Perceptions and Understanding of Transgender Patient Care: A Survey of Practicing Laryngologists.

OBJECTIVES: Transgender individuals face significant health disparities including deficiencies in physician education, knowledge, and comfort with transgender health care. As the prevalence of the transgender population increases more individuals may seek gender-affirming surgery. Herein, we present a survey study which presents data on (1) the current practice patterns, (2) the familiarity with, (3) the perception of, and (4) the future educational goals of transgender health care among laryngologists in the United States. METHODS: A cross-sectional survey study of practicing laryngologists in the United States. RESULTS: A total of 53 laryngologists participated in the study, with 50 (94.3%) coming from an academic practice. Survey response rate was 32.3% (54/167). The number of patients cared for and surgeries performed were significantly associated with self-perceived overall competence (p < 0.001 and p < 0.001), surgical competence (p = 0.013 and p < 0.001), and comfort counseling patients on gender-affirming surgeries (p < 0.001 and p < 0.001). Most obtained training through real-world experience (n = 46, 86.8%), whereas only 11 (20.7%) had formal training in residency or fellowship. Although 37 (70%) of participants felt competent caring for transgender patients, 38 (72%) want to learn more about transgender care, and 49 (93%) support incorporating transgender care into otolaryngology residency/fellowship curricula. CONCLUSION: There is a need for an increased awareness of transgender healthcare issues to address disparities experienced by this diverse population. Many laryngologists report wanting to learn more about this developing part of our field and support incorporating transgender care into training. We attempt to spotlight the degree by which practicing laryngologists are familiar, competent, and comfortable with transgender care. LEVEL OF EVIDENCE: 5 Laryngoscope, 134:3215-3219, 2024.

Patient Attitudes and Barriers to Endoscopy During the COVID-19 Pandemic.

BACKGROUND: After COVID-19 restrictions on nonessential procedures were lifted and safety protocols established, utilization rates of endoscopic procedures remained reduced. AIMS: This study assessed patient attitudes and barriers to scheduling endoscopy during the pandemic. METHODS: A survey was administered to patients with ordered procedures at a hospital-based setting (7/21/2020-2/19/2021) collecting demographic data, body mass index, COVID-19 relevant comorbidities, level of procedural urgency (defined by recommended scheduling window), scheduling and attendance, concerns, and awareness of safety measures. RESULTS: The average respondent was female (63.8%), age 57.6 ± 14, White (72.3%), married (76.7%), insured (99.3%), affluent English speakers (92.3%) and highly educated (at least college 90.2%). Most reported moderate to excellent COVID-19 knowledge (96.6%). Of 1039 procedures scheduled, emergent cases accounted for 5.1%, urgent 55.3% and elective 39.4%. Respondents identified appointment convenience (48.53%) as the most frequent factor impacting scheduling, also noting concern for results (28.4%). Age (p = .022), native language (p = .04), education (p = .007), self-reported COVID knowledge (p = .002), and a desire to be COVID tested pre-procedure (p = .023) were associated with arrival, more commonly in an ambulatory surgical center than hospital (p = .008). Diabetes mellitus (p = .004) and an immunocompromised state (p = .009) were adversely related to attendance. Attitudes towards safety protocols did not affect scheduling. Multivariate analysis demonstrated age, education and COVID knowledgeability were associated with procedure completion. CONCLUSIONS: Safety protocols and urgency levels were not associated with procedure completion. Pre-pandemic barriers to endoscopy persisted as dominant factors amid pandemic concerns.

Morbidity and mortality of multivisceral resection with radical nephrectomy for locally advanced renal cell carcinoma: An analysis of the National Surgical Quality Improvement Program (NSQIP) database.

