Differences in aphasia syndromes between progressive supranuclear palsy-Richardson's syndrome, behavioral variant frontotemporal dementia and Alzheimer's dementia.

Language impairments, hallmarks of speech/language variant progressive supranuclear palsy, also occur in Richardson's syndrome (PSP-RS). Impaired communication may interfere with daily activities. Therefore, assessment of language functions is crucial. It is uncertain whether the Aachen Aphasia Test (AAT) is practicable in PSP-RS, behavioral variant frontotemporal dementia (bvFTD) and Alzheimer's dementia (AD) and language deficits differ in these disorders. 28 PSP-RS, 24 AD, and 24 bvFTD patients were investigated using the AAT and the CERAD-Plus battery. 16-25% of all patients failed in AAT subtests for various reasons. The AAT syndrome algorithm diagnosed amnestic aphasia in 5 (23%) PSP-RS, 7 (36%) bvFTD and 6 (30%) AD patients, Broca aphasia in 1 PSP-RS and 1 bvFTD patient, Wernicke aphasia in 1 bvFTD and 3 (15%) AD patients. However, aphasic symptoms resembled non-fluent primary progressive aphasia in 14 PSP-RS patients. In up to 46% of PSP-RS patients, 61% of bvFTD and 64% of AD patients significant impairments were found in the AAT subtests spontaneous speech, written language, naming, language repetition, language comprehension and the Token subtest. The CERAD-Plus subtest semantic fluency revealed significant impairment in 81% of PSP-RS, 61% of bvFTD, 44% of AD patients, the phonemic fluency subtest in 31, 40 and 31%, respectively. In contrast to bvFTD and AD, severity of language impairment did not correlate with cognitive decline in PSP-RS. In summary, the patterns of aphasia differ between the diagnoses. Local frontal language networks might be impaired in PSP-RS, whereas in AD and bvFTD, more widespread neuropathology might underly language impairment.

Caregiver strain in progressive supranuclear palsy and corticobasal syndromes.

Progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS) progress relentlessly and lead to a need for care. Caregiving is often burdensome. Little is known about the course of caregiver burden (CB) in PSP and CBS patients. Longitudinal analysis of CB in family members caring for PSP and CBS patients. Single-center longitudinal pilot study in 68 newly diagnosed patients with probable PSP and CBS (52 Richardson's syndrome; 1 progressive gait freezing of PSP; 15 CBS). Demographic, educational, occupational parameters, family status, motor functions (UPDRSIII, Hoehn and Yahr Score, Tinetti) and neuropsychological performance (CERAD Plus, Frontal Assessment Battery) were assessed, as well as behavioral and neuropsychiatric impairments (Frontal Behavioral Inventory, Neuropsychiatric Inventory), activities of daily living (ADL) and caregiver burden using the Caregiver Strain Index (CSI), in most patients also the Zarit Burden Interview (ZBI). Patients were followed up every 6 months for up to 2 years. Caregivers reported mild to moderate CB at baseline, which increased by 25-30% in 2 years and was significantly greater in PSP than in CBS. Risk for mental health problems increased over time, especially in female caregivers (depression). Important patient-related factors were apathy, aspontaneity, depression, irritability, disorganization, poor judgment, impairment of language, impairments in ADL, a high educational level of the patient and close family relationship. Behavioral symptoms and impaired ADL are the main patient-related factors of CB in PSP and CBS. CB can be severe and needs to be assessed repeatedly from the time of diagnosis to provide comprehensive support.

Caregiver burden in patients with behavioural variant frontotemporal dementia and non-fluent variant and semantic variant primary progressive aphasia.

