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IntroductionThe methods for diagnosing compartment syndrome non-invasively remain under debate. Bioimpedance measurements offer a promising avenue in clinical practice, detecting subtle changes in organ impedance due to volume shifts. This study explores bioimpedance measurement as a novel, painless method for diagnosing compartment syndrome, potentially enabling continuous monitoring.ObjectiveThis work aims to develop a prototype device for non-invasive diagnosis of compartment syndrome based on bioimpedance changes and assess initial results through in vitro experiments on inanimate biological material. We assume a change in the bioimpedance value after the application of physiological solution.Materials and MethodsBetween 2018 and 2022, a prototype device for diagnosing limb compartment syndrome was collaboratively developed with the Department of Cybernetics and Biomedical Engineering at the Technical University of Ostrava, Czech Republic. This device operates by comparing bioimpedance between two compartments, one of which is pathologically affected (experiencing compartment syndrome). The Bioimpedance Analyzer for Compartment Syndrome (BACS) has been utilized to conduct measurements on inanimate biological material in laboratory settings. Two samples of duck and chicken tissue, as well as piglets, were employed for these experiments. According to the size of sample was compartment syndrome simulated by injecting 20–120 mL saline into one limb (breast) while leaving the other as a control. Invasive intramuscular pressure measurements were conducted post-saline injection using a conventional device (Stryker). Changes in bioimpedance were evaluated following saline application.ResultsThe non-invasive bioimpedance measurement instrument has been developed. It meets the safety requirements of European standard EN 60601-1. Measurement of accuracy showed minimal deviation for both channels (1.08% for the left channel and 1.84% for the right channel) when measuring on resistors. Ten measurements were conducted using the BACS prototype - two on chicken legs, two on duck breasts, two on duck legs, and four on piglets. Compartment syndrome simulation was achieved for all 10 measurements (IMP variance 31–45 mmHg). Following saline application, a notable decrease in bioimpedance was observed in the compartment simulating compartment syndrome (decrease by 12–78 Ω).ConclusionNon-invasive methods could revolutionize limb compartment syndrome diagnosis, offering advantages such as non-invasiveness and continuous monitoring of compartment swelling.

The article deals with an overview of acute extremity compartment syndrome with a focus on the option of non-invasive detection of the syndrome. Acute extremity compartment syndrome (ECS) is an urgent complication that occurs most often in fractures or high-energy injuries. There is still no reliable method for detecting ECS. The only objective measurement method used in clinical practice is an invasive measurement of intramuscular pressure (IMP). The purpose of this paper is to summarize the current state of research into non-invasive measurement methods that could allow simple and reliable continuous monitoring of patients at risk of developing ECS. Clinical trials are currently underway to verify the suitability of the most studied method, near-infrared spectroscopy (NIRS), which is a method for measuring the local oxygenation of muscle compartments. Less explored methods include the use of ultrasound, ultrasound elastography, bioimpedance measurements, and quantitative tissue hardness measurements. Finding a suitable method for continuous non-invasive monitoring of the syndrome would greatly improve the quality of care for patients at risk. ECS must be diagnosed quickly and accurately to prevent irreversible tissue damage that can occur within hours of syndrome onset and may even warrant amputation if neglected.

This paper evaluates the various approaches to strength and stiffness analysis of fracture osteosynthesis using a headless Herbert screw. The problem has been extensively addressed using several scientific approaches, namely the analytical approach, stochastic approach, experimental approach, and (marginally) using the finite elements method. The problem is illustrated on the use of a prototype headless screw Ti: 4.0/1.4 × 30/7 (manufacturer: Medin, Czech Republic) and the surgical treatment of the fifth metatarsal fracture. Mathematical equations for the analytical calculation of the maximum stresses in the screw were established for tensile/compression loading. This problem is also interesting because of its static indetermination in tension and compression; for this reason, it was necessary to use the deformation condition, i.e., the relationship between screw extension and bone contraction. The stochastic (probabilistic) approach, i.e., application of the Monte Carlo method, takes advantage of the mathematical equations derived during the analytical solution by respecting of the natural variabilities and uncertainties. The analytical and stochastic approaches were validated by measurements on porcine bones and by the finite element method. The data measured experimentally were also processed and used for deriving an equation, appropriately approximating the data. The main part of the measurement was to determine the axial force generated during osteosynthesis with a headless screw. The obtained compressional force was used to determine the maximal stress in the screw and bone. Finally, the methods were compared. In this paper, comprehensive and original approaches based on the authors’ experience with multiple methods are presented. Obtained results are necessary for headless screw designers during optimalization of the implants and are also useful for surgeons developing new surgical techniques. This biomechanical problem was solved in cooperation with the engineering industry and physicians to improve the quality of care for patients with trauma in orthopedics and surgery.

This article focuses on a type of surgical implant used in orthopaedics and traumatology—cannulated femoral nails. Femoral nails are used in medical treatment for purposes of osteosynthesis, i.e., when treating various types of complicated fractures, in this case fractures of the femur. The article investigates cases in which a nail has been implanted in the proximal part of the femur for a short time (with the fracture still not healed), compared with cases in which the bone has already healed. According to AO classification, examined fractures are described as AO 31B3 AO 32A3. The main focus is on strength-deformation analysis using the finite element method (FEM), which makes it possible to determine the behaviour of the femur-implant system. FEM analysis was used to compare 1.4441 steel nails made by two manufacturers, Medin (Czech Republic) and Tantum (Germany). Boundary conditions including external loading, prescribed supports and elastic foundation are defined. There were solved FEM analyses for five cases of healed femur and five cases of broken femur both including implants with prescribed collo-diaphyseal angles. The results of the analysis were used to assess stress-deformation states from the perspective of appropriateness for clinical treatment, biomechanical reliability and safety. All examined femoral nails are compared, safe and suitable for patient treatment.

The presented article investigates the biomechanics of the calcaneal nail C-NAILTM by numerical calculations and, partially, experimentally. This nail is widely used in trauma and orthopaedics. A numerical model of implants directly interacting with the bone tissue model obtained from CT scans was calculated. The material properties of the bone tissue can be described by several models; in this work, a non-homogeneous material model with isotropic elements and prescribed elastic modulus was used to provide a more accurate model of the applied force distribution on the individual parts of the implants. The critical areas of the nail and its fixtures were investigated using finite element strength calculations to verify their strength and reliability, contributing to the safety and faster and easier treatment of patients. These analyses suggest that the strength of the calcaneal nail C-NAIL, as well as the stabilization of bone fragments resulting from its use, are sufficient for clinical practice.

Limb asymmetry can, and often does, cause various health problems. Blount bone staples (clips) are used to correct such uneven growth. This article analyzes the performance of a biomechanical staple during bone (tibia) growth arrest. The staples considered in this study were made of 1.4441 stainless steel, the model of tibia consisted of two materials representing corticalis and spongiosis. Hooke’s law was used for modeling materials’ behaviors for finite element analysis (FEA). The maxima of stress and total staple displacement were evaluated using the finite element method and verification of the results, along with the determination of the maximum loading (growing) force that the staples are capable of withstanding, was performed experimentally. The presented method can be used to determine the safety and usability of staples for bone growth arrest. According to our results, the design of Blount staples considered in this paper is safe and suitable for orthopedic treatment.

