4-step Protocol Research Articles

Intraoperative ultrasonography in microsurgical resection of vestibular schwannomas via retrosigmoid approach: surgical technique and proof-of-concept illustrative case series.

Intraoperative ultrasonography (ioUS) is an established tool for the real-time intraoperative orientation and resection control in intra-axial oncological neurosurgery. Conversely, reports about its implementation in the resection of vestibular schwannomas (VS) are scarce. The aim of this study is to describe the role of ioUS in microsurgical resection of VS. ioUS (Craniotomy Transducer N13C5, BK5000, B Freq 8MHz, BK Medical, Burlington, MA, USA) is integrated into the surgical workflow according to a 4-step protocol (transdural preresection, intradural debulking control, intradural resection control, transdural postclosure). Illustrative cases of patients undergoing VS resection through a retrosigmoid approach with the use of ioUS are showed to illustrate advantages and pitfalls of the technique. ioUS allows clear transdural identification of the VS and its relationships with surgically relevant structures of the posterior fossa and of the cerebellopontine cistern prior to dural opening. Intradural ioUS reliably estimates the extent of tumor debulking, thereby helping in the choice of the right moment to start peripheral preparation and in the optimization of the extent of resection in those cases where subtotal resection is the ultimate goal of surgery. Transdural postclosure ioUS accurately depicts surgical situs. ioUS is a cost-effective, safe, and easy-to-use intraoperative adjunctive tool that can provide a significant assistance during VS surgery. It can potentially improve patient safety and reduce complication rates. Its efficacy on clinical outcomes, operative time, and complication rate should be validated in further studies.

Hand hygiene in a clinical setting: Evaluation of an electronic monitoring system in relation to direct observations

This study evaluated the accuracy of an electronic monitoring system for assessing hand hygiene in a clinical setting in relation to direct observations. An electronic monitoring system was installed in a surgical ward at a tertiary hospital in Sweden. Hand hygiene events registered by the system were compared with direct observations conducted simultaneously by a trained observer following the World Health Organization's "My five moments for hand hygiene". A 4-step protocol was developed to evaluate the system's ability to accurately monitor hand hygiene in a clinical setting. A total of 947 opportunities for hand hygiene were observed during June to December 2019. Of these, 484 opportunities were correctly captured by the electronic monitoring system and included in the calculations for accuracy. Sensitivity was 90.2% with a positive predictive value of 95.7%, while specificity was 50.0% with a negative predictive value of 29.0%. Overall accuracy was 87.1%. Evaluating technical systems assessing hand hygiene in clinical settings is hampered by several challenges. The 4-step protocol helped to identify the limitations and strengths of the evaluated electronic monitoring system, revealing high accuracy while also detecting system errors.

Purification and thermodynamic characterization of acid protease with novel properties from Melilotus indicus leaves

A thermostable acid protease from M. indicus leaves was purified 10-fold using a 4-step protocol. We were able to isolate a purified protease fraction with a molecular weight of 50 kDa and exhibited maximal protease activity at pH 4.0 and 40 °C. Structural analysis revealed that the protease is monomeric and non-glycosylated. The addition of epoxy monocarboxylic acid, iodoacetic acid, and dimethyl sulfoxide significantly reduced protease activity while dramatically increasing the inhibition of Mn2+, Fe2+, and Cu2+. The activation energy of the hydrolysis reaction (33.33 kJ mol−1) and activation energy (Ed = 105 kJ mol−1), the standard enthalpy variation of reversible protease unfolding (2.58 kJ/mol) were calculated after activity measurements at various temperatures. Thermal inactivation of the pure enzyme followed first-order kinetics. The half-life (t1/2) of the pure enzyme at 50 °C, 60 °C, and 70 °C was 385, 231, and 154 min, respectively. Thermodynamic parameters (entropy and enthalpy) suggested that the protease was highly thermostable. This is the first report on the thermodynamic parameters of proteases produced by M. indicus. The novel protease appears to be particularly thermostable and may be important for industrial applications based on these thermodynamic properties.

Similar Biomechanics Between the Double-Cortical Button and Docking Techniques for Ulnar Collateral Ligament Reconstruction: A Cadaveric Evaluation.

