders, it is important to have accurate measurement of thyroid hormones, and to acknowledge the diurnal fluctuation found, especially for FT3. Our study highlights the importance of standardization of collection times and implementation of LC-MS/MS in thyroid hormone measurement.

Steroid hormones and hormone antagonists regulate the neural marker neurotrimin in uterine leiomyoma.

OBJECTIVE: To characterize the role of steroid hormone and antihormone exposure on neurotrimin (NTM) expression in human leiomyoma and myometrial tissue and cells. DESIGN: Laboratory study of placebo and ulipristal acetate (UPA)-treated patient tissue. In vitro assessment of immortalized myometrial and leiomyoma cell lines after hormone and antihormone exposure. SETTING: Academic research center. PATIENT(S): Not applicable. INTERVENTIONS(S): Exposure of leiomyoma cell lines to 17ß-E2, medroxyprogesterone acetate (MPA), UPA, and fulvestrant. MAIN OUTCOME MEASURE(S): Messenger RNA expression quantified with the use of RNASeq analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Protein levels quantified by means of Western blot analysis. Immunohistochemistry (IHC) on placebo- and UPA-treated patient uterine tissue specimens. RESULT(S): Expression of NTM in human uterine leiomyoma specimens according to RNASeq was increased compared with myometrium (5.22 ± 0.57-fold), which was confirmed with the use of qRT-PCR (1.95 ± 0.05). Furthermore, NTM protein was elevated in leiomyoma tissue compared with matched myometrium (2.799 ± 0.575). IHC revealed increased staining intensity in leiomyoma surgical specimens compared with matched myometrium of placebo patients. Western blot analysis in immortalized leiomyoma cell lines demonstrated an up-regulation of NTM protein expression (2.4 ± 0.04). Treatment of leiomyoma cell lines with 17ß-E2 yielded a 1.98 ± 0.11-fold increase in NTM protein expression; however, treatment with fulvestrant showed no significant change compared with control. Leiomyoma cell lines demonstrated a 1.91 ± 0.97-fold increase in NTM protein expression after progesterone treatment. RNASeq analysis demonstrated a reduced expression in patient leiomyoma after UPA treatment (0.75 ± 0.14). Treatment of leiomyoma cells with UPA demonstrated a reduced total NTM protein amount (0.54 ± 0.31) in patients, which was confirmed with the use of IHC (UPA10 147.2 ± 9.40, UPA20 182.8 ± 8.98). In vitro studies with UPA treatment revealed a concentration-dependent effect that supported these findings. CONCLUSION(S): NTM, a neural cell adhesion molecule, is increased in leiomyoma compared with myometrium in patient tissue and in vitro models after estrogen and progesterone treatment. Down-regulation of expression occurs after UPA treatment, but not after fulvestrant exposure. CLINICAL TRIAL REGISTRATION NUMBER: NCT00290251.

NAV3, a Tumor Suppressor Gene, Is Decreased in Uterine Leiomyoma Tissue and Cells.

NAV 3 is a tumor suppressor of unknown function in leiomyomas. The objective of this study is to assess NAV3 expression and its potential role in human uterine leiomyomas. NAV3 protein expression was examined in patient leiomyoma and patient-matched myometrial tissue samples by Western blot and immunohistochemistry. NAV3 mRNA and protein expression was assessed in leuprolide acetate- and cetrorelix-treated cell line leiomyoma samples. RNAseq analysis of placebo-treated leiomyoma compared with myometrium demonstrated the presence of transcripts encoding for several neuronal proteins. For NAV3, RNA sequence analysis demonstrated decreased expression in leiomyoma as compared with myometrium (0.86 ± 0.03 fold). Presence of NAV3 mRNA was also decreased in leiomyoma surgical samples (0.43 fold ± 0.05, p = 0.026) compared with patient-matched myometrium. Confirmatory qRT-PCR results on immortalized leiomyoma and myometrial cell lines similarly demonstrated a decrease in expression of NAV3 in leiomyomas (0.28 ± 0.02, p = 0.00075). Immunohistochemical analysis demonstrated a significant decrease in NAV 3 protein in leiomyomas (H-score 154.7 ± 6.2) as compared with myometrium (H-score; 312.5 ± 14.7, p < 0.0001). Leuprolide acetate-treated leiomyoma cells demonstrated an increase in NAV 3 mRNA expression (1.53 ± 0.13, p < 0.0001). Similarly, Western blot analysis on leuprolide-treated leiomyoma cells showed a non-significant increase in NAV 3 protein expression (1.26 ± 0.09, p = 0.063). NAV 3, a tumor suppressor in numerous cancers, is decreased in leiomyoma cells and tissue compared with myometrium, and increased by GnRH analog treatment, suggesting that NAV3 may mediate steroid hormone-independent leiomyoma regulation by GnRH analogs.

