Early Stages of Ex Vivo Collagen Glycation Disrupt the Cellular Interaction and Its Remodeling by Mesenchymal Stem Cells-Morphological and Biochemical Evidence.

Mesenchymal stem cells (MSCs), pivotal for tissue repair, utilize collagen to restore structural integrity in damaged tissue, preserving its organization through concomitant remodeling. The non-enzymatic glycation of collagen potentially compromises MSC communication, particularly upon advancing the process, underlying various pathologies such as late-stage diabetic complications and aging. However, an understanding of the impact of early-stage collagen glycation on MSC interaction is lacking. This study examines the fate of in vitro glycated rat tail collagen (RTC) upon exposure to glucose for 1 or 5 days in contact with MSCs. Utilizing human adipose tissue-derived MSCs (ADMSCs), we demonstrate their significantly altered interaction with glycated collagen, characterized morphologically by reduced cell spreading, diminished focal adhesions formation, and attenuated development of the actin cytoskeleton. The morphological findings were confirmed by ImageJ 1.54g morphometric analysis with the most significant drop in the cell spreading area (CSA), from 246.8 µm2 for the native collagen to 216.8 µm2 and 163.7 µm2 for glycated ones, for 1 day and 5 days, respectively, and a similar trend was observed for cell perimeter 112.9 µm vs. 95.1 µm and 86.2 µm, respectively. These data suggest impaired recognition of early glycated collagen by integrin receptors. Moreover, they coincide with the reduced fibril-like reorganization of adsorbed FITC-collagen (indicating impaired remodeling) and a presumed decreased sensitivity to proteases. Indeed, confirmatory assays reveal diminished FITC-collagen degradation for glycated samples at 1 day and 5 days by attached cells (22.8 and 30.4%) and reduced proteolysis upon exogenous collagenase addition (24.5 and 40.4%) in a cell-free system, respectively. The mechanisms behind these effects remain uncertain, although differential scanning calorimetry confirms subtle structural/thermodynamic changes in glycated collagen.

Altered Mesenchymal Stem Cells Mechanotransduction from Oxidized Collagen: Morphological and Biophysical Observations.

Extracellular matrix (ECM) provides various mechanical cues that are able to affect the self-renewal and differentiation of mesenchymal stem cells (MSC). Little is known, however, how these cues work in a pathological environment, such as acute oxidative stress. To better understand the behavior of human adipose tissue-derived MSC (ADMSC) in such conditions, we provide morphological and quantitative evidence for significantly altered early steps of mechanotransduction when adhering to oxidized collagen (Col-Oxi). These affect both focal adhesion (FA) formation and YAP/TAZ signaling events. Representative morphological images show that ADMSCs spread better within 2 h of adhesion on native collagen (Col), while they tended to round up on Col-Oxi. It also correlates with the lesser development of the actin cytoskeleton and FA formation, confirmed quantitatively by morphometric analysis using ImageJ. As shown by immunofluorescence analysis, oxidation also affected the ratio of cytosolic-to-nuclear YAP/TAZ activity, concentrating in the nucleus for Col while remaining in the cytosol for Col-Oxi, suggesting abrogated signal transduction. Comparative Atomic Force Microscopy (AFM) studies show that native collagen forms relatively coarse aggregates, much thinner with Col-Oxi, possibly reflecting its altered ability to aggregate. On the other hand, the corresponding Young's moduli were only slightly changed, so viscoelastic properties cannot explain the observed biological differences. However, the roughness of the protein layer decreased dramatically, from RRMS equal to 27.95 ± 5.1 nm for Col to 5.51 ± 0.8 nm for Col-Oxi (p < 0.05), which dictates our conclusion that it is the most altered parameter in oxidation. Thus, it appears to be a predominantly topographic response that affects the mechanotransduction of ADMSCs by oxidized collagen.

Efficiency of antenatal care and childbirth services in selected primary health care facilities in rural Tanzania: a cross-sectional study.