This paper represents a multidisciplinary approach to biomechanics (medicine engineering and mathematics) in the field of collum femoris fractures, i.e., of osteosyntheses with femoral/cancellous screws with full or cannulated cross-sections. It presents our new numerical model of femoral screws together with their stochastic (probabilistic, statistical) assessment. In the first part of this article, the new simple numerical model is presented. The model, based on the theory of planar (2D) beams on an elastic foundation and on 2nd-order theory, is characterized by rapid solution. Bending and compression loadings were used for derivation of a set of three 4th-order differential equations. Two examples (i.e., a stainless-steel cannulated femoral screw and full cross-section made of Ti6Al4V material) are presented, explained, and evaluated. In the screws, the internal shearing forces, internal normal forces, internal bending moments, displacement (deflections), slopes, and mechanical stresses are calculated using deterministic and stochastic approaches. For the stochastic approach and a “fully” probabilistic reliability assessment (which is a current trend in science), the simulation-based reliability assessment method, namely, the application of the direct Monte Carlo Method, using Anthill software, is applied. The probabilities of plastic deformations in femoral screws are calculated. Future developments, which could be associated with different configurations of cancellous screws, nonlinearities, experiments, and applications, are also proposed.

Background: The standard ATLS protocol calls for chest drain insertion in patients with hemothorax before performing further diagnostic steps. However, if trauma-induced thoracic aortic rupture is the underlying cause, such drainage can lead to massive bleeding and death of the patient. Case report: This report describes a case of a polytrauma patient (car accident), aged 21, with symmetrical chest and decreased breath sounds dorsally on the left. An urgent CT scan revealed subadventitial Grade III thoracic aortic transection with mediastinal hematoma, a massive left-sided hemothorax with mediastinal shift to the right, and other injuries. Stent-graft implantation with subsequent left hemithorax drainage was urgently performed, during which the patient became increasingly unstable from the circulatory point of view. This traumatic hemorrhagic shock was successfully managed at the ICU. Conclusion: Although hemothorax is a serious condition requiring rapid treatment, the knowledge of its origin is of utmost importance; performing chest drainage without bleeding control can lead to circulatory instability and death of the patient. Hence, where aortic injury can be suspected based on the mechanism of the injury, it is beneficial to perform spiral CT angiography for accurate diagnosis first and, in cases of aortic injury, to control the bleeding prior to drainage.

This article deals with a new original analytical solution of deformation, force and stress states in wood screw joints up to the limit values of pulling out/breaking the screw. The screws are under tension. The wood-to-screw interaction is effectively simplified by introducing several physical model variants using a tangential elastic non-linear foundation. The experimental verification of the proposed models using pull-out tests (i.e., pulling out screws from dry spruce wood in laboratory conditions) confirms the correctness of the proposed models of the elastic linear/non-linear foundation. The validity of the model is also analytically and experimentally verified in the biomechanical model of pulling out screws from the femur of a bovine/human cadaver, which confirms and expands the validity of newly designed screw joint models outside the timber structure area.

This article deals with analysis of the knee continuous passive motion (CPM) splint used for rehabilitation of patients after surgeries and injuries. CPM splints are used for speeding up the treatment and restoration of the joint mobility and to stop complications caused by immobilization. The goal is to solve kinematic, dynamic, deformation and stress parameters of “Artromot K1 Comfort” CPM splint.

At first, the doctors mentioned their own medical experience with treatment of complex pelvic injury in patients with polytrauma and give reasons for necessity of early stabilization of pelvic fractures by means of external fixation, especially with continuous hemorrhage into lesser pelvis region and the retroperitoneum. Afterwards, they used damage control surgery methods including selective embolization. However, this article is focused also on the design of external fixators applied in traumatology and orthopaedics (i.e. skills of engineers). These fixators can be used in the treatment of open and unstable (i.e. complicated) fractures of pelvis and its acetabulum. Two versions (i.e. old and new) are compared. Numerical modelling (i.e. Finite Element Method), together with CAD modelling, experiments, material engineering, and nanotechnology are presented as a support for developing of a new design of external fixators.

Main point in this contribution is the design of external fixators applied in traumatology and orthopaedics. These fixators can be used in the treatment of open and unstable (i.e. complicated) fractures of pelvis and its acetabulum. Numerical modelling (i.e. Finite Element Method), together with CAD modelling, experiments, material engineering, and nanotechnology are presented as a support for developing of a new design of external fixators.

Based on the observation that humans have variable responses of gene expression with the same dose of an adeno-associated vector, we hypothesized that there are deleterious variants in genes coding for processes required for adeno-associated virus (AAV)-mediated gene transfer/expression that may hamper or enhance the effectiveness of AAV-mediated gene therapy. To assess this hypothesis, we evaluated 69,442 whole genome sequences from three populations (European, African/African American, and Qatari) for predicted deleterious variants in 62 genes known to play a role in AAV-mediated gene transfer/expression. The analysis identified 5,564 potentially deleterious mutations of which 27 were classified as common based on an allele frequency ≥1% in at least one population studied. Many of these deleterious variants are predicated to prevent while others enhance effective AAV gene transfer/expression, and several are linked to known hereditary disorders. The data support the hypothesis that, like other drugs, human genetic variability contributes to the person-to-person effectiveness of AAV gene therapy and the screening for genetic variability should be considered as part of future clinical trials., Competing Interests: The authors declare no competing interests., (© 2023.)

Background: Electronic cigarettes are increasing in popularity, but there is only little information on their biologic effects on the oral epithelium, the initial site exposed to electronic cigarette smoke., Methods: We assessed the oral epithelium response to electronic cigarettes by comparing the histology and RNA transcriptome (mRNA and miRNA) of healthy electronic cigarette vapers to nonsmokers. mRNA was assessed based on: (1) genome-wide; (2) genes previously identified as dysregulated in the oral epithelium of electronic cigarette vapers versus nonsmokers; (3) immune and inflammatory-related genes previously identified as dysregulated in the nasal epithelium of electronic cigarette vapers compared to nonsmokers; (4) genes previously identified as dysregulated in the small airway epithelium of nonsmokers following an acute exposure to electronic cigarette; and (5) genes related to the initial steps of COVID-19 infection. In addition, miRNA was assessed genome-wide. Comparisons were performed using analysis of variance, and Benajmini-Hochberg corrected p < 0.05 was considered significant., Results: The histology of the epithelium, lamina propria and basal layer in electronic cigarette vapers appeared normal. Assessment of mRNA and miRNA, based on all gene lists, did not identify any genes significantly modified in the oral epithelium of electronic cigarette vapers in response to electronic cigarette use., Conclusion: An average history of 2 years of vaping results in no detectable histologic or transcriptome abnormalities in the buccal mucosa., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Species determination based on genetic evidence is an indispensable tool in archaeology, forensics, ecology, and food authentication. Most available analytical approaches involve compromises with regard to the number of detectable species, high cost due to low throughput, or a labor-intensive manual process. Here, we introduce "Species by Proteome INvestigation" (SPIN), a shotgun proteomics workflow for analyzing archaeological bone capable of querying over 150 mammalian species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Rapid peptide chromatography and data-independent acquisition (DIA) with throughput of 200 samples per day reduce expensive MS time, whereas streamlined sample preparation and automated data interpretation save labor costs. We confirm the successful classification of known reference bones, including domestic species and great apes, beyond the taxonomic resolution of the conventional peptide mass fingerprinting (PMF)-based Zooarchaeology by Mass Spectrometry (ZooMS) method. In a blinded study of degraded Iron-Age material from Scandinavia, SPIN produces reproducible results between replicates, which are consistent with morphological analysis. Finally, we demonstrate the high throughput capabilities of the method in a high-degradation context by analyzing more than two hundred Middle and Upper Palaeolithic bones from Southern European sites with late Neanderthal occupation. While this initial study is focused on modern and archaeological mammalian bone, SPIN will be open and expandable to other biological tissues and taxa., (© 2022. The Author(s).)