The docking technique is widely used to perform ulnar collateral ligament (UCL) reconstructions because of its high failure torque and reliable clinical outcomes. A double-cortical button technique was recently described, with advantages including the ability to tension the graft at the ulnar and humeral attachments and the creation of single bone tunnels. To compare the biomechanics between the docking and double-button UCL reconstruction techniques using cadaveric specimens. We hypothesized that there would be no difference in postoperative stiffness or maximum strength between the techniques. Controlled laboratory study. Eight matched pairs of cadaveric elbow joints underwent controlled humeral valgus torsion cycles in a test frame. Toe region stiffness, elastic region stiffness, and maximum torque were measured during a 4-step protocol: intact, injured, reconstructed (10 and 1000 cycles), and ramp to failure. Graft strains were calculated using 3-dimensional motion capture. After 10 cycles, intact ligaments from the docking and double-button groupsexhibited mean ± SD elastic torsional stiffness of 1.60 ± 0.49 and 1.64 ± 0.35 N·m/deg (P = .827), while docking (1.10 ± 0.39 N·m/deg) and double-button (1.05 ± 0.29 N·m/deg) reconstructions were lower (P = .754). There were no significant differences in maximum torque between the docking (3.45 ± 1.35 N·m) and double-button (3.25 ± 1.31 N·m) groups (P = .777). Similarly, differences in maximum graft strains were not significant between the docking (8.1% ± 7.2%) and double-button (5.5% ± 3.1%) groups (P = .645). The groups demonstrated similar decreases in these measures after cyclic loading. Ramp-to-failure testing showed no significant differences in ultimate torque between the docking (8.93 ± 3.9 N·m) and double-button (9.56 ± 3.5 N·m) groups (P = .739). The biomechanical behavior of the double-button technique was not significantly different from that of the docking technique. Both reconstruction techniques restored joint stability, but neither fully recapitulated preinjury joint stiffness. With its procedural advantages, results preliminarily support the use of the double-button reconstruction technique for UCL reconstruction as a reliable single-tunnel technique for primary or revision cases.

Embedding Wound Hygiene into a proactive wound healing strategy.

Since a panel published the first consensus document on Wound Hygiene in March 2020, there has been a flurry of activity in support of this newly established concept in proactive wound healing.1 The document concluded that all wounds, particularly hard-to-heal ones, will benefit from Wound Hygiene, which should be initiated at the first referral, following a full holistic assessment to identify the wound aetiology and comorbidities, and then implemented at every dressing change until full healing occurs.1 The consensus has since been bolstered by educational webinars; competency-based skills training and support; development of international Wound Hygiene ambassadors; a survey of 1478 respondents, published in July 2021;2 and a case study supplement, published in January 2022, featuring a range of wound types, anatomies and underlying conditions on the improvements in wound-healing progress that can be achieved.3 Wound Hygiene has gained its own identity and is now a term in and of itself, that encompasses a 4-step protocol of care. It is an antibiofilm approach that is increasingly being used across wound care. The results of the survey2 were particularly encouraging for seeing how far Wound Hygiene has come, and how quickly: More than half (57.4%) had heard of the concept of Wound Hygiene Of those, 75.3% have implemented Wound Hygiene Overall, following implementation of Wound Hygiene, 80.3% of respondents reported improved healing rates.2 However, the top three barriers identified by the survey-lack of confidence, competence and research data-show that there is more to be done to support Wound Hygiene in practice.2 As a result, a consensus panel of international key opinion leaders convened virtually in the summer of 2021 to discuss what has been done so far, the outputs of the survey, and ideas for addressing the unmet needs identified by the results. The result is this publication, which represents an addendum to the initial consensus document, broadening support for implementation of Wound Hygiene. This document will reflect on the reasons Wound Hygiene has been successful in its first two years of implementation, reiterating its DNA: Do not wait to treat hard-to-heal wounds Use a simple 4-step approach Enable all healthcare professionals to implement and use Wound Hygiene. The document will also discuss the evolution of the Wound Hygiene concept, focusing on how and when to implement Wound Hygiene on all tissue types of hard-to-heal wounds, and proposing what these are. The panel has expanded the framework in which Wound Hygiene is used, with the ultimate objective of introducing the concept of 'embedding Wound Hygiene intro a proactive wound healing strategy.' Key inefficiencies are often observed along the journeys of people living with hard-to-heal wounds. The limited number of specialised healthcare professionals and the resulting delays in reaching them may increase the likelihood of a hard-to-heal wound developing. In a world where so much is happening so quickly that we may, at times, feel powerless to drive change, the panel wants to provide further guidance to propel the use of Wound Hygiene. The concept of Wound Hygiene is resonating, and the panel wants you to know that in whatever region you work, in whatever area of clinical practice, you are enabled to make this change. Wielding the 4-step Wound Hygiene protocol consistently is a key action every healthcare professional in every care setting can take to tackle the global wound care crisis. Wound Hygiene has taken off-now, where do we want to land? In a place where Wound Hygiene is practised on all wounds, at every stage, until healing. The panel once again recognises that the community of global healthcare providers should consider their local standards and guidelines when applying the recommendations of this document. To this end, the panel has created a flexible 3-phase framework that situates Wound Hygiene as integral to proactive wound healing. The panel hopes you will continue to implement Wound Hygiene and see the benefits it can bring to people living with wounds, as well as those who care for them.