Cost-related delay in filling prescriptions and health care ratings among medicare advantage recipients.

Despite higher health care needs, older adults often have limited and fixed income. Approximately a quarter of them report not filling or delaying prescription medications due to cost (cost-related prescription delay, CRPD). To ascertain the association between CRPD and satisfaction with health care, secondary analysis of the 2012 Consumer Assessment of Healthcare Providers and Systems (CAHPS) Medicare Advantage Survey was performed.Regression models quantified the association between CRPD and rating of personal doctor, specialist, and overall health care. Models were adjusted for demographic, health-related, and socioeconomic characteristics. 274,996 Medicare Advantage enrollees were mailed the CAHPS survey, of which 101,910 (36.8%) returned a survey that had responses to all the items we analyzed. CRPD was assessed by self-report of delay in filling prescriptions due to cost. Health care ratings were on a 0-10 scale. A score ≤ 5 was considered a poor rating of care.In unadjusted models, CRPD more than doubled the relative risk (RR) for poor ratings of personal doctor (RR 2.34), specialist (RR 2.14), and overall health care (RR 2.40). Adjusting for demographics and health status slightly reduced the RRs to 1.9, but adjusting for low-income subsidy and lack of insurance for medications did not make a difference.CRPD is independently associated with poor ratings of medical care, regardless of health, financial or insurance status. Providers might reduce patients' financial stress and improve patient satisfaction by explicitly discussing prescription cost and incorporating patient priorities when recommending treatments.

Ulipristal acetate decreases active TGF-ß3 and its canonical signaling in uterine leiomyoma via two novel mechanisms.

OBJECTIVE: To characterize the effect of ulipristal acetate (UPA) treatment on transforming growth factor (TGF) canonical and noncanonical signaling pathways in uterine leiomyoma tissue and cells. UPA decreased extracellular matrix in surgical specimens; we characterize the mechanism in this study. DESIGN: Laboratory study. SETTING: University. INTERVENTION(S): Exposure of leiomyoma cell lines to UPA. MAIN OUTCOME MEASURE(S): RNAseq was performed on matched myometrium and leiomyoma surgical specimens of placebo- and UPA-treated patients. Changes in gene expression and protein were measured using quantitative polymerase chain reaction and western immunoblot analysis, respectively. RESULT(S): In surgical specimen, mRNA for TGF-ß3 was elevated 3.75-fold and TGFR2 was decreased 0.50-fold in placebo leiomyomas compared with myometrium. Analysis of leiomyomas from UPA-treated women by western blot revealed significant reductions of active TGF-ß3 (0.64 ± 0.12-fold), p-TGFR2 (0.56 ± 0.23-fold), pSmad 2 (0.54 ± 0.04-fold), and pSmad 3 (0.65 ± 0.09-fold) compared with untreated leiomyomas. UPA treatment demonstrated statistically significant reduction in collagen 1, fibronectin, and versican proteins. Notably, there was a statistically significant increase of the extracellular matrix protein fibrillin in leiomyoma treated with UPA (1.48 ± 0.41-fold). Data from in vitro assays with physiologic concentrations of UPA supported the in vivo findings. CONCLUSION(S): TGF-ß pathway is highly up-regulated in leiomyoma and is directly responsible for development of the fibrotic phenotype. UPA attenuates this pathway by reducing TGF-ß3 message and protein expression, resulting in a reduction in TGF-ß canonical signaling. In addition, UPA significantly increased fibrillin protein expression, which can serve to bind inactive TGF-ß complexes. Therefore, UPA inhibits leiomyoma fibrosis by decreasing active TGF-ß3 and diminishing signaling through the canonical pathway. CLINICAL TRIAL REGISTRATION NUMBER: NCT00290251.