Studies on caregiver burden in patients with frontotemporal lobar degeneration are rare, differ methodologically and show variable results. Single center longitudinal pilot study on caregiver burden and potential risk factors in patients with behavioural variant frontotemporal dementia (bvFTD) and semantic (svPPA) and non-fluent variants (nfvPPA) primary progressive aphasia. Forty-six bvFTD, nine svPPA, and six nfvPPA patients and caring relatives were analysed for up to 2 years using the Mini-Mental State Examination as global measure for cognitive performance, Frontal Assessment Battery (frontal lobe functions), Frontal Behavioural Inventory (personality and behaviour), Neuropsychiatric Inventory (dementia-related neuropsychiatric symptoms), Barthel Index and Lawton IADL Scale (basic and instrumental activities of daily living), the Caregiver Strain Index (CSI), and in most participants also the Zarit Burden Interview (ZBI). CSI baseline sum scores were highest in bvFTD (mean ± SD 5.5 ± 3.4, median 5, IQR 6), intermediate in svPPA (2.9 ± 2.3; 3; 3.5) and low in nfvPPA (1.6 ± 2.1; 1; 2). Similar differences of caregiver burden were found using the ZBI. During follow-up, CSI and ZBI sum scores deteriorated in svPPA, not in bvFTD and nfvPPA, and correlated significantly with personality and behaviour, neuropsychiatric symptoms, caregiver age, and instrumental, but not basic activities of daily living, Mini-Mental State Examination scores or frontal lobe functions. This study reveals differences in caregiver burden in variants of frontotemporal lobar degeneration. Caregivers should be systematically asked for caregiver burden from the time of the diagnosis to provide comprehensive support in time.

Impact of labile metal nanoparticles on cellular homeostasis. Current developments in imaging, synthesis and applications.

BACKGROUND: The use of nanomaterials is constantly increasing in electronics, cosmetics, food additives, and is emerging in advanced biomedical applications such as theranostics, bio-imaging and therapeutics. However their safety raises concerns and requires appropriate methods to analyze their fate in vivo. SCOPE OF REVIEW: In this review, we describe the current knowledge about the toxicity of labile metal (ZnO, CuO and Ag) nanoparticles (NPs) both at the organism and cellular levels, and describe the pathways that are triggered to maintain cellular homeostasis. We also describe advanced elemental imaging approaches to analyze intracellular NP fate. Finally, we open the discussion by presenting recent developments in terms of synthesis and applications of Ag and CuO NPs. MAJOR CONCLUSIONS: Labile metal nanoparticles (MeNPs) release metal ions that trigger a cellular response involving biomolecules binding to the ions followed by regulation of the redox balance. In addition, specific mechanisms are set up by the cell in response to physiological ions such as Cu(I) and Zn(II). Among all types of NPs, labile MeNPs induce the strongest inflammatory responses which are most probably due to the combined effects of the NPs and of its released ions. Interestingly, recent developments in imaging technologies enable the intracellular visualization of both the NPs and their ions and promise new insights into nanoparticle fate and toxicity. GENERAL SIGNIFICANCE: The exponential use of nanotechnologies associated with the difficulties of assessing their impact on health and the environment has prompted scientists to develop novel methodologies to characterize these nanoobjects in a biological context.

Self-Organization of Long-Lasting Human Endothelial Capillary-Like Networks Guided by DLP Bioprinting.

Tissue engineering holds great promise for regenerative medicine, drug discovery, and as an alternative to animal models. However, as soon as the dimensions of engineered tissue exceed the diffusion limit of oxygen and nutriments, a necrotic core forms leading to irreversible damage. To overcome this constraint, the establishment of a functional perfusion network is essential. In this work, digital light processing bioprinting is used to encapsulate endothelial progenitor cells (EPCs) in 3D light-cured hydrogel scaffolds to guide them toward vascular network formation. In these scaffolds, EPCs proliferate and self-organize within a few days into branched tubular structures with predefined geometry, forming capillary-like vascular tubes or trees of diameters in the range of 10 to 100 µm. Presenting a confluent monolayer wall of cells strongly connect by tight junctions around a central lumen-like space, these structures can be microinjected with a fluorescent dye and are stable for several weeks in vitro. These endothelial structures can be recovered and manipulated in an alginate patch without altering their shape or viability. This approach opens new opportunities for future applications, such as stacking with other cell sheets or multicellular constructs to yield bioengineered tissue with higher complexity and functionality.

In vitro proliferation and long-term preservation of functional primary rat hepatocytes in cell fibers.