The club cell, a small airway epithelial (SAE) cell, plays a central role in human lung host defense. We hypothesized that subpopulations of club cells with distinct functions may exist. The SAE of healthy nonsmokers and healthy cigarette smokers were evaluated by single-cell RNA sequencing, and unsupervised clustering revealed subpopulations of SCGCB1A1 + KRT5 lo MUC5AC - club cells. Club cell heterogeneity was supported by evaluations of SAE tissue sections, brushed SAE cells, and in vitro air-liquid interface cultures. Three subpopulations included: (1) progenitor; (2) proliferating; and (3) effector club cells. The progenitor club cell population expressed high levels of mitochondrial, ribosomal proteins, and KRT5 relative to other club cell populations and included a differentiation branch point leading to mucous cell production. The small proliferating population expressed high levels of cyclins and proliferation markers. The effector club cell cluster expressed genes related to host defense, xenobiotic metabolism, and barrier functions associated with club cell function. Comparison of smokers vs. nonsmokers demonstrated that smoking limited the extent of differentiation of all three subclusters and altered SAM pointed domain-containing Ets transcription factor (SPDEF)-regulated transcription in the effector cell population leading to a change in the location of the branch point for mucous cell production, a potential explanation for the concomitant reduction in effector club cells and increase in mucous cells in smokers. These observations provide insights into both the makeup of human SAE club cell subpopulations and the smoking-induced changes in club cell biology., (© 2021. The Author(s).)

Background: Lysophosphatidic acid (LPA), generated extracellularly by the action of autotaxin and phospholipase A2, functions through LPA receptors (LPARs) or sphingosine-1-phosphate receptors (S1PRs) to induce pro-fibrotic signaling in the lower respiratory tract of patients with idiopathic pulmonary fibrosis (IPF). We hypothesized that LPA induces changes in small airway epithelial (SAE) basal cells (BC) that create cross-talk between the BC and normal human lung fibroblasts (NHLF), enhancing myofibroblast formation., Methods: To assess LPA-induced signaling, BC were treated with LPA for 2.5 min and cell lysates were analyzed by phosphokinase array and Western blot. To assess transcriptional changes, BC were treated with LPA for 3 h and harvested for collection and analysis of RNA by quantitative polymerase chain reaction (qPCR). To assess signaling protein production and function, BC were washed thoroughly after LPA treatment and incubated for 24 h before collection for protein analysis by ELISA or functional analysis by transfer of conditioned medium to NHLF cultures. Transcription, protein production, and proliferation of NHLF were assessed., Results: LPA treatment induced signaling by cAMP response element-binding protein (CREB), extracellular signal-related kinases 1 and 2 (Erk1/2), and epithelial growth factor receptor (EGFR) resulting in elevated expression of connective tissue growth factor (CTGF), endothelin-1 (EDN1/ET-1 protein), and platelet derived growth factor B (PDGFB) at the mRNA and protein levels. The conditioned medium from LPA-treated BC induced NHLF proliferation and increased NHLF expression of collagen I (COL1A1), smooth muscle actin (ACTA2), and autotaxin (ENPP2) at the mRNA and protein levels. Increased autotaxin secretion from NHLF correlated with increased LPA in the NHLF culture medium. Inhibition of CREB signaling blocked LPA-induced changes in BC transcription and translation as well as the pro-fibrotic effects of the conditioned medium on NHLF., Conclusion: Inhibition of CREB signaling may represent a novel target for alleviating the LPA-induced pro-fibrotic feedback loop between SAE BC and NHLF.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic fibrotic lung disease with an irreversible decline of lung function. "Bronchiolization", characterized by ectopic appearance of airway epithelial cells in the alveolar regions, is one of the characteristic features in the IPF lung. Based on the knowledge that club cells are the major epithelial secretory cells in human small airways, and their major secretory product uteroglobin (SCGB1A1) is significantly increased in both serum and epithelial lining fluid of IPF lung, we hypothesize that human airway club cells contribute to the pathogenesis of IPF. By assessing the transcriptomes of the single cells from human lung of control donors and IPF patients, we identified two SCGB1A1+ club cell subpopulations, highly expressing MUC5B, a significant genetic risk factor strongly associated with IPF, and SCGB3A2, a marker heterogeneously expressed in the club cells, respectively. Interestingly, the cellular proportion of SCGB1A1+MUC5B+ club cells was significantly increased in IPF patients, and this club cell subpopulation highly expressed genes related to mucous production and immune cell chemotaxis. In contrast, though the cellular proportion did not change, the molecular phenotype of the SCGB1A1+SCGB3A2high club cell subpopulation was significantly altered in IPF lung, with increased expression of mucins, cytokine and extracellular matrix genes. The single cell transcriptomic analysis reveals the cellular and molecular heterogeneity of club cells, and provide novel insights into the biological functions of club cells in the pathogenesis of IPF., Competing Interests: K. Quast, S. Visvanathan, JS Fine, MJ Thomas are employees of Boehringer Ingelheim Pharmaceuticals; all other authors declare no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: The human small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of most inherited and acquired lung disorders. Little is known about the molecular phenotypes of the specific cell populations comprising the SAE in humans, and the contribution of SAE specific cell populations to the risk for lung diseases., Methods: Drop-seq single-cell RNA-sequencing was used to characterize the transcriptome of single cells from human SAE of nonsmokers and smokers by bronchoscopic brushing., Results: Eleven distinct cell populations were identified, including major and rare epithelial cells, and immune/inflammatory cells. There was cell type-specific expression of genes relevant to the risk of the inherited pulmonary disorders, genes associated with risk of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis and (non-mutated) driver genes for lung cancers. Cigarette smoking significantly altered the cell type-specific transcriptomes and disease risk-related genes., Conclusions: This data provides new insights into the possible contribution of specific lung cells to the pathogenesis of lung disorders.

Rationale: Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease (COVID-19), a predominantly respiratory illness. The first step in SARS-CoV-2 infection is binding of the virus to ACE2 (angiotensin-converting enzyme 2) on the airway epithelium. Objectives: The objective was to gain insight into the expression of ACE2 in the human airway epithelium. Methods: Airway epithelia sampled by fiberoptic bronchoscopy of trachea, large airway epithelia (LAE), and small airway epithelia (SAE) of nonsmokers and smokers were analyzed for expression of ACE2 and other coronavirus infection-related genes using microarray, RNA sequencing, and 10x single-cell transcriptome analysis, with associated examination of ACE2 -related microRNA. Measurements and Main Results: 1 ) ACE2 is expressed similarly in the trachea and LAE, with lower expression in the SAE; 2 ) in the SAE, ACE2 is expressed in basal, intermediate, club, mucus, and ciliated cells; 3 ) ACE2 is upregulated in the SAE by smoking, significantly in men; 4 ) levels of miR-1246 expression could play a role in ACE2 upregulation in the SAE of smokers; and 5 ) ACE2 is expressed in airway epithelium differentiated in vitro on air-liquid interface cultures from primary airway basal stem/progenitor cells; this can be replicated using LAE and SAE immortalized basal cell lines derived from healthy nonsmokers. Conclusions: ACE2 , the gene encoding the receptor for SARS-CoV-2, is expressed in the human airway epithelium, with variations in expression relevant to the biology of initial steps in SARS-CoV-2 infection.

