Brush cells in the human fetal trachea

We performed a morphologic examination of human fetal lung tissue, using scanning and transmission electron microscopy, in order to establish the presence of brush cells in extrapulmonary and intrapulmonary airways, and developing acinar epithelium. Brush cells, characterized by a border of regular straight microvilli containing a filamentous core, were observed within the tracheal epithelium of a 19-20 week gestational age fetus. These cells constituted 0.5% of the total epithelial cell population. Brush cells were not seen within the bronchial, bronchiolar or developing acinar epithelium. Our study shows that brush cells occur infrequently but normally in the developing tracheal epithelium of the second trimester fetus.     

Components of the phosphatidylserine endoplasmic reticulum to plasma membrane transport mechanism as targets for KRAS inhibition in pancreatic cancer

KRAS is mutated in 90% of human pancreatic ductal adenocarcinomas (PDACs). To function, KRAS must localize to the plasma membrane (PM) via a C-terminal membrane anchor that specifically engages phosphatidylserine (PtdSer). This anchor-binding specificity renders KRAS–PM localization and signaling capacity critically dependent on PM PtdSer content. We now show that the PtdSer lipid transport proteins, ORP5 and ORP8, which are essential for maintaining PM PtdSer levels and hence KRAS PM localization, are required for KRAS oncogenesis. Knockdown of either protein, separately or simultaneously, abrogated growth of KRAS-mutant but not KRAS–wild-type pancreatic cancer cell xenografts. ORP5 or ORP8 knockout also abrogated tumor growth in an immune-competent orthotopic pancreatic cancer mouse model. Analysis of human datasets revealed that all components of this PtdSer transport mechanism, including the PM-localized EFR3A-PI4KIIIα complex that generates phosphatidylinositol-4-phosphate (PI4P), and endoplasmic reticulum (ER)–localized SAC1 phosphatase that hydrolyzes counter transported PI4P, are significantly up-regulated in pancreatic tumors compared to normal tissue. Taken together, these results support targeting PI4KIIIα in KRAS-mutant cancers to deplete the PM-to-ER PI4P gradient, reducing PM PtdSer content. We therefore repurposed the US Food and Drug Administration–approved hepatitis C antiviral agent, simeprevir, as a PI4KIIIα inhibitor In a PDAC setting. Simeprevir potently mislocalized KRAS from the PM, reduced the clonogenic potential of pancreatic cancer cell lines in vitro, and abrogated the growth of KRAS-dependent tumors in vivo with enhanced efficacy when combined with MAPK and PI3K inhibitors. We conclude that the cellular ER-to-PM PtdSer transport mechanism is essential for KRAS PM localization and oncogenesis and is accessible to therapeutic intervention.

RAS proteins are small GTPases that switch between active GTP-bound and inactive GDP-bound states, regulating cell proliferation, differentiation, and apoptosis. RAS is regulated by guanine nucleotide exchange factors that promote GDP–GTP exchange, thereby activating RAS, and GTPase-activating proteins (GAPs), which stimulate intrinsic RAS GTPase activity to return it to its inactive state. Approximately 20% of human cancers express oncogenic RAS with mutations at residues 12, 13, or 61 (1), which prevent RASGAPs from stimulating GTP hydrolysis, rendering RAS constitutively active. The RAS isoforms, HRAS, NRAS, KRAS4A, and KRAS4B (hereafter referred to as KRAS), have near-identical G-domains, which are implicated in guanine nucleotide binding and effector interaction. However, they have different C termini and membrane anchors, which contribute to their differential signaling outputs (2). KRAS is the most-frequently mutated isoform in cancer and hence represents the major clinical concern, especially in pancreatic, colon, and non–small cell lung cancers (NSCLCs) in which mutant KRAS is expressed in ∼90%, ∼50%, and ∼25% of cases, respectively (3).RAS proteins must localize to the plasma membrane (PM) and organize into nanoclusters for biological activity (4–8), whereby RAS.GTP recruits its effectors to PM nanoclusters, leading to downstream pathway activation. KRAS interacts with the PM via its C-terminal membrane anchor that comprises a farnesyl-cysteine-methyl-ester and a polybasic domain (PBD) of six contiguous lysines (9–11). Together, the KRAS PBD sequence and prenyl group define a combinatorial code for lipid binding, resulting in a membrane anchor that specifically interacts with asymmetric species of phosphatidylserine (PtdSer) that contain one saturated and one desaturated acyl chain (8, 12–14). Since PtdSer binding specificity is hardwired into its anchor structure, KRAS–PM interactions are PtdSer dependent. KRAS that partitions into the cytosol following endocytosis is captured by PDEδ, which, upon interacting with ARL2, is released to the recycling endosome (RE) for forward transport back to the PM (15). Capture of KRAS by the RE is again PtdSer dependent; therefore, abrogating PtdSer delivery to the PM will reduce PM and RE PtdSer content, abrogating both KRAS PM binding and KRAS recycling back to the PM. In sum, KRAS–PM localization, nanoclustering, and signaling capacity are all exquisitely dependent on PM PtdSer levels.Previous attempts at preventing KRAS–PM localization to inhibit its function include the development of farnesyltransferase inhibitors (FTIs), which inhibit the first posttranslational processing step that generates the KRAS membrane anchor. FTIs were clinically unsuccessful since KRAS can alternatively be geranylgeranylated by geranylgeranyl transferase1 when cells are treated with FTIs, allowing for continued PM localization (2, 16, 17). We recently leveraged the dependence of KRAS on PM PtdSer to inhibit KRAS signaling by targeting the cellular machinery that actively maintains PM PtdSer levels (18). Genetic knockdown (KD) of ORP5 or ORP8, two lipid transporters that function at endoplasmic reticulum (ER)–PM membrane contact sites to transport PtdSer to the PM (Fig. 1), mislocalized KRAS from the PM and reduced nanoclustering of any remaining KRAS. Consequently, ORP5/8 KD decreased proliferation and anchorage-independent growth of multiple KRAS-dependent pancreatic cancer cell lines. In this study, we examine the effects of ORP5/8 genetic KD and knockout (KO) on tumor growth in vivo and provide compelling evidence that these proteins are essential for tumor maintenance in KRAS-dependent pancreatic cancer. ORP5/8 function by exchanging phosphoinositide-4-phosphate (PI4P) synthesized on the PM by PI4KIIIα for PtdSer synthesized in the ER (19, 20). We demonstrate both in vitro and in vivo that PI4KIIIα inhibitors can potently inhibit oncogenic KRAS function. One such inhibitor is simeprevir, a US Food and Drug Administration (FDA)–approved antiviral agent used for the treatment of hepatitis C, that may have potential for repurposing as a therapeutic for mutant KRAS-driven cancers.Open in a separate windowFig. 1.ORP5 and ORP8 transport PtdSer to the PM. ORP5 and ORP8 exchange ER PtdSer with PM PI4P. This is driven by a PI4P concentration gradient whereby PM PI4P levels are kept high by PI4KIIIα and low at the ER by SAC1P, which hydrolyzes PI4P. ORP, oxysterol-binding protein-related protein; PI4KIIIα, class III PI4 kinase alpha; and SAC1P, SAC1-like phosphatidylinositide phosphatase.     