BACKGROUND: Cost studies are paramount for demonstrating how resources have been spent and identifying opportunities for more efficient use of resources. The main objective of this study was to assess the actual dimension and distribution of the costs of providing antenatal care (ANC) and childbirth services in selected rural primary health care facilities in Tanzania. In addition, the study analyzed determining factors of service provision efficiency in order to inform health policy and planning. METHODS: This was a retrospective quantitative cross-sectional study conducted in 11 health centers and dispensaries in Lindi and Mtwara rural districts. Cost analysis was carried out using step down cost accounting technique. Unit costs reflected efficiency of service provision. Multivariate regression analysis on the drivers of observed relative efficiency in service provision between the study facilities was conducted. Reported personnel workload was also described. RESULTS: The health facilities spent on average 7 USD per capita in 2009. As expected, fewer resources were spent for service provision at dispensaries than at health centers. Personnel costs contributed a high approximate 44% to total costs. ANC and childbirth consumed approximately 11% and 12% of total costs; and 8% and 10% of reported service provision time respectively. On average, unit costs were rather high, 16 USD per ANC visit and 79.4 USD per childbirth. The unit costs showed variation in relative efficiency in providing the services between the health facilities. The results showed that efficiency in ANC depended on the number of staff, structural quality of care, process quality of care and perceived quality of care. Population-staff ratio and structural quality of basic emergency obstetric care services highly influenced childbirth efficiency. CONCLUSIONS: Differences in the efficiency of service provision present an opportunity for efficiency improvement. Taking into consideration client heterogeneity, quality improvements are possible and necessary. This will stimulate utilization of ANC and childbirth services in resource-constrained health facilities. Efficiency analyses through simple techniques such as measurement of unit costs should be made standard in health care provision, health managers can then use the performance results to gauge progress and reward efficiency through performance based incentives.

Morphological and Quantitative Evidence for Altered Mesenchymal Stem Cell Remodeling of Collagen in an Oxidative Environment-Peculiar Effect of Epigallocatechin-3-Gallate.

Mesenchymal stem cells (MSCs) are involved in the process of extracellular matrix (ECM) remodeling where collagens play a pivotal role. We recently demonstrated that the remodeling of adsorbed collagen type I might be disordered upon oxidation following its fate in the presence of human adipose-derived MSC (ADMSCs). With the present study we intended to learn more about the effect of polyphenolic antioxidant Epigallocatechin-3-gallate (EGCG), attempting to mimic the conditions of oxidative stress in vivo and its putative prevention by antioxidants. Collagen Type I was isolated from mouse tail tendon (MTC) and labelled with FITC before being oxidized according to Fe2+/H2O2 protocol. FITC-collagen remodeling by ADMSC was assessed morphologically before and after EGCG pretreatment and confirmed via detailed morphometric analysis measuring the anisotropy index (AI) and fluorescence intensity (FI) in selected regions of interest (ROI), namely: outside the cells, over the cells, and central (nuclear/perinuclear) region, whereas the pericellular proteolytic activity was measured by de-quenching fluorescent collagen probes (FRET effect). Here we provide morphological evidence that MTC undergoes significant reorganization by the adhering ADMSC and is accompanied by a substantial activation of pericellular proteolysis, and further confirm that both processes are suppressed upon collagen oxidation. An important observation was that this abrogated remodeling cannot be prevented by the EGCG pretreatment. Conversely, the detailed morphometric analysis showed that oxidized FITC-collagen tends to accumulate beneath cells and around cell nuclei, suggesting the activation of alternative routes for its removal, such as internalization and/or transcytosis. Morphometric analysis also revealed that both processes are supported by EGCG pretreatment.

Crohn's Disease Complicated by Ileosigmoid Fistula - Synchronous Resection or Primary Sigmoid Repair, One or Two-stage Procedure? A systematic review of the literature and prospective case series.