Graded coronavirus disease 2019 vaccine administration: A safe alternative to vaccine avoidance

Graded coronavirus disease 2019 vaccine administration: A safe alternative to vaccine avoidance

The National Inpatient Sample: A Primer for Neurosurgical Big Data Research and Systematic Review

The National Inpatient Sample (NIS) (the largest all-payer inpatient database in the United States) is an important instrument for big data analysis of neurosurgical inquiries. However, earlier research has determined that many NIS studies are limited by common methodological pitfalls. In this study, we provide the first primer of NIS methodological procedures in the setting of neurosurgical research and review all reported neurosurgical studies using the NIS. We designed a protocol for neurosurgical big data research using the NIS, based on our subject matter expertise, NIS documentation, and input and verification from the Healthcare Cost and Utilization Project. We subsequently used a comprehensive search strategy to identify all neurosurgical studies using the NIS in the PubMed and MEDLINE, Embase, and Web of Science databases from inception to August 2021. Studies underwent qualitative categorization (years of NIS studied, neurosurgical subspecialty, age group, and thematic focus of study objective) and analysis of longitudinal trends. We identified a canonical, 4-step protocol for NIS analysis: study population selection; defining additional clinical variables; identification and coding of outcomes; and statistical analysis. Methodological nuances discussed include identifying neurosurgery-specific admissions, addressing missing data, calculating additional severity and hospital-specific metrics, coding perioperative complications, and applying survey weights to make nationwide estimates. Inherent database limitations and common pitfalls of NIS studies discussed include lack of disease process-specific variables and data after the index admission, inability to calculate certain hospital-specific variables after 2011, performing state-level analyses, conflating hospitalization charges and costs, and not following proper statistical methodology for performing survey-weighted regression. In a systematic review, we identified 647 neurosurgical studies using the NIS. Although almost 60% of studies were reported after 2015, <10% of studies analyzed NIS data after 2015. The average sample size of studies was 507,352 patients (standard deviation= 2,739,900). Most studies analyzed cranial procedures (58.1%) and adults (68.1%). The most prevalent topic areas analyzed were surgical outcome trends (35.7%) and health policy and economics (17.8%), whereas patient disparities (9.4%) and surgeon or hospital volume (6.6%) were the least studied. We present a standardized methodology to analyze the NIS, systematically review the state of the NIS neurosurgical literature, suggest potential future directions for neurosurgical big data inquiries, and outline recommendations to improve the design of future neurosurgical data instruments.

A Dutch Prediction Tool to Assess the Risk of Incidental Gallbladder Cancers in Cholecystectomies for Benign Gallstone Disease