Primary hepatocytes are essential cellular resources for drug screening and medical transplantation. While culture systems have already succeeded in reconstituting the biomimetic microenvironment of primary hepatocytes, acquiring additional capabilities to handle them easily as well as to expand them remains unmet needs. This paper describes a culture system for primary rat hepatocytes, based on cell fiber technology, that brings scalability and handleability. Cell fibers are cell-laden core-shell hydrogel microfibers; in the core regions, cells are embedded in extracellular matrix proteins, cultured three-dimensionally, and exposed to soluble growth factors in the culture medium via the hydrogel shells. By encapsulating primary rat hepatocytes within cell fibers, we first demonstrated their proliferation while maintaining their viability and their hepatic specific functions for up to thirty days of subsequent culture. We then demonstrated the efficiency of proliferating primary rat hepatocytes in cell fibers not only as cell-based sensors to detect drugs that damage hepatic functions and hepatocellular processes but also as transplants to improve the plasma albumin concentrations of congenital analbuminemia. Our culture system could therefore be included in innovative strategies and promising developments in applying primary hepatocytes to both pharmaceutical and medical fields.

Impaired cellular immune response to diphtheria and tetanus vaccines in children after thoracic transplantation.

Safety and immunogenicity of diphtheria and tetanus booster vaccination were evaluated in 28 children after thoracic transplantation. Adverse events were documented in a patient diary. Blood was collected prior to and four wk after vaccination. Specific antibody concentrations were measured by ELISA. Lymphocytes were investigated for expression of activation markers (CD25, HLA-DR) by flow cytometry and proliferation assays with and without stimulation. Post-vaccination antibody titers were higher than prevaccination (p < 0.001), with more patients having protective antibody levels against diphtheria (p < 0.02) and tetanus (p < 0.001). There was no increased proliferation in non-stimulated or stimulated cultures after vaccination. The number of T-lymphocytes activated by the vaccination antigens was similar pre- and post-vaccination, whereas HLA-DR-expression on stimulated and non-stimulated CD4(+) T-cells increased significantly. Increase in antibodies was negatively correlated with tacrolimus dose, and impaired cellular immunity was associated with higher tacrolimus dose and steroid use. Adverse events were similar to the general population; serious adverse events and rejection did not occur. Vaccination with inactivated vaccines can be performed safely in immunosuppressed children after thoracic transplantation and induces protective antibody levels in the majority of patients. Impaired induction of specific cellular immunity is correlated with intensity of immunosuppression and may explain reduced sustainability of antibodies.

Trends in 3D bioprinting for esophageal tissue repair and reconstruction.

In esophageal pathologies, such as esophageal atresia, cancers, caustic burns, or post-operative stenosis, esophageal replacement is performed by using parts of the gastrointestinal tract to restore nutritional autonomy. However, this surgical procedure most often does not lead to complete functional recovery and is instead associated with many complications resulting in a decrease in the quality of life and survival rate. Esophageal tissue engineering (ETE) aims at repairing the defective esophagus and is considered as a promising therapeutic alternative. Noteworthy progress has recently been made in the ETE research area but strong challenges remain to replicate the structural and functional integrity of the esophagus with the approaches currently being developed. Within this context, 3D bioprinting is emerging as a new technology to facilitate the patterning of both cellular and acellular bioinks into well-organized 3D functional structures. Here, we present a comprehensive overview of the recent advances in tissue engineering for esophageal reconstruction with a specific focus on 3D bioprinting approaches in ETE. Current biofabrication techniques and bioink features are highlighted, and these are discussed in view of the complexity of the native esophagus that the designed substitute needs to replace. Finally, perspectives on recent strategies for fabricating other tubular organ substitutes via 3D bioprinting are discussed briefly for their potential in ETE applications.

Construction of functional biliary epithelial branched networks with predefined geometry using digital light stereolithography.