Capitalizing on liver tropism of adeno-associated viral (AAV) vectors, intravenous vector administration is commonly used to genetically modify hepatocytes, a strategy currently in clinical trials for a number of liver-based hereditary disorders. Although hepatocytes are known to exhibit extensive phenotypic heterogeneity influenced by liver zonation and dietary cycle, there is little data available for the tropism capacity, as well as the potential transcriptional dysregulation, of AAV vectors for specific liver cell types. To assess these issues, we employed single-cell RNA sequencing of the mouse liver after intravenous administration of the liver tropic AAVrh.10 vector to characterize cell-specific AAV-mediated transgene expression and transcriptome dysregulation. Wild-type 8-week-old male C57Bl/6 mice under normal feed cycle were randomly divided into three groups and intravenously administered phosphate-buffered saline (PBS), AAVrh.10Null (no transgene), or AAVrh.10mCherry (marker gene). Overall, a total of 46,500 liver cells were sequenced. The single-cell transcriptomic profiles were grouped into three separate clusters of hepatocytes ( Ttr -enriched "Hep1," Tat -enriched "Hep2," and Alb -enriched "Hep3") and multiple other cell types. The hepatocyte diversity was driven by glucose and lipid homeostasis signaling. Assessment of the transgene expression demonstrated that AAVrh.10 is primarily Hep1-tropic, with a 10-gene signature positively correlated with AAVrh.10-mediated transgene expression. The transgene expression was less in Hep2 and Hep3 cells with a high receptor tyrosine kinase phenotype. Importantly, AAVrh.10 vector interactions with the liver markedly altered the transcriptional patterns of all cell types, with modified genes enriched in pathways of complement and coagulation cascade, cytochrome P450, peroxisome, antigen processing and presentation, and endoplasmic reticulum protein processing. These observations provide insights into the liver cell-specific consequences of AAV-mediated liver gene transfer, far beyond the well-known organ-specific expression of the vector-delivered transgene.

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disorder caused by repetitive trauma to the central nervous system (CNS) suffered by soldiers, contact sport athletes, and civilians following accident-related trauma. CTE is a CNS tauopathy, with trauma-induced inflammation leading to accumulation of hyperphosphorylated forms of the microtubule-binding protein Tau (pTau), resulting in neurofibrillary tangles and progressive loss of neurons. At present, there are no therapies to treat CTE. We hypothesized that direct CNS administration of an adeno-associated virus (AAV) vector coding for an anti-pTau antibody would generate sufficient levels of anti-pTau in the CNS to suppress pTau accumulation thus interrupting the pathogenic process. Using a serotype AAVrh.10 gene transfer vector coding for a monoclonal antibody directed against pTau, we demonstrate the feasibility of this strategy in a murine CTE model in which pTau accumulation was elicited by repeated traumatic brain injury (TBI) using a closed cortical impact procedure over 5 days. Direct delivery of AAVrh.10 expression vectors coding for either of the two different anti-pTau antibodies to the hippocampus of these TBI mice significantly reduced pTau levels across the CNS. Using doses that can be safely scaled to humans, the data demonstrate that CNS administration of AAVrh.10anti-pTau is effective, providing a new strategy to interrupt the CTE consequences of TBI.

Background: The pathology of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and most lung cancers involves the small airway epithelium (SAE), the single continuous layer of cells lining the airways ≥ 6th generations. The basal cells (BC) are the stem/progenitor cells of the SAE, responsible for the differentiation into intermediate cells and ciliated, club and mucous cells. To facilitate the study of the biology of the human SAE in health and disease, we immortalized and characterized a normal human SAE basal cell line., Methods: Small airway basal cells were purified from brushed SAE of a healthy nonsmoker donor with a characteristic normal SAE transcriptome. The BC were immortalized by retrovirus-mediated telomerase reverse transcriptase (TERT) transduction and single cell drug selection. The resulting cell line (hSABCi-NS1.1) was characterized by RNAseq, TaqMan PCR, protein immunofluorescence, differentiation capacity on an air-liquid interface (ALI) culture, transepithelial electrical resistance (TEER), airway region-associated features and response to genetic modification with SPDEF., Results: The hSABCi-NS1.1 single-clone-derived cell line continued to proliferate for > 200 doubling levels and > 70 passages, continuing to maintain basal cell features (TP63 + , KRT5 + ). When cultured on ALI, hSABCi-NS1.1 cells consistently formed tight junctions and differentiated into ciliated, club (SCGB1A1 + ), mucous (MUC5AC + , MUC5B + ), neuroendocrine (CHGA + ), ionocyte (FOXI1 + ) and surfactant protein positive cells (SFTPA + , SFTPB + , SFTPD + ), observations confirmed by RNAseq and TaqMan PCR. Annotation enrichment analysis showed that "cilium" and "immunity" were enriched in functions of the top-1500 up-regulated genes. RNAseq reads alignment corroborated expression of CD4, CD74 and MHC-II. Compared to the large airway cell line BCi-NS1.1, differentiated of hSABCi-NS1.1 cells on ALI were enriched with small airway epithelial genes, including surfactant protein genes, LTF and small airway development relevant transcription factors NKX2-1, GATA6, SOX9, HOPX, ID2 and ETV5. Lentivirus-mediated expression of SPDEF in hSABCi-NS1.1 cells induced secretory cell metaplasia, accompanied with characteristic COPD-associated SAE secretory cell changes, including up-regulation of MSMB, CEACAM5 and down-regulation of LTF., Conclusions: The immortalized hSABCi-NS1.1 cell line has diverse differentiation capacities and retains SAE features, which will be useful for understanding the biology of SAE, the pathogenesis of SAE-related diseases, and testing new pharmacologic agents.

Background: KRAS is a GTPase that activates pathways involved in cell growth, differentiation and survival. In normal cells, KRAS-activity is tightly controlled, but with specific mutations, the KRAS protein is persistently activated, giving cells a growth advantage resulting in cancer. While a great deal of attention has been focused on the role of mutated KRAS as a common driver mutation for lung adenocarcinoma, little is known about the role of KRAS in regulating normal human airway differentiation., Methods: To assess the role of KRAS signaling in regulating differentiation of the human airway epithelium, primary human airway basal stem/progenitor cells (BC) from nonsmokers were cultured on air-liquid interface (ALI) cultures to mimic the airway epithelium in vitro. Modulation of KRAS signaling was achieved using siRNA-mediated knockdown of KRAS or lentivirus-mediated over-expression of wild-type KRAS or the constitutively active G12 V mutant. The impact on differentiation was quantified using TaqMan quantitative PCR, immunofluorescent and immunohistochemical staining analysis for cell type specific markers. Finally, the impact of cigarette smoke exposure on KRAS and RAS protein family activity in the airway epithelium was assessed in vitro and in vivo., Results: siRNA-mediated knockdown of KRAS decreased differentiation of BC into secretory and ciliated cells with a corresponding shift toward squamous cell differentiation. Conversely, activation of KRAS signaling via lentivirus mediated over-expression of the constitutively active G12 V KRAS mutant had the opposite effect, resulting in increased secretory and ciliated cell differentiation and decreased squamous cell differentiation. Exposure of BC to cigarette smoke extract increased KRAS and RAS protein family activation in vitro. Consistent with these observations, airway epithelium brushed from healthy smokers had elevated RAS activation compared to nonsmokers., Conclusions: Together, these data suggest that KRAS-dependent signaling plays an important role in regulating the balance of secretory, ciliated and squamous cell differentiation of the human airway epithelium and that cigarette smoking-induced airway epithelial remodeling is mediated in part by abnormal activation of KRAS-dependent signaling mechanisms.