Introduction: A selective histopathologic (Sel-HP) policy of gallbladder specimens following cholecystectomy is increasingly implemented in low incidence countries. However, the fear of missing incidental gallbladder cancer (iGBC) persists and thereby restrict further implementation of a Sel-HP policy. This study aims to develop a diagnostic prediction model for selecting gallbladders in need of histopathologic examination after cholecystectomy. Method: A registration based retrospective cohort study of nine hospitals in the Southern part of the Netherlands between 2004 and 2014 was used. Five potential clinical predictors for gallbladder cancer (GBC) were selected based on previous literature and expert opinion. The prediction model was internally validated by bootstrapping. Discriminative capacity and accuracy were tested by assessing the area under the receiver operating characteristic curve (AUC) Nagelkerke Pseudo-R2 and Brier score. Finally, a clinical 4-step protocol was formulated using radiological, clinical indications correlated with high susceptibility for GBC, the clinical prediction model and finally the macroscopic examination of the gallbladder. Results: The prediction model was developed using a cohort of 22025 gallbladders containing 75 GBC cases. A model was developed including the following variables: age, sex, urgency, type of surgery and indication for surgery. The AUC was 90.3% (95% confidence interval 86.2% – 94.4%), indicating a good discriminative ability of the model. The calibration plot indicated good calibration of the predicted probabilities. Conclusions: We developed a clinical prediction model for selecting gallbladder specimens for histopathologic examination after cholecystectomy for ruling out GBC. This protocol will support surgeons in selecting appropriate gallbladders specimens for selective pathologic policy.

The Effect of Early Mobilization on Respiratory Parameters of Mechanically Ventilated Patients With Respiratory Failure.

The effect of early mobilization on hemodynamic parameters of patients under mechanical ventilation has been associated with positive results and yet its effect on specific respiratory parameters is less well appreciated. This article reports the results of a study of a randomized clinical trial of intensive care unit patients receiving mechanical ventilation. The findings of this study confirmed that a 4-step protocol for early mobilization can improve Pao2, O2 saturation, Pao2/Fio2 (fraction of inspired oxygen) ratio, and pulmonary compliance. The value of interdisciplinary collaboration supporting early mobilization was confirmed.

A novel approach for studying mast cell–driven disorders: Mast cells derived from induced pluripotent stem cells

Mast cells (MCs) are considered the main effectors in allergic reactions and well known for their contribution to the pathogenesis of various inflammatory diseases, urticaria, and mastocytosis. To study their functions invitro, human primary MCs are isolated directly from several tissues or differentiated from hematopoietic progenitors. However, these techniques bear several disadvantages and challenges including low proliferation capacity, donor-dependent heterogeneity, and the lack of a continuous cell source. To address this, we developed a novel strategy for the rapid and efficient differentiation of MCs from human-induced pluripotent stem cells (hiPSCs). A 4-step protocol for the generation of hiPSC-derived MCs, based on the use of 3 hiPSC lines, was established and validated by comparison with human skin MCs and peripheral hematopoietic stem cell-derived MCs. hiPSC-MCs share phenotypic and functional characteristics of human skin MCs and peripheral hematopoietic stem cell-derived MCs. They display stable expression of the MC-associated receptors CD117, FcεRIα, and Mas-related G protein-coupled receptor X2 and degranulate in response to IgE/anti-IgE and substance P. This novel hiPSC-based approach provides a sustainable and homogeneous source for a rapid and highly productive generation of phenotypically mature, functional MCs, and its principle allows for the investigation of disease- and patient-specific MC populations.

Isocyanate derivatization coupled with phospholipid removal microelution-solid phase extraction for the simultaneous quantification of (S)-metoprolol and (S)-α-hydroxymetoprolol in human plasma with LC–MS/MS

A sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS) assay was developed and validated for the quantification of (S)-metoprolol (MET) and its main metabolite, (S)-α-hydroxymetoprolol (OH-MET). Human plasma samples (50 μL) were spiked with both analytes and their deuterated internal standards (IS) (S)-MET-(d7) and α-OH-MET-(d5). Phospholipid removal microelution-solid phase extraction (PRM-SPE) was performed using a 4-step protocol with Oasis PRiME MCX μElution 96-well cartridges. The eluates were reconstituted in 100 μL of acetonitrile with 50 μg/mL (S)-α-methylbenzyl isocyanate (MBIC) for chiral derivatization. After 60 min at room temperature, the reaction was quenched using 100 μL of water 2 % formic acid. Chromatographic separation of the derivatized analytes was performed on a Kinetex phenyl-hexyl core-shell stationary phase with an elution gradient. Mobile phases were composed of a mixture of water and methanol, with ammonium formate and formic acid as buffers. Total runtime was 15 min. Analyte detection was performed by an AB/SCIEX 4000 QTRAP mass spectrometer with multiple reaction monitoring. Chromatograms showed MBIC successfully reacted with racemic MET, α-OH-MET, and their respective IS. Detection by positive electrospray ionization did not reveal derivatized by-products. Quantification ranges were validated for (S)-MET and (S)-α-OH-MET between 0.5–500 and 1.25−500 ng/mL, respectively, with correlation coefficients (r2) >0.9906. The PRM-SPE assay showed low matrix effects (86.9–104.0 %) and reproducible recoveries (69.4–78.7 %) at low, medium, and high quality control (QC) levels. Precision and accuracy were all comprised between 85–115 % for all three QCs, and between 80–120 % for the lower limit of quantification, for intra- and inter-day values (n = 6, 3 consecutive days). Non-derivatized analytes were stable at room temperature, after 3 freeze-thaw cycles, and stored for 30 days at −80 °C (n = 4). Reinjection reproducibility of a previously validated batch was achieved after 8 days under auto-sampler conditions, indicating the stability of (S)-MET and (S)-α-OH-MET derivatives. Its clinical use was established in a cohort of 50 patients and could be used to further investigate the clinical impact of (S)-MET concentrations.

Simulated clinical adjustment and intra-oral polishing of two translucent, monolithic zirconia dental ceramics: An in vitro investigation of surface roughness

ObjectiveTo assess the surface roughness and residual flaws in two translucent zirconia substrates following simulated clinical adjustment and intra-oral finishing/polishing. Materials and methodsSpecimens were prepared from two translucent (5Y-ZP/8Y-ZP) and one conventional (3Y-TZP) zirconia substrates (n = 84 p/g). Arithmetic mean roughness parameter (Ra) was determined for all experimental groups at four stages (n = 21 p/g): (1) as-sintered, (2) surface grinding using a zirconia cutting diamond bur, (3) polishing using 1-step, 2-step or 4-step intra-oral polishing kits, and (4) laboratory polishing/glazing. Scanning electron microscopy (SEM) was used to evaluate residual surface flaws. Data were statistically analysed using a two-way ANOVA test, multiple and pairwise comparisons were performed using a Bonferroni post hoc test. ResultsRa was significantly higher for as-sintered and ground 5Y-ZP/8Y-ZP compared to 3Y-TZP (p ≤ 0.001). Material type and polishing protocol had statistically significant effect on Ra (p < 0.001). The 4-step polishing resulted in the lowest Ra in 3Y-TZP/5Y-ZP (p ≤ 0.001). No significant differences were observed between different polishing protocols in 8Y-ZP (p≥0.655). Glazing significantly reduced Ra in all materials compared to other polishing protocols (p ≤ 0.001) except for the 4-step protocol in 3Y-TZP (p = 0.195). SEM revealed pronounced surface flaws in ground 5Y-ZP/8Y-ZP. Grinding of 3Y-TZP induced ductile material removal and thereby, minimal cohesive material loss. The 4-step polishing protocol resulted in the fewest and shallowest surface flaws in all groups. Glazing of 3Y-TZP resulted in the most homogenous surface contrary to 5Y-ZP/8Y-ZP. ConclusionsThe finest surface finish was achieved using the 4-step polishing protocol in all studied materials. This protocol might be inadequate however for polishing of ground 5Y-ZP/8Y-ZP as a result of the pronounced surface damage induced by the grinding process. The glazing of 5Y-ZP/8Y-ZP might not be as predictable as for 3Y-TZP. Clinical significanceThe higher susceptibility to surface damage, as a result of increasing dopant concentration, may render polishing of ground, translucent zirconia more complicated compared to the conventional 3Y-TZP counterparts.