Evaluation of the cost-effectiveness of ovulation suppression with progestins compared with GnRH analogs in assisted reproduction cycles.

RESEARCH QUESTION: Is ovulation suppression with progestins, requiring a freeze-all approach and subsequent frozen embryo transfer resulting from progestenic endometrial changes, cost-effective compared with gonadotropin releasing hormone analogues (GnRH) during assisted reproduction cycles. DESIGN: Cost-effectiveness analysis derived from a PubMed literature search of average US costs of GnRH agonist and antagonist IVF cycles. RESULTS: In all fresh IVF cycle models, progestin cycles were more expensive owing to the additional costs of increased gonadotropin use, embryo freezing and subsequent frozen embryo transfer (FET). The average cost per live birth with progestins ($32,466-$56,194) was higher than fresh IVF cycles with short (flare) GnRH agonist ($4,447-$12,797 higher) and GnRH antagonist ($1,542-$9,893 higher). When analyzing an initial embryo transfer plus additional FET in patients not initially pregnant, progestin cycles were still more expensive per live birth compared with conventional protocols. When planned freeze only cycles were analyzed, progestins became more cost-effective per live birth compared with antagonist cycles ($2,079 lower) but remained more expensive than short agonist cycles ($823 more expensive). CONCLUSIONS: Ovulation inhibition in IVF using progestins requires a freeze-only approach of embryos, and thus progestin use was not cost-effective compared with fresh embryo transfer cycles. Progestins, however, may be cost-effective compared with GnRH antagonist in planned freeze only cycles such as in preimplantation genetic testing or fertility preservation.

Promoting Health Equity through De-Implementation Research.

Ensuring equitable access to quality health care historically has focused on gaps in care, where patients fail to receive the high-value care that will benefit them, something termed underuse. But providing high-quality health care sometimes requires reducing low-value care that delivers no benefit or where known harms outweigh expected benefits. These situations represent health care overuse. The process involved in reducing low-value care is known as de-implementation. In this article, we argue that de-implementation is critical for advancing equity for several reasons. First, medical overuse is associated with patient race, ethnicity, and socioeconomic status. In some cases, the result is even double jeopardy, where racial and ethnic minorities are at higher risk of both overuse and underuse. In these cases, more traditional efforts focused exclusively on underuse ignore half of the problem. Second, overuse of preventive care and screening is often greater for more socioeconomically advantaged patients. Within insured populations, this means more socioeconomically disadvantaged patients subsidize overuse. Finally, racial and ethnic minorities may have different experiences of overuse than Whites in the United States. This may make efforts to de-implement overuse particularly fraught. We therefore provide several actions for closing current research gaps, including: adding subgroup analyses in studies of medical overuse; specifying and measuring potential mechanisms related to equity (eg, double jeopardy vs thermostat models of overuse); and testing de-implementation strategies that may mitigate bias.

Defining thresholds for abnormal premature progesterone levels during ovarian stimulation for assisted reproduction technologies.

OBJECTIVE: To evaluate methodologies to establish abnormal progesterone (P) levels on the day of trigger for recommending freeze only cycles. DESIGN: Threshold analysis and cost analysis. SETTING: Private ART practice. PATIENT(S): Fresh autologous ART. INTERVENTIONS(S): None. MAIN OUTCOME MEASURE(S): Live birth. RESULT(S): Fourteen established statistical methodologies for generating clinical thresholds were evaluated. These methods were applied to 7,608 fresh ART transfer cycles to generate various P thresholds which ranged widely from 0.4 to 3.0 ng/mL. Lower thresholds ranged from 0.4 to 1 ng/mL and classified the majority of cycles as abnormal as well as required very large number needed to treat (NNT) to increase one live birth. Frozen embryo transfer was cost-effective when P was ≥1.5 ng/mL, with 12% of the population having an abnormal test result and an NNT of 13. Statistical and cost-effective thresholds clustered between 1.5 and 2.0 ng/mL. CONCLUSION(S): Statistically significant thresholds for P were demonstrated as low as 0.4 ng/mL but resulted in a very large NNT to increase one live birth. A clinical benefit to a freeze-only approach was demonstrated above P thresholds ranging from 1.5 to 2.0 ng/dL. At these thresholds, elevated P has a demonstrable and clinically significant negative effect and captures a smaller percentage of the patient population at higher risk for fresh transfer failure, thus making freeze-only a cost-effective option.