INTRODUCTION: Locally advanced renal cell carcinoma (RCC) can rarely invade into adjacent abdominal viscera without clinical evidence of distant metastases. The role of multivisceral resection (MVR) of involved adjacent organs at the time of radical nephrectomy (RN) remains poorly described and quantified. Using a national database, we aimed to evaluate the association between RN+MVR and 30-day postoperative complications. METHODS AND MATERIALS: We conducted a retrospective cohort study of adult patients undergoing RN for RCC with and without MVR between 2005 and 2020 using the ACS-NSQIP database. The primary outcome was a composite of any of the following 30-day major postoperative complications: mortality, reoperation, cardiac event, and neurologic event. Secondary outcomes included individual components of the composite primary outcome, as well as infectious and venous thromboembolic complications, unplanned intubation and ventilation, transfusion, readmission, and prolonged length of stay (LOS). Groups were balanced using propensity score matching. Likelihood of complications was assessed by conditional logistic regression adjusted for unbalanced total operation time. Postoperative complications were compared by Fisher's exact test among subtypes of resection. RESULTS: A total of 12,417 patients were identified: 12,193 (98.2%) undergoing RN alone and 224 (1.8%) undergoing RN+MVR. Patients undergoing RN+MVR were more likely to experience major complications (odds ratio [OR] 2.46; 95% confidence interval [CI] 1.28-4.74). However, there was no significant association between RN+MVR and postoperative mortality (OR 2.49; 95% CI 0.89-7.01). RN+MVR was associated with higher rates of reoperation (OR 7.85; 95% CI 2.38-25.8), sepsis (OR 5.45; 95% CI 1.83-16.2), surgical site infection (OR 4.41; 95% CI 2.14-9.07), blood transfusion (OR 2.24; 95% CI 1.55-3.22), readmission (OR 1.78; 95% CI 1.11-2.84), infectious complications (OR 2.62; 95% CI 1.62-4.24), and longer hospital stay (5 days [IQR 3-8] vs. 4 days [IQR 3-7]; OR 2.31 [95% CI 2.13-3.03]).  There was no heterogeneity in the association between subtype of MVR and major complication rate. CONCLUSION: Undergoing RN+MVR is associated with an increased risk of 30-day postoperative morbidity, including infectious complications, reoperation, blood transfusion, prolonged LOS, and readmission.

Comparison of keratometry using SS-OCT and dual Scheimpflug-Placido imaging in postmyopic corneal refractive surgery eyes.

PURPOSE: To compare simulated (SimK) and total (True-K) keratometry and corneal astigmatism values between the IOLMaster 700 (IOLM) and Galilei G4 (G4) devices in postmyopic laser refractive surgery eyes. SETTING: Methodist Eye Associates, Blanton Eye Institute, Houston Methodist Hospital, Houston, Texas. DESIGN: Retrospective cohort study. METHODS: A chart review was conducted on patients with prior myopic laser-assisted in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK), undergoing phacoemulsification at a single institution from May 2019 through January 2022, who underwent imaging with both the IOLM and G4. Exclusion criteria were prior radial keratotomy, keratoectatic diseases, and inability to obtain a reliable image. Mean, flat, and steep SimK and True-K (TK from the IOLM and TCP IOL from the G4) values and astigmatism magnitude were compared. RESULTS: 50 eyes of 50 patients were included. The mean difference in SimK and True-K between devices (IOLM - G4) was -0.04 (95% CI -0.13 to 0.06; P > .05) diopters (D) and 1.14 (95% CI 1.02 to 1.25; P < .05) D, respectively. The IOLM measured steeper True-K values than the G4. There were no statistically significant differences between devices for all other SimK values, whereas for True-K there were significant differences in flat K and steep K ( P < .05), but not astigmatism magnitude. CONCLUSIONS: Despite an overall good correlation in postmyopic laser refractive surgery eyes in keratometry and astigmatism measurements, there is a significant difference in True-K, with the IOLM measuring steeper values by about 1.0 D compared with the G4, similar to prior studies on nonrefractive surgery eyes.

Pancreatic head vs pancreatic body/tail cancer: Are they different?