SU32 - PATIENT-SPECIFIC AD IN VITRO MODELS FOR THE ANALYSIS OF LATE-ONSET ALZHEIMER'S DISEASE

Background Alzheimer´s Disease (AD) is the most common form of dementia. Environmental and genetic factors contribute to the risk for AD, but the underlying disease mechanisms are poorly understood. AD is genetically complex and shows heritability of up to 80%. Glia cells including astrocytes and microglia are affected in AD patients contributing to the tau pathology and the accumulation of neurotoxic amyloid as well. In recent years, Genome-Wide Association Studies (GWAS) allowed the identification of DNA variations associated with an elevated risk for AD. Together with other groups, we identified a number of AD susceptibility genes including CD33, SORL1, ABCA7 and TREM2, which highly recommends that the immune system plays a major role in onset, progression and treatment of AD. Reprogramming of EBV-transformed patient specific cell lines for the generation of induced pluripotent stem cells (iPS) enables the patient-specific differentiation into neurons, astrocytes, and microglia. Patient-specific cells can be used as a model to functionally characterize disease associated variants. Methods Blood was collected from AD patients carrying risk variants in AD susceptibility genes for the generation of iPS cells. SNP variants in those genes were genotyped based on their potential function according to literature and genomic localization (exonic non-synonymous, binding domain, or promoter affecting SNPs). Reprogrammed iPS cells carried the genetic background of a certain AD patient. Pluripotency was characterized by alkaline phosphatase staining, the expression of pluripotency markers, and the differentiation into the three germ layers. Crucial pluripotency markers are OCT4, SOX2, NANOG, KLF4, MYC, and LIN28. AD iPS cells were differentiated into astrocytes and microglia and the differentiation status was characterized by the expression of crucial glia cell markers. Further, AD susceptibility genes recently published in GWAS were analysed. Results The protein expression was successfully induced shown by immunofluorescence and western-blot analysis. Cells were also screened for the most efficient induction of the three germ layers and the induction of neural cell fates including glia cell fates. The established AD-specific iPS cell lines from AD patients represent a powerful tool for the analysis of molecular and cellular disease mechanisms. We established 4-step protocol for the generation of AD-specific microglia enabling the focused analysis of AD-associated risk variants. The protocol was verified by morphology, FACS analysis, immunofluorescence and RNA expression of crucial microglia marker, including hematopoetic lineage markers and the induction of well-described microglia markers including IBA1. The disease-specific analysis of the genetic background of AD patients represents a completely new approach for the understanding of AD genetics and AD-associated risk variants. Discussion Together, combining molecular genetics of AD for the investigation of risk variants and iPS cell technology for the generation of patient- and disease-specific stem cells provides a promising approach to characterize known disease mechanisms, to deepen the understanding of known disease mechanisms, and to discover unknown disease aspects.

4-Step Protocol for Disparities in STEMI Care and Outcomes in Women

BackgroundWomen with ST-segment elevation myocardial infarction (STEMI) receive suboptimal care and have worse outcomes than men. Whether strategies to reduce STEMI care variability impact disparities in the care and outcomes of women with STEMI is unknown. ObjectivesThe study assessed the care and outcomes of men versus women with STEMI before and after implementation of a comprehensive STEMI protocol. MethodsOn July 15, 2014, the authors implemented: 1) emergency department catheterization lab activation; 2) STEMI Safe Handoff Checklist; 3) immediate transfer to an immediately available catheterization lab; and 4) radial first approach to percutaneous coronary intervention (PCI). The authors prospectively studied consecutive patients with STEMI and assessed guideline-directed medical therapy (GDMT) before PCI, median door-to-balloon time (D2BT), in-hospital adverse events, and 30-day mortality stratified by sex before (January 1, 2011 to July 14, 2014; control group) and after (July 15, 2014 to December 31, 2016) implementation of the STEMI protocol. ResultsOf 1,272 participants (68% men, 32% women), women were older with more comorbidities than men. In the control group, women had less GDMT (77% vs. 69%; p = 0.019) and longer D2BT (median 104 min; [interquartile range (IQR): 79 to 133] min vs. 112 [IQR: 85 to 147] min; p = 0.023). Women had more in-hospital stroke, vascular complications, bleeding, transfusion, and death. In the comprehensive 4-step STEMI protocol, sex disparities in GDMT (84% vs. 80%; p = 0.32), D2BT (89 [IQR: 68 to 106] min vs. 91 [IQR: 68 to 114] min; p = 0.15), and in-hospital adverse events resolved. The absolute sex difference in 30-day mortality decreased from the control group (6.1% higher in women; p = 0.002) to the comprehensive 4-step STEMI protocol (3.2% higher in women; p = 0.090). ConclusionsA systems-based approach to STEMI care reduces sex disparities and improves STEMI care and outcomes in women.