Oocyte cryopreservation for women with GATA2 deficiency.

PURPOSE: To describe controlled ovarian stimulation (COS) in a population of women with GATA2 deficiency, a genetic bone marrow failure syndrome, prior to allogeneic hematopoietic stem cell transplant METHODS: This is a retrospective case series of nine women with GATA2 deficiency who underwent oocyte preservation at a research institution. Main outcomes measured include baseline fertility characteristics ((antimullerian hormone (AMH) and day 3 follicle-stimulating hormone (FSH) and estradiol (E2)) and total doses of FSH and human menopausal gonadotropins (HMG), E2 on day of trigger, and total number of metaphase II oocytes retrieved. RESULTS: The mean age was 24 years [16-32], mean AMH was 5.2 ng/mL [0.7-10], and day 3 mean FSH was 5.1 U/L [0.7-8.1], and E2 was 31.5 pg/mL [< 5-45]. The mean dose of FSH was 1774 IU [675-4035], and HMG was 1412 IU [375-2925] with a mean E2 of 2267 pg/mL [60.7-4030] on day of trigger. The mean total of metaphase II oocytes was 7.7 [0-15]. One patient was diagnosed with a deep vein thrombosis (DVT) with pulmonary embolism (PE) during COS. CONCLUSION: This study is the first to analyze the outcomes of COS in women with GATA2 deficiency. The response to ovarian stimulation suggests that oocyte cryopreservation should be considered prior to gonadotoxic therapy. However, due to the risk of potentially life-threatening complications, it is prudent that patients are properly counseled of the risks and are evaluated by a multi-disciplinary medical team prior to COS.

Multiple A2E treatments lead to melanization of rod outer segment-challenged ARPE-19 cells.

PURPOSE: Daily phagocytosis of outer segments (OSs) and retinoid recycling by the RPE lead to the accumulation of storage bodies in the RPE containing autofluorescent lipofuscin, which consists of lipids and bisretinoids such as A2E and its oxidation products. Accumulation of A2E and its oxidation products is implicated in the pathogenesis of several retinal degenerative diseases. However, A2E accumulates in the RPE during normal aging. In this study, we used a cell model to determine the homeostatic mechanisms of RPE cells in response to A2E accumulation. METHODS: To distinguish between pathologic and normal responses of the RPE to A2E accumulation, we treated established ARPE-19 cells (cultured for 3 weeks after reaching confluence) with low micromolar amounts of A2E for several weeks. We compared the lysosomal function, lysosomal pH, degree of OS digestion, and melanization of the treated cells to untreated control cells in response to a challenge of purified rod OSs (ROSs). A2E was analyzed with high-performance liquid chromatography (HPLC); and A2E and melanin were identified with mass spectrometry. RESULTS: We found that post-confluent ARPE-19 cells took up and accumulated A2E under dim light conditions. Spectral analysis of the HPLC separations and mass spectrometry showed that A2E-fed cells contained A2E and oxidized A2E (furan-A2E). A2E accumulation led to a modest increase (up to 0.25 unit) in lysosomal pH in these cells. The specific activity of cathepsin D and lysosomal acid phosphatase was reduced in the A2E-treated cells, but ROS degradation was not impaired. We found that, upon challenge with ROSs, melanin pigment was induced in the lysosomal fraction of the A2E-treated ARPE-19 cells. Thus, the ARPE-19 cells responded to the A2E treatment and ROS challenge by producing a melanin-containing lysosome fraction. We speculate that this prevents them from becoming impaired in OS processing. CONCLUSIONS: We used a modified ARPE-19 cell model in which melanization was elicited as a response to chronic accumulation of A2E. We found that although A2E treatment led, as has been previously reported, to modest lysosomal alkalinization and lysosomal impairment of ARPE-19 cells, a potential homeostatic mechanism may involve production of a special type of lysosomes containing melanin.