BACKGROUND: The impact of pancreatic tumor location on patient survival has been studied in large national data-based analyses which yielded controversial results. AIM: To explore if pancreatic head cancer (PHC) and pancreatic body/tail cancer (PBTC) have different overall survival (OS), molecular signature and response to chemotherapy. METHODS: We retrospectively queried patient records from July 2016 to June 2020 in our institution. Patient demographics, cancer stage on diagnosis, tumor location, somatic mutations, treatment, and survival are recorded and analyzed. A test is considered statistically significant if the P value was < 0.05. RESULTS: We reviewed 101 patients with complete records, among which 67 (66.34%) were PHC and 34 (33.66%) were PBTC. More PHC were diagnosed at younger age [61.49 vs 68.97, P = 0.010], earlier stages (P = 0.006) and underwent surgical resection (P = 0.025). There were no significant differences among all mutations and pathways studied except for TP53 mutations (37.0% in PHC vs 70.0% in PBTC, P = 0.03). OS was not statistically different between PHC and PBTC (P = 0.636) in the overall population and in subgroups according to surgical resection status or stages. In terms of response to chemotherapy, chemotherapy regimens (FOLFIRINOX-based vs gemcitabine-based) didn't impact disease free interval in those who had surgical resection in either PHC (P = 0.546) or PBTC (P = 0.654), or the duration of response to first line palliative treatment in those with advanced disease in PHC (P = 0.915) or PBTC (P = 0.524). CONCLUSION: Even though PHC and PBTC have similar poor OS and response to chemotherapy, the different presentations and molecular profiles indicate they are different diseases. Utilization of molecular profiling to develop targeted therapy for individualization of treatment is needed.

ARHGEF3 Regulates Skeletal Muscle Regeneration and Strength through Autophagy.

Skeletal muscle regeneration after injury is essential for maintaining muscle function throughout aging. ARHGEF3, a RhoA/B-specific GEF, negatively regulates myoblast differentiation through Akt signaling independently of its GEF activity in vitro. Here, we report ARHGEF3's role in skeletal muscle regeneration revealed by ARHGEF3-KO mice. These mice exhibit indiscernible phenotype under basal conditions. Upon acute injury, however, ARHGEF3 deficiency enhances the mass/fiber size and function of regenerating muscles in both young and regeneration-defective middle-aged mice. Surprisingly, these effects occur independently of Akt but via the GEF activity of ARHGEF3. Consistently, overexpression of ARHGEF3 inhibits muscle regeneration in a Rho-associated kinase-dependent manner. We further show that ARHGEF3 KO promotes muscle regeneration through activation of autophagy, a process that is also critical for maintaining muscle strength. Accordingly, ARHGEF3 depletion in old mice prevents muscle weakness by restoring autophagy. Taken together, our findings identify a link between ARHGEF3 and autophagy-related muscle pathophysiology.

Complications associated with L4-5 anterior retroperitoneal trans-psoas interbody fusion: a single institution series.

BACKGROUND: Lateral lumbar interbody fusion (LLIF), first described in the literature in 2006 by Ozgur et al., involves direct access to the lateral disc space via a retroperitoneal trans-psoas tubular approach. Neuromonitoring is vital during this approach since the surgical corridor traverses the psoas muscle where the lumbar plexus lies, risking injury to the lumbosacral plexus that could result in sensory or motor deficits. The risk of neurologic injury is especially higher at L4-5 due to the anatomy of the plexus at this level. Here we report our single-center clinical experience with L4-5 LLIF. METHODS: A retrospective chart review of all patients who underwent an L4-5 LLIF between May 2016 and March 2019 was performed. Baseline demographics and clinical characteristics, such as body mass index (BMI), medical comorbidities, surgical history, tobacco status, operative time and blood loss, length of stay (LOS), and post-op complications were recorded. RESULTS: A total of 220 (58% female and 42% male) cases were reviewed. The most common presenting pathology was spondylolisthesis. The average age, BMI, operative time, blood loss, and LOS were 64.6 years, 29 kg/m2, 214 min, 75 cc, and 2.5 days respectively. A review of post-operative neurologic deficits revealed 31.4% transient hip flexor weakness and 4.5% quadricep weakness on the approach side. At 3-week follow-up, 9.1% of patients experienced mild hip flexor weakness (4 or 4+/5), 0.9% reported mild quadricep weakness, and 9.5% reported anterior thigh dysesthesias; 93.2% of patients were discharged home and 2.3% were readmitted within the first 30 days post discharge. Female sex, higher BMI and longer operative time were associated with hip flexor weakness. CONCLUSIONS: LLIF at L4-5 is a safe, feasible, and versatile approach to the lumbar spine with an acceptable approach-related sensory and motor neurologic complication rates.