Ultrasonographic Evaluation of the Anatomy of the Foal Foot From 0 to 7 Months

This study aimed to provide an ultrasonographic description of the anatomical development of the foal foot from 7 months pre- to 7 months postpartum. The right forelimbs of 20 foals (age range, 7-month fetus to 7-month foal) without orthopedic disease and which died for reasons unrelated to this study were examined by an experienced ultrasound operator, and reference images were obtained for every age. A 4-step protocol was used to assess images of the complete foal foot, and these were compared with macroscopic dissection, performed using a standardized protocol. It was concluded that ultrasound images correlated well with macroscopic dissection.

Improvement of mesenchymal stem cell differentiation into the endoderm lineage by four step sequential method in biocompatible biomaterial.

Introduction: The goal of the study described here, was to investigate the potential of umbilical cord derived mesenchymal stem cell (UC-MSCs) into hepatocyte like cells in a sequential 2D and 3D differentiation protocols as alternative therapy. Methods: Mesenchymal stem cells (MSCs) were isolated from the umbilical cord (UC) and CD markers were analyzed by flow cytometry. For hepatic differentiation of UC-MSCs, cells were induced with a sequential 4-step protocol in 3D and 2D culture system. Urea concentration and albumin secretion into the culture medium was quantified by ELISA. Gene expression levels of AFP, ALB, and CK18 were determined by RT-PCR. Data were statistically analyzed by the SPSS software. The difference between the mean was considered significant when p < 0.05. Results: Growth factor dependent morphological changes from elongated fibroblast-like cells to round epithelial cell morphology were observed in 2D culture. Cell proliferation analysis showed round-shaped morphology with clear cytoplasm and nucleus on the alginate scaffold in 3D culture. The mean valuses of albumin production and urea secretion were significantly higher in the 3D Culture system when compared with the 2D culture (p = 0.005 vs p = 0.001), respectively. Treatment of cells with TSA in the final step of differentiation induced an increased expression of CK18 and a decreased expression of αFP in both the 3D and 2D cultures (p = 0.026), but led to a decreased albumin gene expression, and an increased expression in the 2D culture (p = 0.001). Conclusion: Findings of the present study indicated that sequential exposure of UC-MSCs with growth factors in 3D culture improves hepatic differentiation.

In vitro Differentiation of Human Adipose Tissue-Derived Stem Cells into Islet-Like Clusters Promoted by Islet Neogenesis-Associated Protein Pentadecapeptide

Human adipose tissue-derived stem cells (hASCs) are considered an ideal tool for the supply of insulin-producing cells to treat diabetes mellitus, with high differentiation efficiency. Islet neogenesis-associated protein (INGAP) is an initiator of islet neogenesis, and the peptide sequence comprising amino acids 104-118, named INGAP pentadecapeptide (INGAP-PP), has been shown to increase β-cell mass in animals and human pathological states. Here, we report a novel 4-step method to promote hASCs to differentiate into islet-like clusters (ILCs) more efficiently by adding INGAP-PP. The hASCs were isolated, purified and differentiated using a 4-step protocol including trichostatin A, INGAP-PP/scrambled peptide (Scrambled-P), dexamethasone, nicotinamide, glucagon-like peptide-1, transforming growth factor β1 and exendin-4. Results showed that ILCs in the INGAP-PP group were more similar to the fresh islets with regard to both size and morphology and expressed significantly higher levels of both insulin and C-peptide than those in the Scrambled-P group. Moreover, the ILCs from the INGAP-PP group secreted higher levels of insulin and C-peptide than those from the Scrambled-P group in response to both a low (5.6 m<smlcap>M</smlcap>) and high (25 m<smlcap>M</smlcap>) glucose challenge and secreted 6 times more hormones under the high-glucose challenge. Real-time PCR and immunocytochemistry showed that ILCs of the INGAP-PP group expressed human pancreatic endocrine hormones and transcription factors. Transplantation of ILCs into diabetic rats partially reversed diabetes and prolonged their life span. In conclusion, the INGAP-PP protocol can efficiently induce hASCs to differentiate into ILCs in vitro, and thus hASCs could be a promising source of cells for transplantation to treat diabetes mellitus.