Use-dependent dendritic regrowth is limited after unilateral controlled cortical impact to the forelimb sensorimotor cortex.

Compensatory neural plasticity occurs in both hemispheres following unilateral cortical damage incurred by seizures, stroke, and focal lesions. Plasticity is thought to play a role in recovery of function, and is important for the utility of rehabilitation strategies. Such effects have not been well described in models of traumatic brain injury (TBI). We examined changes in immunoreactivity for neural structural and plasticity-relevant proteins in the area surrounding a controlled cortical impact (CCI) to the forelimb sensorimotor cortex (FL-SMC), and in the contralateral homotopic cortex over time (3-28 days). CCI resulted in considerable motor deficits in the forelimb contralateral to injury, and increased reliance on the ipsilateral forelimb. The density of dendritic processes, visualized with immunostaining for microtubule-associated protein-2 (MAP-2), were bilaterally decreased at all time points. Synaptophysin (SYN) immunoreactivity increased transiently in the injured hemisphere, but this reflected an atypical labeling pattern, and it was unchanged in the contralateral hemisphere compared to uninjured controls. The lack of compensatory neuronal structural plasticity in the contralateral homotopic cortex, despite behavioral asymmetries, is in contrast to previous findings in stroke models. In the cortex surrounding the injury (but not the contralateral cortex), decreases in dendrites were accompanied by neurodegeneration, as indicated by Fluoro-Jade B (FJB) staining, and increased expression of the growth-inhibitory protein Nogo-A. These studies indicate that, following unilateral CCI, the cortex undergoes neuronal structural degradation in both hemispheres out to 28 days post-injury, which may be indicative of compromised compensatory plasticity. This is likely to be an important consideration in designing therapeutic strategies aimed at enhancing plasticity following TBI.

Aromatic lipophilic spin traps effectively inhibit RPE65 isomerohydrolase activity.

We previously showed that RPE65 does not specifically produce 11-cis-retinol only but also 13-cis-retinol, supporting a carbocation or radical cation mechanism of isomerization. The intrinsic properties of conjugated polyene chains result in facile formation of radical cations in oxidative conditions. We hypothesized that such radical intermediates, if involved in the mechanism of RPE65, could be stabilized by spin traps. We tested a variety of hydrophilic and lipophilic spin traps for their ability to inhibit RPE65 isomerohydrolase activity. We found that the aromatic lipophilic spin traps such as N-tert-butyl-α-phenylnitrone (PBN), 2,2-dimethyl-4-phenyl-2H-imidazole-1-oxide (DMPIO), and nitrosobenzene (NB) strongly inhibit RPE65 isomerohydrolase activity in vitro.

Proteomic analyses of corneal tissue subjected to alkali exposure.

PURPOSE: To determine whether exposure to alkaline chemicals results in predictable changes in corneal protein profile. To determine whether protein profile changes are indicative of severity and duration of alkali exposure. METHODS: Enucleated bovine and porcine (n = 59 each) eyes were used for exposure to sodium, ammonium, and calcium hydroxide, respectively. Eyes were subjected to fluorescein staining, 5-bromo-2'-deoxy-uridine (BrdU) labeling. Excised cornea was subjected to protein extraction, spectrophotometric determination of protein amount, dynamic light scattering and SDS-PAGE profiling, mass spectrometric protein identification, and iTRAQ-labeled quantification. Select identified proteins were subjected to Western blot and immunohistochemical analyses. RESULTS: Alkali exposure resulted in lower protein extractability from corneal tissue. Elevated aggregate formation was found with strong alkali exposure (sodium hydroxide>ammonium, calcium hydroxide), even with a short duration of exposure compared with controls. The protein yield after exposure varied as a function of postexposure time. Protein profiles changed because of alkali exposure. Concentration and strength of the alkali affected the profile change significantly. Mass spectrometry identified 15 proteins from different bands with relative quantification. Plexin D1 was identified for the first time in the cornea at a protein level that was further confirmed by Western blot and immunohistochemical analyses. CONCLUSIONS: Exposure to alkaline chemicals results in predictable and reproducible changes in corneal protein profile. Stronger alkali, longer durations, or both, of exposure resulted in lower yields and significant protein profile changes compared with controls.