Lanthionine synthetase C-like protein 2 (LanCL2) is important for adipogenic differentiation.

Adipogenic differentiation is a highly regulated process that is necessary for metabolic homeostasis and nutrient sensing. The expression of PPARγ and the subsequent activation of adipogenic genes is critical for the process. In this study, we identified lanthionine synthetase C-like protein 2 (LanCL2) as a positive regulator of adipogenesis in 3T3-L1 cells. Knockdown of LanCL2, but not LanCL1, inhibited adipogenic differentiation, and this effect was not mediated through cAMP or Akt signaling pathways. The expression of early adipogenic markers CCAAT enhancer binding protein ß (C/EBPß) and C/EBPδ remained intact in LanCL2 knockdown cells, but levels of late adipogenic markers PPARγ and C/EBPα were suppressed. The addition of the naturally occurring PPARγ activator 15-deoxy-Δ12,14-prostaglandin J2 or conditioned medium from differentiating cells did not restore differentiation, implying that LanCL2 may not be involved in the production of a secreted endogenous PPARγ ligand. Pulldown assays demonstrated a direct physical interaction between LanCL2 and PPARγ. Consistent with a regulatory role of LanCL2, luciferase reporter assays revealed that full transcriptional activation by PPARγ was dependent on LanCL2. Taken together, our study reveals a novel role of LanCL2 in adipogenesis, specifically involved in PPARγ-mediated transactivation of downstream adipogenic genes.

hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in α1-antitrypsin deficiency.

BACKGROUND & AIMS: α1-Antitrypsin deficiency (A1ATD) is an autosomal recessive disorder caused by mutations in the SERPINA1 gene. Individuals with the Z variant (Gly342Lys) retain polymerised protein in the endoplasmic reticulum (ER) of their hepatocytes, predisposing them to liver disease. The concomitant lack of circulating A1AT also causes lung emphysema. Greater insight into the mechanisms that link protein misfolding to liver injury will facilitate the design of novel therapies. METHODS: Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes provide a novel approach to interrogate the molecular mechanisms of A1ATD because of their patient-specific genetic architecture and reflection of human physiology. To that end, we utilised patient-specific hiPSC hepatocyte-like cells (ZZ-HLCs) derived from an A1ATD (ZZ) patient, which faithfully recapitulated key aspects of the disease at the molecular and cellular level. Subsequent functional and "omics" comparisons of these cells with their genetically corrected isogenic-line (RR-HLCs) and primary hepatocytes/human tissue enabled identification of new molecular markers and disease signatures. RESULTS: Our studies showed that abnormal A1AT polymer processing (immobilised ER components, reduced luminal protein mobility and disrupted ER cisternae) occurred heterogeneously within hepatocyte populations and was associated with disrupted mitochondrial structure, presence of the oncogenic protein AKR1B10 and two upregulated molecular clusters centred on members of inflammatory (IL-18 and Caspase-4) and unfolded protein response (Calnexin and Calreticulin) pathways. These results were validated in a second patient-specific hiPSC line. CONCLUSIONS: Our data identified novel pathways that potentially link the expression of Z A1AT polymers to liver disease. These findings could help pave the way towards identification of new therapeutic targets for the treatment of A1ATD. LAY SUMMARY: This study compared the gene expression and protein profiles of healthy liver cells and those affected by the inherited disease α1-antitrypsin deficiency. This approach identified specific factors primarily present in diseased samples which could provide new targets for drug development. This study also demonstrates the interest of using hepatic cells generated from human-induced pluripotent stem cells to model liver disease in vitro for uncovering new mechanisms with clinical relevance.