Rationale: Little is known about human club cells, dome-shaped cells with dense cytoplasmic granules and microvilli that represent the major secretory cells of the human small airways (at least sixth-generation bronchi)., Objectives: To define the ontogeny and biology of the human small airway epithelium club cell., Methods: The small airway epithelium was sampled from the normal human lung by bronchoscopy and brushing. Single-cell transcriptome analysis and air-liquid interface culture were used to assess club cell ontogeny and biology., Measurements and Main Results: We identified the club cell population by unbiased clustering using single-cell transcriptome sequencing. Principal component gradient analysis uncovered an ontologic link between KRT5 (keratin 5) + basal cells and SCGB1A1 (secretoglobin family 1A member 1) + club cells, a hypothesis verified by demonstrating in vitro that a pure population of human KRT5 + SCGB1A1 - small airway epithelial basal cells differentiate into SCGB1A1 + KRT5 - club cells on air-liquid interface culture. Using SCGB1A1 as the marker of club cells, the single-cell analysis identified novel roles for these cells in host defense, xenobiotic metabolism, antiprotease, physical barrier function, monogenic lung disorders, and receptors for human viruses., Conclusions: These observations provide novel insights into the molecular phenotype and biologic functions of the human club cell population and identify basal cells as the human progenitor cells for club cells.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder occurring in 1:10,000 to 1:20,000 live births. In >95% of the cases, CAH results from mutations in the CYP21A2 gene, encoding the adrenal steroid enzyme 21-hydroxylase (21OH). Cardinal phenotypic features of CAH include genital ambiguity and sexual precocity, and in severe cases, neonatal salt loss and death. Current standard of care consists of lifelong oral steroid replacement to reverse the cortisol deficiency. Although significant advances in the treatment of CAH have been made, the burden of a lifelong therapeutic intervention is not ideal for quality of life. Gene therapy for CAH by adeno-associated virus (AAV) vectors has been shown to efficiently transduce the adrenal cortex, restoring normal steroidogenesis in the short term. However, adrenocortical cells are continuously renewed by stem cells located at the adrenal capsule, which differentiate as they centripetally migrate towards the adrenal medulla where they undergo apoptosis. In this context, we hypothesized that AAV-mediated genetic correction of the adrenal cortex will work short term but will eventually lead to a loss of correction. To test this hypothesis, we administered intravenously an AAV serotype rh.10 gene transfer vector (AAVrh.10-21OH-HA) to 21-hydroxylase deficient mice (21OH -/- ). The data demonstrates that a single intravenous administration efficiently transduces adrenocortical cells leading to 21OH-HA expression and restoration of normal steroidogenesis. However, the duration of therapeutic efficacy lasted for only 8 weeks, accompanied by loss of 21OH-HA expression in the adrenal gland. Analysis in immunodeficient mice confirmed that the disappearance of transgene expression was not due to an antiviral/transgene immune response. Taken together, these results demonstrate that a single treatment with an adeno-associated viral vector expressing a functional copy of the mutated gene can only transiently treat adrenocortical hereditary disorders and that strategies to genetically modify the adrenocortical stem cells population will likely be required.

Due to high levels of expression in aggressive tumors, high mobility group AT-hook 1 (HMGA1) has recently attracted attention as a potential anti-tumor target. However, HMGA1 is also expressed in normal somatic progenitor cells, raising the question: how might systemic anti-HMGA1 therapies affect the structure and function of normal tissue differentiation? In the present study, RNA sequencing data demonstrated HMGA1 is highly expressed in human airway basal stem/progenitor cells (BC), but decreases with BC differentiation in air-liquid interface cultures (ALI). BC collected from nonsmokers, healthy smokers, and smokers with chronic obstructive pulmonary disease (COPD) displayed a range of HMGA1 expression levels. Low initial expression levels of HMGA1 in BC were associated with decreased ability to maintain a differentiated ALI epithelium. HMGA1 down-regulation in BC diminished BC proliferation, suppressed gene expression related to normal proliferation and differentiation, decreased airway epithelial resistance, suppressed junctional and cell polarity gene expression, and delayed wound closure of airway epithelium following injury. Furthermore, silencing of HMGA1 in airway BC in ALI increased the expression of genes associated with airway remodeling in COPD including squamous, epithelial-mesenchymal transition (EMT), and inflammatory genes. Together, the data suggests HMGA1 plays a central role in normal airway differentiation, and thus caution should be used to monitor airway epithelial structure and function in the context of systemic HMGA1-targeted therapies., Competing Interests: CONFLICTS OF INTEREST None, for all authors.

The majority of dairy goat farms in Bavaria operate pasture-based systems. Endoparasites are therefore a common problem affecting health and productivity of these herds. Pooled faecal samples from 37 commercial dairy goat farms in Bavaria were examined by modified McMaster, flotation, sedimentation and Baermann funnel techniques. In addition, a questionnaire was used to gather information on farmers' perceptions and parasite management efforts. The average trichostrongyle faecal egg count across the 37 farms was 620 epg, with a median of 450 epg (1st quartile: 135 epg; 3rd quartile: 930 epg; range: < 30 to 3090 epg). Fasciola hepatica eggs were detected on four farms, Moniezia expansa eggs on one, Muellerius capillaris larvae on 13 and Dicrocoelium dendriticum eggs in none of the samples. Following coproculture third stage larvae of trichostrongyle species were identified morphologically. Sufficient larval numbers were obtained from samples from 23 farms. Haemonchus spp. was the most abundant larval genus and accounted for 30.4% of all larvae examined (n = 4868), followed by Trichostrongylus spp. (27.5%), Teladorsagia spp. (21.8%) and Oesophagostomum spp./Chabertia spp. (19.0%; these two genera were not differentiated). Further nematodes were identified according to their egg morphology: Nematodirus spp were present on nine farms, Skrjabinema spp. on nine, Trichuris spp. on five and Strongyloides spp. were not detected in any of the samples. The questionnaire results indicated a widespread lack of farmer awareness of appropriate parasite management and treatment measures. Farmer and veterinary education is therefore important to avoid future resistance problems caused by under-dosing or inappropriate treatments.