Cholangiocytes, biliary epithelial cells, are known to spontaneously self-organize into spherical cysts with a central lumen. In this work, we explore a promising biocompatible stereolithographic approach to encapsulate cholangiocytes into geometrically controlled 3D hydrogel structures to guide them towards the formation of branched tubular networks. We demonstrate that within the appropriate mix of hydrogels, normal rat cholangiocytes can proliferate, migrate, and organize into branched tubular structures with walls consisting of a cell monolayer, transport fluorescent dyes into the luminal space, and show markers of epithelial maturation such as primary cilia and continuous tight junctions. The resulting structures have dimensions typically found in the intralobular and intrahepatic biliary tree and are stable for weeks, without any requirement of bulk supporting material, thereby offering total access to the external side of these biliary epithelial constructs.

Self-Organogenesis from 2D Micropatterns to 3D Biomimetic Biliary Trees.

BACKGROUND AND AIMS: Globally, liver diseases account for 2 million deaths per year. For those with advanced liver disease the only curative approach is liver transplantation. However, less than 10% of those in need get a liver transplant due to limited organ availability. To circumvent this challenge, there has been a great focus in generating a bioengineered liver. Despite its essential role in liver functions, a functional biliary system has not yet been developed. In this framework, exploration of epithelial cell self-organogenesis and microengineering-driven geometrical cell confinement allow to envision the bioengineering of a functional biomimetic intrahepatic biliary tract. APPROACH: three-dimensional (3D) bile ducts were built in vitro by restricting cell adhesion to two-dimensional (2D) patterns to guide cell self-organization. Tree shapes mimicking the configuration of the human biliary system were micropatterned on glass slides, restricting cell attachment to these areas. Different tree geometries and culture conditions were explored to stimulate self-organogenesis of normal rat cholangiocytes (NRCs) used as a biliary cell model, either alone or in co-culture with human umbilical endothelial cells (HUVECs). RESULTS: Pre-seeding the micropatterns with HUVECs promoted luminogenesis with higher efficiency to yield functional branched biliary tubes. Lumen formation, apico-basal polarity, and preservation of the cholangiocyte phenotype were confirmed. Moreover, intact and functional biliary structures were detached from the micropatterns for further manipulation. CONCLUSION: This study presents physiologically relevant 3D biliary duct networks built in vitro from 2D micropatterns. This opens opportunities for investigating bile duct organogenesis, physiopathology, and drug testing.

Lack of serologic immunity against vaccine-preventable diseases in children after thoracic transplantation.

We investigated whether children after heart- (HTx) or heart-lung transplantation (HLTx) show protective antibody levels against recommended vaccinations, whether vaccination schedules are completed and which factors influence serologic immunity. We performed a cross sectional ELISA - quantification of specific antibodies in 46 patients after pediatric thoracic Tx. Findings were correlated to vaccination history, age at Tx, clinical course and immunosuppressive regimen. We found protective antibody levels against diphtheria in 74% of patients, against tetanus in 22%, against Haemophilus influenzae type b in 30% and against Streptococcus pneumoniae in 59%. Antibody concentrations against live attenuated vaccines were significantly lower in children transplanted in the first 2 years of life. Antibodies were absent for measles in 55% of late - and 81% of early transplanted children, for mumps in 66%/94%, for rubella in 30%/56% and for Varicella in 34%/63%. We found significant correlation of low antibody concentrations and age at Tx. Patients without protective antibody concentrations had significantly longer use of steroids. Vaccination schedules were incomplete or delayed in the majority of patients associated with more days in hospital pre-Tx. Our study shows that closer adherence to pretransplantation vaccination schedules and also post-transplantation monitoring of antibody levels are required in transplant patients.

Convergence of microengineering and cellular self-organization towards functional tissue manufacturing.

Technical progress in materials science and bioprinting has for the past few decades fostered considerable advances in medicine. More recently, the understanding of the processes of self-organization of cells into three-dimensional multicellular structures and the study of organoids have opened new perspectives for tissue engineering. Here, we review microengineering approaches for building functional tissues, and discuss recent progress in the understanding of morphogenetic processes and in the ability to steer them in vitro. On the basis of biological and technical considerations, we emphasize the achievements and remaining challenges of bringing together microengineering and morphogenesis. Our viewpoint underlines the importance of cellular self-organization for the success of tissue engineering in therapeutic applications. We reason that directed self-organization, at the convergence of microengineering and cellular self-organization, is a promising direction for the manufacturing of complex functional tissues.