Valores séricos de ácido fólico en un grupo de pacientes mayores de 18 años de edad en un hospital de alta complejidad / Serum Folic Acid Values in a Group of Patients over 18 Years of Age in a Highly Complex Hospital

Objetivo: establecer las concentraciones séricas de ácido fólico en un grupo de pacientes adultos. Métodos: medición del ácido fólico con inmunoanálisis en 1797 sujetos. Se calcularon medidas de tendencia central e índice de confianza al 95 %. Se compararon grupos etarios y sexos con pruebas no paramétricas. Resultados: la media global de ácido fólico sérico fue: 11,61 ng'ml (DE: 6,05). La media en mujeres fue de 11,87 ng'ml (DE: 6,09). La media en hombres fue de 11,31 ng'ml (DE: 6). Se reportaron los percentiles 5, 50 y 95 por sexo y edad. En el 2 % de los casos se encontró ácido fólico < 2 ng'ml (2 % de hombres y 3 % de mujeres). Discusión: las concentraciones séricas de ácido fólico son mayores en mujeres que en hombres (p = 0,024), lo que contrasta con la noción de déficit del micronutriente en el sexo femenino. Conclusiones: las cantidades descritas son un comparador adecuado para mediciones en muestras de población general.

Objective: lo establish the serum levels of folie acid in a group of adult patients. Methods: Measurement of folie acid using ¿mmunoassay in 1797 subjeets. Níeasures of central tendeney and 95% CI were calculated. Age groups and genders were compared with non-parametric tests. Results: The overall mean serum folate was: 11.61 ng'ml (SD: 6.05). The average for women was: 11.87 ng'ml (SD: 6.09). The average for men was: 11.31 ngml (SD: 6). Percentiles 5, 50, and 95 by sex and age are reported. In 2% of cases folie acid was found < 2 ng'ml (2% of men and 3% women). Discussion: Serum levels of folie acid are higher in women than in men (p=0.024), which contrasts with the notion of micronutrient defíciency in remides. Condusions: The presented measurements are an appropriate comparator described for measurements in general population samples.

Interactions between N-linked glycosylation and polymerisation of neuroserpin within the endoplasmic reticulum.

The neuronal serpin neuroserpin undergoes polymerisation as a consequence of point mutations that alter its conformational stability, leading to a neurodegenerative dementia called familial encephalopathy with neuroserpin inclusion bodies (FENIB). Neuroserpin is a glycoprotein with predicted glycosylation sites at asparagines 157, 321 and 401. We used site-directed mutagenesis, transient transfection, western blot, metabolic labelling and ELISA to probe the relationship between glycosylation, folding, polymerisation and degradation of neuroserpin in validated cell models of health and disease. Our data show that glycosylation at N157 and N321 plays an important role in maintaining the monomeric state of neuroserpin, and we propose this is the result of steric hindrance or effects on local conformational dynamics that can contribute to polymerisation. Asparagine residue 401 is not glycosylated in wild type neuroserpin and in several polymerogenic variants that cause FENIB, but partial glycosylation was observed in the G392E mutant of neuroserpin that causes severe, early-onset dementia. Our findings indicate that N401 glycosylation reports lability of the C-terminal end of neuroserpin in its native state. This C-terminal lability is not required for neuroserpin polymerisation in the endoplasmic reticulum, but the additional glycan facilitates degradation of the mutant protein during proteasomal impairment. In summary, our results indicate how normal and variant-specific N-linked glycosylation events relate to intracellular folding, misfolding, degradation and polymerisation of neuroserpin.

Increased ERK signalling promotes inflammatory signalling in primary airway epithelial cells expressing Z α1-antitrypsin.

Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling. However, whether endogenous expression in primary bronchial epithelial cells has similar consequences remains unclear. Moreover, the mechanism of NF-κB activation has not yet been elucidated. Here, we report excessive NF-κB signalling in resting primary bronchial epithelial cells from ZZ patients compared with wild-type (MM) controls, and this appears to be mediated by mitogen-activated protein/extracellular signal-regulated kinase, EGF receptor and ADAM17 activity. Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin. Treatment of ZZ primary bronchial epithelial cells with purified plasma M α1-antitrypsin attenuates this inflammatory response, opening up new therapeutic options to modulate airway inflammation in the lung.

p53 and translation attenuation regulate distinct cell cycle checkpoints during endoplasmic reticulum (ER) stress.

Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G(1) phase, although recent studies have identified a novel ER stress G(2) checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G(2) delay involving CHK1 and a later induction of G(1) arrest associated both with the induction of p53 target genes and loss of cyclin D(1). We show that substitution of p53/47 for p53 impairs the ER stress G(1) checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G(2) progression prior to ultimate G(1) arrest.

Endoplasmic reticulum polymers impair luminal protein mobility and sensitize to cellular stress in alpha1-antitrypsin deficiency.

UNLABELLED: Point mutants of alpha1 -antitrypsin (α1AT) form ordered polymers that are retained as inclusions within the endoplasmic reticulum (ER) of hepatocytes in association with neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. These inclusions cause cell damage and predispose to ER stress in the absence of the classical unfolded protein response (UPR). The pathophysiology underlying this ER stress was explored by generating cell models that conditionally express wild-type (WT) α1AT, two mutants that cause polymer-mediated inclusions and liver disease (E342K [the Z allele] and H334D) and a truncated mutant (Null Hong Kong; NHK) that induces classical ER stress and is removed by ER-associated degradation. Expression of the polymeric mutants resulted in gross changes in the ER luminal environment that recapitulated the changes observed in liver sections from individuals with PI*ZZ α1AT deficiency. In contrast, expression of NHK α1AT caused electron lucent dilatation and expansion of the ER throughout the cell. Photobleaching microscopy in live cells demonstrated a decrease in the mobility of soluble luminal proteins in cells that express E342K and H334D α1AT, when compared to those that express WT and NHK α1AT (0.34 ± 0.05, 0.22 ± 0.03, 2.83 ± 0.30, and 2.84 ± 0.55 µm(2) /s, respectively). There was no effect on protein mobility within ER membranes, indicating that cisternal connectivity was not disrupted. Polymer expression alone was insufficient to induce the UPR, but the resulting protein overload rendered cells hypersensitive to ER stress induced by either tunicamycin or glucose depletion. CONCLUSION: Changes in protein diffusion provide an explanation for the cellular consequences of ER protein overload in mutants that cause inclusion body formation and α1AT deficiency.

Embriopatía por isotretinoína con microtia-anotia y cardiopatía: Presentación de un caso / Isotretinoin embryopathy with microtia-anotia and congenital heart disease: Case report

La isotretinoína es un retinoide derivado de la vitamina A utilizado para el tratamiento del acné noduloquístico y refractario, pero ha sido catalogado como un medicamento teratogénico. Se ha comunicado un espectro de defectos congénitos que incluyen malformaciones craneofaciales, defectos cardíacos y defectos en el sistema nervioso con la exposición prenatal a este medicamento. Se presenta el caso de una recién nacida con antecedente de exposición prenatal a isotretinoína con defectos congénitos craneofaciales que incluyen parálisis facial, anotia derecha y microtia izquierda, y cardiopatía compleja.

Isotretinoin is a retinoid that derivates from vitamin A. It is indicated for recalcitrant nodular acne treatment, but it has been classifed as teratogenic. A wide spectrum of birth defects including craniofacial, heart and nervous system malformations have been described associated to prenatal exposure to this drug. We report the case of a newborn with a history of prenatal exposure to isotretinoin with craniofacial defects, including facial paralysis, right anotia, left microtia and complex heart disease.

The serpinopathies studying serpin polymerization in vivo.

The serpinopathies result from point mutations in members of the serine protease inhibitor or serpin superfamily. They are characterized by the formation of ordered polymers that are retained within the cell of synthesis. This causes disease by a "toxic gain of function" from the accumulated protein and a "loss of function" as a result of the deficiency of inhibitors that control important proteolytic cascades. The serpinopathies are exemplified by the Z (Glu342Lys) mutant of α1-antitrypsin that results in the retention of ordered polymers within the endoplasmic reticulum of hepatocytes. These polymers form the intracellular inclusions that are associated with neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. A second example results from mutations in the neurone-specific serpin-neuroserpin to form ordered polymers that are retained as inclusions within subcortical neurones as Collins' bodies. These inclusions underlie the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. There are different pathways to polymer formation in vitro but not all form polymers that are relevant in vivo. It is therefore essential that protein-based structural studies are interpreted in the context of human samples and cell and animal models of disease. We describe here the biochemical techniques, monoclonal antibodies, cell biology, animal models, and stem cell technology that are useful to characterize the serpin polymers that form in vivo.

Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

Effectiveness of regular reporting of spirometric results combined with a smoking cessation advice by a primary care physician on smoking quit rate in adult smokers: a randomized controlled trial. ESPIROTAB study.

BACKGROUND: Undiagnosed airflow limitation is common in the general population and is associated with impaired health and functional status. Smoking is the most important risk factor for this condition. Although primary care practitioners see most adult smokers, few currently have spirometers or regularly order spirometry tests in these patients. Brief medical advice has shown to be effective in modifying smoking habits in a large number of smokers but only a small proportion remain abstinent after one year. The aim of this study is to evaluate the effectiveness of regular reporting of spirometric results combined with a smoking cessation advice by a primary care physician on smoking quit rate in adult smokers. METHODS/DESIGN: Intervention study with a randomized two arms in 5 primary care centres. A total of 485 smokers over the age of 18 years consulting their primary care physician will be recruited.On the selection visit all participants will undergo a spirometry, peak expiratory flow rate, test of smoking dependence, test of motivation for giving up smoking and a questionnaire on socio-demographic data. Thereafter an appointment will be made to give the participants brief structured advice to give up smoking combined with a detailed discussion on the results of the spirometry. After this, the patients will be randomised and given appointment for follow up visits at 3, 6, 12 and 24 months. Both arms will receive brief structured advice and a detailed discussion of the spirometry results at visit 0. The control group will only be given brief structured advice about giving up smoking on the follow up. Cessation of smoking will be tested with the carbon monoxide test. DISCUSSION: Early identification of functional pulmonary abnormalities in asymptomatic patients or in those with little respiratory symptomatology may provide "ideal educational opportunities". These opportunities may increase the success of efforts to give up smoking and may improve the opportunities of other preventive actions to minimise patient risk. Comparing adult smokers in the intervention group with those in the control group, a minimum improvement expected with respect to the rates of smoking cessation would represent a large number of avoided morbimortality. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01296295.

Increased levels of citrullinated antithrombin in plasma of patients with rheumatoid arthritis and colorectal adenocarcinoma determined by a newly developed ELISA using a specific monoclonal antibody.

Citrullination is a post-translational modification that plays essential roles in both physiological processes and disease. Recent studies have found increased levels of citrullinated antithrombin in patients with rheumatoid arthritis and in different malignant tumours. Antithrombin, the main haemostatic serpin, loses its anticoagulant function via citrullination, which might contribute to the pathogenesis or thrombotic side effects of these disorders. We have developed a specific monoclonal antibody against citrullinated antithrombin. We determined the levels of citrullinated antithrombin and anti-FXa activity in plasma from 66 donors, 17 patients with rheumatoid arthritis and 77 patients with colorectal adenocarcinoma (42 suffering from venous thrombosis). Healthy subjects had negligible amounts of citrullinated antithrombin in plasma (7.9 ± 22.1 ng/ml), while it significantly increased in patients with rheumatoid arthritis or adenocarcinoma (159.7 ± 237.6 ng/ml and 36.8 ± 66.1 ng/ml), levels that, however, did not modify the plasma anticoagulant activity. Moreover, we did not find association between citrullinated antithrombin and the thrombotic risk in patients with adenocarcinoma. In conclusion, we have developed an antibody specific for citrullinated antithrombin that allows its quantification in biological samples, offering a new tool for the analysis of citrullination in different diseases. We confirm increased levels of citrullinated antithrombin in plasma of patients with rheumatoid arthritis and adenocarcinoma. This modification, probably local, could have pathological consequences in both disorders, but only affects a minor fraction of plasma antithrombin, resulting in no significant reduction of global anticoagulant activity. This result explains the absence of association of this marker with an increased risk of thrombosis in patients with colorectal adenocarcinoma.