Cationic lipids are promising non-viral vectors for the cellular delivery of nucleic acids. Important considerations for the development of new delivery vectors are enhanced uptake efficiency, low toxicity and traceability. Traceable gene transfer systems however typically require the inclusion of a labeled excipient, and highly sensitive imaging instrumentation to detect the presence of the label. Recently, we reported the synthesis and characterization of colored, polyene cationic phospholipidoids composed of a rigid, polyenoic acid of predetermined dimension (C20:5 and C30:9) paired with flexible saturated alkyl chains of varying lengths (12:0, 14:0, 16:0, 18:0, 20:0 carbons). Herein, the potential of these cationic phospholipids as siRNA carriers was evaluated through standard liposomal formulations in combination with a neutral helper lipid DOPE. The polyene-based lipids were compared with a standard cationic lipid for siRNA-delivery into luciferase expressing HR5-CL11 cells. Within the series of lipids screened, knockdown results indicated that polyene cationic phospholipids paired with longer saturated alkyl chains are more effective as gene transfer agents, and perform comparably with the commercial lipid EPC. Furthermore, the chromophore associated with the polyene chain allowed tracking of the siRNA delivery using direct observation. The polyene lipoplexes were tracked on both a macroscopic and microscopic level either as a single-component or as a multi-component lipoplex formulation. When combined with a reference EPC, effective knockdown and tracking abilities were combined in a single preparation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

With the assistance of an ink-jet printer, solvent (the "ink") can be controllably and reproducibly printed onto electrospun nanofiber meshes (the "paper") to generate various micropatterns and subsequently guide distinct cellular organization and phenotype expression. In combination with the nanofiber-assisted layer-by-layer cell assembly, the patterned electrospun meshes will define an instructive microenvironment for guided tissue formation., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cationic glycol phospholipids were synthesized introducing chromophoric, rigid polyenoic C20:5 and C30:9 chains next to saturated flexible alkyl chains of variable lengths C6-20:0. Surface properties and liposome formation of the amphiphilic compounds were determined, the properties of liposome/DNA complexes (lipoplexes) were established using three formulations (no co-lipid, DOPE as a co-lipid, or cholesterol as a co-lipid), and the microstructure of the best transfecting compounds inspected using small angle X-ray diffraction to explore details of the partially ordered structures of the systems that constitute the series. Transfection and cytotoxicity of the lipoplexes were evaluated by DNA delivery to Chinese hamster ovary (CHO-K1) cells using the cationic glycerol phospholipid 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) as a reference compound. The uncontrollable self-association of the molecules in water resulted in aggregates and liposomes of quite different sizes without a structure-property relationship. Likewise, adding DNA to the liposomes gave rise to unpredictable sized lipoplexes, which, again, transfected without a structure-activity relationship. Nevertheless, one compound among the novel lipids (C30:9 chain paired with a C20:0 chain) exhibited comparable transfection efficiency and toxicity to the control cationic lipid EPC. Thus, the presence of a rigid polyene chain in this best performing achiral glycol lipid did not have an influence on transfection compared with the chiral glycerolipid reference ethyl phosphocholine EPC with two flexible saturated C14 chains., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Wound healing and cancer metastasis share a common starting point, namely, a change in the phenotype of some cells from stationary to motile. The term, epithelial-to-mesenchymal transition (EMT) describes the changes in molecular biology and cellular physiology that allow a cell to transition from a sedentary cell to a motile cell, a process that is relevant not only for cancer and regeneration, but also for normal development of multicellular organisms. The present review compares the similarities and differences in cellular response at the molecular level as tumor cells enter EMT or as keratinocytes begin the process of re-epithelialization of a wound. Looking toward clinical interventions that might modulate these processes, the mechanisms and outcomes of current and potential therapies are reviewed for both anti-cancer and pro-wound healing treatments related to the pathways that are central to EMT. Taken together, the comparison of re-epithelialization and tumor EMT serves as a starting point for the development of therapies that can selectively modulate different forms of EMT., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The success of nucleic acid delivery requires the development of safe and efficient delivery vectors that overcome cellular barriers for effective transport. Herein we describe the synthesis of a series of novel, single-chain rigid cationic carotenoid lipids and a study of their preliminary in vitro siRNA delivery effectiveness and cellular toxicity. The efficiency of siRNA delivery by the single-chain lipid series was compared with that of known cationic lipid vectors, 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) and 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (EPC) as positive controls. All cationic lipids (controls and single-chain lipids) were co-formulated into liposomes with the neutral co-lipid, 1,2-dioleolyl-sn-glycerol-3-phosphoethanolamine (DOPE). Cationic lipid-siRNA complexes of varying (+/-) molar charge ratios were formulated for delivery into HR5-CL11 cells. Of the five single-chain carotenoid lipids investigated, lipids 1, 2, 3 and 5 displayed significant knockdown efficiency with HR5-CL11 cells. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/-) molar charge ratios studied. This novel, single-chain rigid carotenoid-based cationic lipid represents a new class of transfection vector with excellent cell tolerance, accompanied with encouraging siRNA delivery efficiency.

Background: Whereas cilia damage and reduced cilia beat frequency have been implicated as causative of reduced mucociliary clearance in smokers, theoretically mucociliary clearance could also be affected by cilia length. Based on models of mucociliary clearance predicting that cilia length must exceed the 6-7 microm airway surface fluid depth to generate force in the mucus layer, we hypothesized that cilia height may be decreased in airway epithelium of normal smokers compared to nonsmokers., Methodology/principal Findings: Cilia length in normal nonsmokers and smokers was evaluated in aldehyde-fixed, paraffin-embedded endobronchial biopsies, and air-dried and hydrated samples were brushed from human airway epithelium via fiberoptic bronchoscopy. In 28 endobronchial biopsies, healthy smoker cilia length was reduced by 15% compared to nonsmokers (p<0.05). In 39 air-dried samples of airway epithelial cells, smoker cilia length was reduced by 13% compared to nonsmokers (p<0.0001). Analysis of the length of individual, detached cilia in 27 samples showed that smoker cilia length was reduced by 9% compared to nonsmokers (p<0.05). Finally, in 16 fully hydrated, unfixed samples, smoker cilia length was reduced 7% compared to nonsmokers (p<0.05). Using genome-wide analysis of airway epithelial gene expression we identified 6 cilia-related genes whose expression levels were significantly reduced in healthy smokers compared to healthy nonsmokers., Conclusions/significance: Models predict that a reduction in cilia length would reduce mucociliary clearance, suggesting that smoking-associated shorter airway epithelial cilia play a significant role in the pathogenesis of smoking-induced lung disease.

Epithelial-mesenchymal transition (EMT) is essential for organogenesis and is triggered during carcinoma progression to an invasive state. Transforming growth factor-beta (TGF-beta) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT. The SNAIL1-SMAD3/4 complex was targeted to the gene promoters of CAR, a tight-junction protein, and E-cadherin during TGF-beta-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1-SMAD3/4 transcriptional complex represents a mechanism of gene repression during EMT.

Bone marrow contains distinct microenvironments that regulate hematopoietic stem cells (HSCs). The endosteal HSC niche includes osteoblasts, mineral, and extracellular matrix proteins that interact through various molecular signals to control HSCs. Sonic hedgehog (Shh) is a morphogen involved in the regulation of skeletal development and hematopoiesis, but the effects of Shh on bone in relation to the HSC niche are not well understood. We demonstrate that systemic overexpression of Shh in mice increases osteoblast number with the resultant formation of new trabeculae in the femoral diaphysis. Suggestive of a functional change in the hematopoietic niche, numbers of Lin(-) Sca-1(+) c-Kit(+) cells with hematopoietic progenitor function expand, although cells with in vivo repopulating capacity in the wild-type environment do not increase. Instead, Shh mediates a decrease in number of bone marrow lymphocytes accompanied by a decreased expression of stromal-derived growth factor 1 (SDF-1) and a decrease in Flk2-expressing Lin(-) Sca-1(+) c-Kit(+) cells, indicating a modulation of early lymphopoiesis. This is caused by a microenvironment-induced mechanism as Shh treatment of bone marrow recipients, but not donors, results in a dramatic depletion of lymphocytes. Together, these data suggest that Shh mediates alterations in the bone marrow hematopoietic niche affecting the early lymphoid differentiation.