Molecular epidemiology of spinocerebellar ataxias in Cuba: Insights into SCA2 founder effect in Holguin

The objective of this study was to determine the prevalence of hereditary ataxias in Cuba, with a special focus on the clinical and molecular features of SCA2. Clinical assessments were performed by neurological examinations and application of the SARA scale. Molecular analyses of genes SCA1–3, SCA6, SCA17 and DRPLA identified 753 patients with SCA and 7173 asymptomatic relatives, belonging to 200 unrelated families. 86.79% of all SCA patients were affected with SCA2. In the Holguin province, the average population prevalence of SCA2 is 40.18 × 10⁵ inhabitants, with the remarkable figure of 141.66 × 10⁵ in the Baguanos municipality. The high prevalence of the SCA2 mutation in Holguin reflects most likely a founder effect. The stabilization of the prevalence along time suggests the existence of premutated chromosomes with pure CAG, acting as reservoir for further expansions. CAG repeat length correlated inversely with age at onset, accounting for 80% of the variability. Genetic anticipation was observed in the 80% of transmissions. Repeat instability was greater in paternal transmissions whereas CAG expansions without anticipation was observed in 10.97% suggesting the effect of CAA interruptions in the CAG segment, which decrease the toxicity of the abnormal ataxin-2, and/or other protective factors.     

The risk of developing coronary artery disease or congestive heart failure, and overall mortality, in type 2 diabetic patients receiving rosiglitazone, pioglitazone, metformin, or sulfonylureas: a retrospective analysis

Oral anti-diabetic agents have been associated with adverse cardiovascular events in type 2 diabetes (DM2). We investigated the risk of coronary artery disease (CAD), congestive heart failure (CHF), and mortality using multivariable Cox models in a retrospective cohort of 20,450 DM2 patients from our electronic health record (EHR). We observed no differences in CAD risk among the agents. Metformin was associated with a reduced risk of CHF (HR 0.76, 95% CI 0.64–0.91) and mortality (HR 0.54, 95% CI 0.46–0.64) when compared to sulfonylurea. Pioglitazone was also associated with a lower risk of mortality when compared to sulfonylurea (HR 0.59, 95% CI 0.43–0.81). No other significant differences were found between the oral agents. In conclusions, our results did not identify an increased CAD risk with rosiglitazone in clinical practice. However, the results do reinforce a possible increased risk of adverse events in DM2 patients prescribed sulfonylureas.     