Electronic sorting and recovery of single live cells from microlitre sized samples.

Sorting and recovering specific live cells from samples containing less than a few thousand cells have become major hurdles in rare cell exploration such as stem cell research, cell therapy and cell based diagnostics. We describe here a new technology based on a microelectronic chip integrating an array of over 100,000 independent electrodes and sensors which allow individual and parallel single cell manipulation of up to 10,000 cells while maintaining viability and proliferation capabilities. Manipulation is carried out using dynamic dielectrophoretic traps controlled by an electronic interface. We also demonstrate the capabilities of the chip by sorting and recovering individual live fluorescent cells from an unlabeled population.

Functional analysis of genomic DNA, cDNA, and nucleotide sequence of the mature C-type natriuretic peptide gene in vascular cells.

OBJECTIVE: The aim of this study was to investigate the effect of various C-type natriuretic peptide (CNP) sequences (genomic DNA [CNPDNA], cDNA derived from mRNA [CNPcDNA], and sequence coding for 22 amino acids of the mature CNP [CNP22aa]) on the growth of primary porcine vascular cells. METHODS AND RESULTS: Gene transfer was performed with cationic lipid DOCSPER or linear polyethylenimine. All 3 CNP sequences led to significant inhibition of smooth muscle cell (SMC) proliferation. In contrast, significant stimulation of cell growth was observed in endothelial cells (ECs) using CNPDNA or CNPcDNA but not CNP22aa. In a porcine restenosis model, a significant reduction in neointima hyperplasia was found 3 months after application of the CNPcDNA vector compared with the control transfection. CONCLUSIONS: The results demonstrate that the first intron in the CNP sequence does not contain any additional enhancer-binding sites. However, the signal sequence is indispensable for secretion of CNP and its appropriate physiological function. Furthermore, the results show for the first time the therapeutic effect of CNP using liposome-mediated gene transfer and local adventitial delivery. Advantages of the CNP gene are its dual effects with inhibition of SMC proliferation and simultaneous promotion of EC growth. Functional analysis of various C-type natriuretic peptide (CNP) sequences on growth of vascular cells. For the first time, dual therapeutic effects of CNP with inhibition of smooth muscle cell proliferation and stimulation of re-endothelialization were demonstrated in a pig restenosis model using liposome-mediated gene transfer and local adventitial delivery.

Optimization of nonviral transfection: variables influencing liposome-mediated gene transfer in proliferating vs. quiescent cells in culture and in vivo using a porcine restenosis model.

Cationic liposomes/DNA complexes are widely used vectors for delivering genes in clinical and experimental trials. Relatively low transfer efficiencies in vivo compared with viral gene transfer may be improved using local application. In addition, markedly increased transfer efficiency may be achieved in vitro and in vivo via optimization of known variables influencing liposomal transfection. Lipofection under different conditions was performed in various cell lines and primary porcine smooth muscle cells. Optimized conditions found in vitro were verified in vivo using a porcine restenosis model. Toxicity was monitored analyzing cell metabolism. Transfer efficiency and safety were determined using morphometry, histology, galactosidase assays, PCR, and RT-PCR. The most important variables enabling maximum transfer efficiency were firstly the appropriate selection of cationic lipids for the cell type to be transfected, secondly the DNA/liposome ratio chosen, which depended on the cell type and cationic lipids used, and thirdly the state of proliferation of the targeted cells. Transfection in vivo demonstrated two- to fivefold higher transfer efficiencies when transfer conditions were extrapolated from optimization experiments in stationary cells compared with the use of conditions established in proliferating cells. Application of the therapeutic gene for cecropin using optimized transfer conditions resulted in a significantly reduced neointima formation compared with the transfection using a control gene for ss-galactosidase. Thus, in this vascular model, initial optimization of lipofection in stationary cells in culture followed by local delivery in vivo and with selection of a suitable therapeutic gene led to markedly improved transfer efficiencies, gene expression, and biological effect. Stationary cell cultures simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments. In addition, the advantages of liposomes are easy handling, low toxicity, and the lack of carcinogenicity or immunogenic reactions.