In general, virus infections of the brain are rather rare in the immune competent host. However, neurotropic viruses have developed mechanisms to exploit weaknesses in immunological defense mechanisms that eventually allow them to reach and infect CNS neurons. Once in the CNS, these viruses can induce significant neuronal dysfunction and degeneration of specific neuronal populations, sometimes leading to devastating, life-threatening consequences for the host. Here, we examine viruses with the ability to infect neurons and their resulting pathologies, their modes of entry to the CNS, and the cellular and molecular alterations that these viruses induce in neuronal cells. We also discuss the importance of various pathogenic events associated with viral infection of neurons and elaborate on the implications of recent findings suggesting that neuronal cells affected by viruses undergo a "dying back" pattern of degeneration. Finally, findings of virus-induced alterations in kinase activity are discussed in the context of recent evidence linking abnormalities in kinase signaling to the pathogenesis of major human neurodegenerative conditions.

Cystic fibrosis is characterized by deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) transporter. The packaging constraints of adeno-associated viral (AAV) vectors preclude delivery of both an active promoter and CFTR cDNA to target cells. We hypothesized that segmental trans-splicing, in which two AAV vectors deliver the 5' and 3' halves of the CFTR cDNA, could mediate splicing of two pre-mRNAs into a full-length, functional CFTR mRNA. Using a segmental trans-splicing 5' donor-3' acceptor pair that split the CFTR cDNA between exons 14a and 14b, cotransfection of donor and acceptor plasmids into CFTR(-) cells resulted in full-length CFTR message and protein. Microinjection of plasmids into CFTR(-) cells produced cAMP-activated Cl(-) conductance. Vectors created with an engineered human serotype, AAV6.2, were used to deliver CFTR donor and acceptor constructs, resulting in full-length CFTR mRNA and protein as well as cAMP-activated Cl(-) conductance in CFTR(-) cells, including human CF airway epithelial IB3-1 cells. Thus, segmental trans-splicing can be used with AAV vectors to mediate expression of CFTR, a strategy potentially applicable to individuals with CF.

Cigarette smoking is the major risk factor for emphysema, a disorder of the lung parenchyma characterized by destruction of the alveolar walls. Current concepts of the pathogenesis of emphysema hold that the destruction of the lung parenchyma results, in part, from a local imbalance of proteases and antiproteases. Based on the knowledge that human alveolar macrophages express ADAM 10, a protease capable of destroying basement membrane collagen but not previously implicated in emphysema, we used adenovirus-mediated lung expression of ADAM 10 in a mouse model to assess whether an increased burden of ADAM 10 was capable of inducing emphysema. To assess this, the human ADAM 10 cDNA under control of a constitutive promoter was inserted into an adenovirus gene transfer vector (AdhADAMlO), and the vector (10(11) particle units) administered to the respiratory tract of wild type C57BI/6 mice. Lung levels of ADAM 10 mRNA and protein were upregulated following AdhADAMlO administration. After 8 weeks, quantitative morphometry of the lung parenchyma demonstrated that AdhADAMlO administration induced emphysema (mean linear intercept of 60.6 + 1.3 microm compared with 55.6 + 0.8 in mice treated with a control vector, p < 0.003). These results suggest a role of ADAM 10 in the pathogenesis of emphysema, adding to the list of proteases expressed in the lung that are capable of contributing to the development of lung destruction.

Autophagy is an intracellular pathway that contributes to the degradation and recycling of unfolded proteins. Based on the knowledge that autophagy affects glycogen metabolism and that alpha(1)-antitrypsin (AAT) deficiency is associated with an autophagic response in the liver, we hypothesized that the conformational abnormalities of the Z-AAT protein interfere with hepatocyte glycogen storage and/or metabolism. Compared with wild-type mice (WT), the Z-AAT mice had lower liver glycogen stores (P < 0.001) and abnormal activities of glycogen-related enzymes, including acid alpha-glucosidase (P < 0.05) and the total glycogen synthase (P < 0.05). As metabolic consequences, PiZ mice demonstrated lower blood glucose levels (P < 0.05), lower body weights (P < 0.001), and lower fat pad weights (P < 0.001) compared with WT. After the stress of fasting or partial hepatectomy, PiZ mice had further reduced liver glycogen and lower blood glucose levels (both P < 0.05 compared WT). Finally, PiZ mice exhibited decreased survival after partial hepatectomy (P < 0.01 compared with WT), but this was normalized with postoperative dextrose supplementation. In conclusion, these observations are consistent with the general concept that abnormal protein conformation and degradation affects other cellular functions, suggesting that diseases in the liver might benefit from metabolic compensation if glycogen metabolism is affected.

The intracellular trafficking of adenovirus capsids has been described mainly through observations of trafficking by capsids from subgroup C adenoviruses in transformed cell lines. The basic elements of the trafficking pathway include high affinity binding of the adenovirus capsid to receptors at the cell surface, internalization by endocytosis, lysis of the endosomal membrane resulting in escape to the cytosol, trafficking along microtubules, binding to the nuclear envelope, and insertion of the viral genome through the nuclear pore. The net effect of this basic pathway is to deliver the adenovirus genome to the nucleus in a highly efficient manner with greater than 80% of the genome reaching the nucleus in approximately 1 h. However, exceptions to this trafficking pattern have been noted, including: (1) variations based on adenovirus serotype; (2) variations based on target cell type; and (3) variations based on cell physiology. This review summarizes the classical adenovirus infection pathway along with the exceptions to that trafficking pathway, providing an overview of intracellular trafficking of adenovirus.

Human embryonic stem cells (hESCs) theoretically represent an unlimited supply of normal differentiated cells to engineer diseased tissues to regain normal function. However, before hESCs can be useful as human therapeutics, technologies must be developed to provide them with the specific signals required to differentiate in a controlled fashion, to regulate and/or shut down the growth of hESCs and their progeny once they have been transferred to the recipient, and to circumvent the recognition of non-autologous hESC-derived cells as foreign. In the context that gene therapy technologies represent strategies to deliver biological signals to address all of these challenges, this review sets out a framework for combined gene transfer/hESC therapies. We discuss how hESCs are derived, characterized, and differentiated into specific cell lineages, and we summarize the characteristics of the 500 hESC lines reported to date. The successes and failures of gene transfer to hESCs are reviewed for both non-viral and viral vectors, as are the challenges to successful use of gene transfer in developing hESC therapy. We also consider gene transfer as a means of facilitating growth and isolation of genetically modified hESCs and as a mechanism for mitigating adverse effects associated with administration of hESCs or their derivatives. Finally, we evaluate the challenges that are likely to be encountered in translating the promise of hESCs to the clinic.