Diabetic Foot: Surgical Approach in Emergency

Introduction. Critical limb lschemia (CLI) and particularly diabetic foot (DF) are still considered “Cinderella” in our departments. Anyway, the presence of arterial obstructive disease increases the risk of amputation by itself; when it is associated with foot infection, the risk of amputation is greatly increased. Methods. From January 2007 to December 2011, 375 patients with DF infection and CLI have been admitted to our Unit; from 2007 to 2009, 192 patients (Group A) underwent surgical debridement of the lesion followed by a delayed revascularization; from 2010 to 2011, 183 patients (Group B) were treated following a new 4-step protocol: (1) early diagnosis with a 24 h on call DF team; (2) urgent treatment of severe foot infection with an aggressive surgical debridement; (3) early revascularization within 24 hours; (4) definitive treatment: wound healing, reconstructive surgery, and orthesis. We reported rates of mortality, major amputation, and foot healing at 6 months of followup. Results. The majority of patients in both groups were male; no statistical differences in medical history and clinical condition were reported at the baseline. The main difference between the two groups was the mean time from debridement to revascularization (3 days in Group A and 24 hours in Group B). After 6 months of follow-up, mortality was 11% in Group A versus 4.4% in Group B. Major amputation rate was 39.6% and 24.6% in Groups A and B, respectively. Wound healing was achieved in 17.8% in Group A and 20.8% in Group B. Conclusions. This protocol requires a lot of professional skills that should to reach the goal to avoid major amputations in patients with DF. Only an interdisciplinary integrated DF team and an early intervention may significantly impact the outcome of our patients: “Time is Tissue”!

Development of a diagnosis disclosure model for perinatally HIV-infected children in Thailand

While disclosure of HIV status to perinatally HIV-infected children has become an increasingly important clinical issue, specific disclosure guidelines are lacking. We developed a pediatric HIV diagnosis disclosure model to support caretakers. All HIV-infected children greater than 7-years-old at two participating hospitals in Bangkok, Thailand, and their caretakers, were offered disclosure according to the 4-step protocol: (1) screening; (2) readiness assessment; (3) disclosure; and (4) follow-up. Disclosure occurred after agreement of both providers and caretakers. Among 438 children who were screened, 398 (89%) were eligible. Readiness assessment was completed for 353 (91%) of eligible children and 216 (61%) were determined ready. Disclosure was done for 186 children. The mean age at eligibility screening was 10.5 years (range: 6.8–15.8 years); the mean age at disclosure was 11.7 years (range: 7.6–17.7 years). The mean duration between eligibility screening and disclosure was 15.2 months. There were no significant negative behavioral or emotional outcomes reported in children following disclosure. This HIV diagnosis disclosure model was feasible to implement and had no negative outcomes. As the time for preparation process was over 1 year for most cases, the disclosure process can be initiated as early as age 7 to allow enough time for disclosure to be completed by the age of adolescence.

Improving test properties for neonatal cystic fibrosis screening in the Netherlands before the nationwide start by May 1st 2011

When new technical possibilities arise in health care, often attunement is needed between different actors from the perspectives of research, health care providers, patients, ethics and policy. For cystic fibrosis (CF) such a process of attunement in the Netherlands started in a committee of the Health Council on neonatal screening in 2005. In the balancing of pros and cons according to Wilson and Jungner criteria, the advantages for the CF patient were considered clear, even though CF remains a severe health problem with treatment. Nevertheless, screening was not started then, mainly since the specificity of the tests available at that time was considered too low. Many healthy infants would have been referred for sweat testing and much uncertainty would arise in their parents. Also the limited sensitivity for immigrants and the detection of less severe phenotypes and carriers were considered problematic. The Health Council recommended a pilot screening project which was subsequently performed in some provinces, leading to a 4-step protocol: IRT, PAP, screening for a CFTR mutation panel, and sequencing of the CFTR gene. This would lead to the identification of 23 cases of classical CF, two infants with less severe forms and 12 carriers per year in the Netherlands. Thus many CF patients can be diagnosed early, while limiting the number of referrals, the number of infants with less severe forms diagnosed and the number of carriers identified. Technical solutions were found to limit the ethical problems. A nationwide program using this four step protocol started by 1 May 2011.