Clinical stage T1c prostate cancer: pathologic outcomes following radical prostatectomy in black and white men

BACKGROUND: The incidence of prostate cancer in black men is 50% to 70% higher than among age-matched white men. Black men have a twofold higher mortality rate and overall tend to have higher serum prostate-specific antigen (PSA) levels than white men. To determine whether racial differences exist in men whose prostate cancer was diagnosed based solely on an elevated serum PSA level, we compared clinical and pathologic features in black and white men undergoing radical prostatectomy (RP) for clinical stage T1c prostate cancer. METHODS: We used a prospectively collected database to identify all men undergoing RP for clinical T1c prostate cancer between July 1995 and October 2000. A total of 129 consecutive men (56 black men and 73 white men) were compared for age at diagnosis, serum PSA level, biopsy Gleason score, pathologic stage, RP specimen Gleason score, incidence of lymph node metastasis, and incidence of positive surgical margins. RESULTS: Statistically significant differences were not found by race in patients' ages, serum PSA levels, biopsy Gleason score, pathologic stage, incidence of lymph node metastases, or incidence of positive surgical margins. The RP specimen Gleason score was more heterogeneous in black men than white men (P=0.02). CONCLUSIONS: Racial differences in the incidence and mortality rate of prostate cancer are well known, but differences in the clinical and pathologic features between black and white men with prostate cancer identified solely based on an elevated serum PSA level with negative results on digital rectal examination (clinical stage T1c ) have been poorly studied. Our results suggest that men with clinical stage T1c prostate cancer have similar clinical and pathologic findings regardless of race. These results suggest that early-detection programs using serum PSA testing for prostate cancer in black men potentially can result in improvements in prostate cancer outcomes in this high-risk group.     

Scleral fixation of posterior chamber intraocular lenses using fascia lata to cover the knots

BACKGROUND AND OBJECTIVE: To describe the use of fascia lata to cover the polypropylene knots of scleral fixated posterior chamber intraocular lenses (PCIOL). PATIENTS AND METHODS: Fascia lata was used to cover the knots of scleral fixated PCIOL in 5 eyes with significant scleral thinning. Four of the 5 eyes had the PCIOL insertion and the fascia lata patching in the same setting. The fifth eye previously had scleral fixated PCIOL with late suture erosion through a partial thickness scleral flap. RESULTS: There was no suture exposure or graft thinning throughout a follow-up period of 8 to 16 months. The eyes tolerated the fascia lata well with no early or late postoperative complications. CONCLUSION: Fascia lata provides an effective means to cover the knots of scleral fixated PCIOL, especially in aphakic patients with significant scleral thinning.     

Steroid therapy for idiopathic retroperitoneal fibrosis: dose and duration

PURPOSE: Idiopathic retroperitoneal fibrosis is an uncommon disease of unknown etiology that may involve the ureters and other retroperitoneal structures. Surgical ureterolysis as well as medical treatment with steroids have been used to treat these patients. However, there is no agreement as to the dose and duration of steroid. We adopted a regimen of long-term steroid use in this prospective study. MATERIALS AND METHODS: We treated 12 patients with idiopathic retroperitoneal fibrosis with a regimen of steroids during a 10-year period. Tissue diagnosis was established by biopsy of all lesions and ureteral obstruction was managed with insertion of a nephrostomy tube or a ureteral stent. The initial dose of prednisolone was 60 mg. on alternate days for 2 months and was tapered during the following 2 months to a daily dose of 5 mg. The total duration of prednisolone use was 2 years. RESULTS: Of the 12 patients 11 who completed this treatment regimen have been followed for a duration of 26 to 132 months (median 63.1) after discontinuation of treatment. Good response in the form of relief of symptoms and regression of the mass occurred in 9 cases and there were 2 failures. In 1 case the retroperitoneal mass did not regress and surgical ureterolysis was required. In the 2nd case symptoms recurred after discontinuation of steroid and a further small dose of steroids was required. Function deteriorated in 1 of 19 functioning renal units. No steroid related serious side effects developed. CONCLUSIONS: This regimen of steroid may be used as the primary mode of treatment for the majority of patients with idiopathic retroperitoneal fibrosis with minimal complications. Patients with idiopathic retroperitoneal fibrosis should be followed periodically for the rest of their lives.     

Comparisons of nomograms and urologists' predictions in prostate cancer

When applying nomograms to a clinical setting it is essential to know how their predictions compare with clinicians'. Comparisons exist outside of the prostate cancer literature. We reviewed these comparisons and conducted 2 experiments comparing predictions of clinicians with prostate cancer nomograms. By using Medline, we searched studies from January 1966 to July 1999 that compared human predictions with nomogram predictions. Next, we conducted 2 experiments: (1) 17 urologists were presented with 10 case vignettes and asked to predict the 5-year recurrence-free probabilities for each patient; (2) case presentations of 63 prostate cancer patients (including full clinical histories with complete diagnostic data and surgical findings) were made to a group of 25 clinicians who were asked to predict organ-confined disease. We found 22 published studies comparing human experts with nomograms, greater than half (13 of 22) showed the nomogram performing above the level of the human expert. Our first experiment showed urologist modification of 165 nomogram predictions led to a decrease in prediction accuracy (c-index decreased from.67 to.55, P <.05). In our second experiment, clinician predictions of organ-confined disease were comparable to the nomogram (area under the receiver operating characteristic curve [AUC] 0.78 and 0.79, respectively). A mixed-model suggests the nomogram did not augment clinician prediction accuracy (doctor excess error 1.4%, P =.75, 95% confidence interval [CI]: -10.9% to 8.2%). Our data suggest that nomograms do not seem to diminish predictive accuracy and they may be of significant benefit in certain clinical decision making settings.