To identify genes participating in human airway epithelial repair, we used bronchoscopy and brushing to denude the airway epithelium of healthy individuals, sequentially sampled the same region 7 and 14 days later, and assessed gene expression by Affymetrix microarrays with TaqMan RT-PCR confirmation. Histologically, the injured area was completely covered by a partially redifferentiated epithelial layer after 7 days; by 14 days the airway epithelium was very similar to the uninjured state. At day 7 compared with resting epithelium, there were substantial differences in gene expression pattern, with a distinctive airway epithelial "repair transcriptome" of actively proliferating cells in the process of redifferentiation. The repair transcriptome at 7 days was dominated by cell cycle, signal transduction, metabolism and transport, and transcription genes. Interestingly, the majority of differentially expressed cell cycle genes belonged to the G2 and M phases, suggesting that the proliferating cells were relatively synchronized 1 wk following injury. At 14 days postinjury, the expression profile was similar to that of resting airway epithelium. These observations provide a baseline of the functional gene categories participating in the process of normal human airway epithelial repair that can be used in future studies of injury and repair in airway epithelial diseases.

The earliest morphologic evidence of changes in the airways associated with chronic cigarette smoking is in the small airways. To help understand how smoking modifies small airway structure and function, we developed a strategy using fiberoptic bronchoscopy and brushing to sample the human small airway (10th-12th order) bronchial epithelium to assess gene expression (Affymetrix HG-U133A and HG-133 Plus 2.0 array) in phenotypically normal smokers (n = 16, 25 +/- 7 pack-years) compared to matched nonsmokers (n = 17). Compared to samples from large (second to third order) bronchi, the small airway samples had a higher proportion of ciliated cells, but less basal, undifferentiated, and secretory cells, and contained Clara cells. Even though the smokers were phenotypically normal, microarray analysis of gene expression of the small airway epithelium of the smokers compared to the nonsmokers demonstrated up- and downregulation of genes in multiple categories relevant to the pathogenesis of chronic obstructive lung disease (COPD), including genes coding for cytokines/innate immunity, apoptosis, mucin, response to oxidants and xenobiotics, and general cellular processes. In the context that COPD starts in the small airways, these gene expression changes in the small airway epithelium in phenotypically normal smokers are candidates for the development of therapeutic strategies to prevent the onset of COPD.

Neuroendocrine differentiation is a common feature of lung cancer and increased numbers of neuroendocrine cells and their peptides have been described in chronic smokers. To understand the effects of cigarette smoking on the gene expression profile of neuroendocrine cells, microarray analysis with TaqMan confirmation was used to assess airway epithelial samples obtained by fiberoptic bronchoscopy from 81 individuals [normal nonsmokers, normal smokers, smokers with early chronic obstructive lung disease (COPD), and smokers with established COPD]. Of 11 genes considered to be neuroendocrine cell specific, only ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a member of the ubiquitin proteasome pathway, was consistently up-regulated in smokers compared with nonsmokers. Up-regulation of UCHL1 at the protein level was observed with immunohistochemical analysis of bronchial biopsies of smokers compared with nonsmokers. UCHL1 expression was evident only in neuroendocrine cells of the airway epithelium in nonsmokers; however, UCHL1 was also expressed in ciliated epithelial cells in smokers. This observation may add further weight to recent observations that ciliated cells are capable of transdifferentiating to other airway epithelial cells. In the context that UCHL1 is involved in the degradation of unwanted, misfolded, or damaged proteins within the cell and is overexpressed in >50% of lung cancers, its overexpression in chronic smokers may represent an early event in the complex transformation from normal epithelium to overt malignancy.

Neutralization of adenovirus (Ad) by anti-Ad neutralizing antibodies in serum involves formation of Ad-immune complexes that prevent the virus from interacting with target cells. We hypothesized that Ad-immune complexes likely contain viable Ad vectors which, although no longer capable of gaining access to receptors on target cells, may be able to express transgenes in cells bearing Fc receptors for immunoglobulins, i.e., that antibody-based "neutralization" of Ad vectors may be circumvented by the Fc receptor pathway. To test this hypothesis, we expressed the Fcgamma receptor IIA (FcgammaR) in A549 lung epithelial cells or human dermal fibroblasts and evaluated gene transfer in the presence of human neutralizing anti-Ad serum. FcgammaR-expressing cells bound and internalized copious amounts of Ad, with a distinct population of internalized Ad trafficking to the nucleus. The dose-response curves for inhibition of gene transfer revealed that FcgammaR-expressing cells required a more-than-10-fold higher concentration of anti-Ad serum to achieve 50% inhibition of Ad-encoded beta-galactosidase expression compared with non-FcgammaR-expressing cells. The discrepancy between neutralization of Ad during infection of FcgammaR-expressing cells and neutralization of Ad during infection of non-FcgammaR-expressing cells occurred with either heat-inactivated or non-heat-inactivated sera, was blocked by addition of purified Fc domain protein, and did not require the cytoplasmic domain of FcgammaR, suggesting that immune complex internalization proceeded via endocytosis rather than phagocytosis. FcgammaR-mediated infection by Ad-immune complexes did not require expression of the coxsackie virus-Ad receptor (CAR) since similar data were obtained when CAR-deficient human dermal fibroblasts were engineered to express FcgammaR. However, interaction of the Ad penton base with cell surface integrins contributed to the difference in neutralization between FcgammaR-expressing and non-FcgammaR-expressing cells. The data indicate that complexes formed from Ad and anti-Ad neutralizing antibodies, while compromised with respect to infection of non-FcgammaR-expressing target cells, maintain the potential to transfer genes to FcgammaR-expressing cells, with consequent expression of the transgene. The formation of Ad-immune complexes that can target viable virus to antigen-presenting cells may account for the success of Ad-based vaccines administered in the presence of low levels of neutralizing anti-Ad antibody.

von Willebrand disease (VWD), the most common inherited bleeding disorder in the U.S. population, is caused by defects in the expression and processing of von Willebrand factor (VWF), a blood glycoprotein required for normal hemostasis that mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet glycoproteins and to constituents of exposed connective tissue. To assess whether VWF deficiency can be corrected by gene transfer, a plasmid expressing the intact 8.4-kb murine VWF coding sequence, directed by the cyto-megalovirus immediate/early promoter/enhancer, was delivered through hydrodynamic tail vein injection into VWF knockout mice (VWF(-/-)) that exhibit defects in hemostasis, including highly prolonged bleeding time and spontaneous bleeding events, closely mimicking severe human VWD. VWF antigen levels in plasma from animals receiving VWF cDNA, but not control animals, revealed normalized levels of circulating VWF that persisted for at least 1 week after injection. Western blot analysis of plasma from animals receiving VWF cDNA, but not control animals, revealed high molecular-weight multimers with patterns similar to those observed in wild-type mice. Reverse transcription-polymerase chain reaction (RT-PCR) on RNA isolated from the livers of animals receiving VWF cDNA, but not control animals, demonstrated that VWF was expressed in the liver, and immunohistochemical analysis of the livers of treated VWF(-/-) mice revealed VWF-specific staining throughout the liver parenchyma but not in endothelial cells. Plasma from treated VWF(-/-) mice, but not control VWF(-/-) mice, supported the hypothesis that murine platelets aggregate in the presence of botrocetin. Although levels of circulating factor VIII in untreated VWF(-/-) mice were less than 10% those in wild-type mice, levels of factor VIII in VWF(-/-) animals treated with VWF cDNA, but not in control animals, were normalized to values in wild-type mice, indicating the restoration of factor VIII carrier function for VWF in treated mice that persisted for at least 1 week at higher doses of VWF cDNA. Most important, bleeding time was normalized by 48 hours after the delivery of VWF cDNA, but not by the control plasmid. These data suggest that with the use of gene transfer of VWF cDNA, VWF protein can be expressed, processed, and secreted in a physiologically active form; thus, it may be possible to correct VWD using gene transfer.