Heart transplantation: an approach to treating primary cardiac sarcoma?

Treating rare, primary cardiac soft-tissue sarcomas (C-STS) with heart transplantation (HTx) is controversial. Conventional tumor resection only partially alleviates the disease, and patients die of local recurrence of the tumor or of distant metastases. Heart transplantation offers the opportunity to eradicate the primary malignancy completely. In our experience of 4 patients with C-STS indicates, HTx followed by post-operative chemotherapy does not affect the long-term outcome. However, pre-operative chemotherapy can regress tumors in chemosensitive C-STS and potentially eradicate early micrometastases. Consecutive HTx for responders may then offer a chance of long-term survival.

Vascular endothelial growth factor response in porcine coronary and peripheral arteries using nonsurgical occlusion model, local delivery, and liposome-mediated gene transfer.

Angiogenesis and arteriogenesis play an important role in advanced vascular occlusive diseases. Whether angiogenesis or arteriogenesis predominate depends on the preexisting collateral vessel network, the type and location of occlusion, and different developmental origin of the arteries. Angiogenesis and arteriogenesis were investigated following vascular endothelial growth factor (VEGF) treatment in different arteries important in occlusive arterial diseases using a newly developed porcine arterial occlusion model. Porcine coronary and peripheral arteries were occluded interventionally using blinded stent grafts. Gene transfer was performed using a needle injection catheter and cationic lipid DOCSPER as gene carrier. DNA and gene expression in arterial tissue was examined using polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR. Vessel development was determined by angiography, immunohistochemistry, and measurement of capillary density. The transfected gene and its expression were found 3 months following application. In tissue adjacent to coronary arteries, there was significantly enhanced capillary density but no increase in angiographic score. In contrast, tissue surrounding peripheral arteries demonstrated no enhancement of capillary density but an enhancement in angiographic score. These results demonstrate differential responses to VEGF treatment in coronary and peripheral arteries resulting predominantly in either angiogenesis or arteriogenesis. Further investigation of VEGF signaling pathway is necessary for better understanding of the processes of vascular development, which may have potential impact on the design of cardiovascular therapeutics.

Interleukin-6 and procalcitonin in serum of children undergoing cardiac surgery with cardiopulmonary bypass.

OBJECTIVE: The aim of our study was to investigate the systemic inflammatory response in children with congenital heart disease undergoing surgical correction with cardiopulmonary bypass. We wanted to discuss interleukin 6 and procalcitonin as components of the systemic inflammatory response syndrome to cardiopulmonary bypass and evaluate postoperative kinetics of these parameters in case of an uncomplicated course. METHODS: Procalcitonin and interleukin 6 were determined before and after cardiopulmonary bypass surgery in 37 children on the day of surgery, the first and fourth postoperative day. The increased procalcitonin and interleukin 6 levels were evaluated in relationship to intraoperative variables such as duration of aortic cross clamping, incisional trauma and cardiac bypass temperature. RESULTS: Peak levels of procalcitonin were detected on the first postoperative day, while interleukin 6 reached its highest values on the day of surgery. In contrast to interleukin 6 the median values of procalcitonin differed significantly between short versus long aortic clamping time and atriotomy versus ventriculotomy. Interleukin 6 reached normal levels on the fourth postoperative day, while procalcitonin was still clearly above normal. CONCLUSIONS: Serum concentrations of procalcitonin and interleukin 6 were influenced by systemic inflammatory response syndrome following cardiac surgery with cardiopulmonary bypass. Even in case of an uncomplicated course both parameters were elevated for at least four days. While procalcitonin serum concentrations were dependent on aortic clamping time or incisional trauma, interleukin 6 showed no significant relation with these intraoperative variables.

Improved visualization and quantitative analysis of drug effects using micropatterned cells.

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck (1-2). To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.