INTRODUCTION: Although ileosigmoid fistulas (ISFs) in Crohn's disease (CD) are rare they can be quite challenging, especially for the inexperienced surgeons. Furthermore, current guidelines offer no clear recommendation regarding the surgical strategy in such cases. A systematic review of the literature to determine the best surgical strategy and a prospective case series are presented herein. MATERIALS AND METHODS: The systematic review was performed according to PRISMA guidelines. A single-center prospective data-base from January 1, 2014 to August 20, 2019 is presented. Age, duration of CD, and the rates of ISF, emergency, preoperative diagnosis, type of surgery, type of stoma, and complications were analyzed and a prospective case series. RESULTS: Eleven of 69 papers with a total of 505 patients were included in the systematic analysis. The rate of ISF was 3-5% of all CD patients. The combined preoperative detection rate of all modalities was 71%. Primary repair was performed in 42% of the cases; the rate of stoma was 31.5% with a similar proportion in primary repair and sigmoid resection.In the presented series, 35 of 176 patients with CD were operated (51% in an emergency setting). There were 7 cases with ISFs (4% of all and 20% of the operated patients). Preoperative diagnosis was made at 57%. Primary repair was performed in 71%, and a two-stage intervention with a stoma - in 58% of patients. CONCLUSIONS: Primary repair should be attempted in all cases in which the sigmoid colon is disease-free or is not involved in the adja-cent abscess. The synchronous resections are not a mandatory indication for the stoma, but rather a tailored approach is recommended with an evaluation of the risk factors. Based on the available literature, no clear recommendation regarding the type of stoma can be made.

The AaLplus near-peer teaching program in Family Medicine strengthens basic medical skills-A five-year retrospective study.

BACKGROUND: Basic medical skills such as history taking and physical examination are essential components of clinical work profiles, but nevertheless have been neglected by conventional preclinical curricula. The near-peer-teaching program AaLplus [living anatomy plus] teaches basic medical skills, especially history taking, physical examination, and venepuncture, to preclinical students. It is a highly popular compulsory course in the first four semesters (320 students/year, 9h/semester) at Heidelberg University and ends with a formative Objective Structured Clinical Examination (OSCE) during which students receive structured in-depth feedback on their performance. AaLplus is part of the Department of General Practice's longitudinal curriculum for Family Medicine. OBJECTIVES: This study aims to assess whether the AaLplus program has positive effects on students' clinical skill development and subjective confidence in history taking, physical examination and venepuncture. METHODS: From 2015 to 2019, we asked all AaLplus participants to rate the program and self-assess their medical skills on 5-point Likert scales (min 1, max 5). In 4-station OSCEs, trained tutors rated the students' performance in all taught skills using standardized checklists. RESULTS: From 2015 to 2019 n = 1534 questionnaires returned (response rate = 98.6%, 52.7% females). After course completion, students felt able to take a patient's history (mean 3.97, SD = 0.75) and perform physical examinations (means range 3.82-4.36, SDs range 0.74-0.89) as well as venepuncture (mean 4.12, SD = 0.88). A large majority of students claimed they acquired these skills in the AaLplus program. During OSCE, 81.9% passed anamnesis, 93.1% passed physical examination, and 95.4% passed venepuncture (of n = 1556). Students mostly rated the feedback they received during the OSCE as "helpful" or "very helpful" (means for different stations 4.69-4.76, SDs 0.50-0.70). CONCLUSIONS: AaLplus is a positive example of a peer teaching program in the preclinical stage of medical studies. It successfully trains junior students in essential medical abilities and increases their confidence in their skills. A high percentage of students pass the formative OSCE and evaluate it positively. Consistently high ratings indicate the program's routine viability. Further studies are needed to analyze if programs like AaLplus could have an impact on the number of graduates choosing career in Family Medicine.

Rs5918ITGB3 Polymorphism, Smoking, and BMI as Risk Factors for Early Onset and Recurrence of DVT in Young Women.

OBJECTIVE: To evaluate the contribution of rs5918ITGB3 on the incidence and recurrence of deep venous thrombosis (DVT) in women and the relationship with body mass index (BMI) and smoking and to compare with data in men. RESULTS: Rs5918(C) polymorphism in ITGB3 gene was assessed in 224 patients diagnosed with DVT and 216 controls. Thrombophilic genetic variant rs5918(C) was significantly pronounced in women (χ2 =7.565, P = .008) and total patients (χ2 = 9.266, P = .002) but not in men. Women patients (<45 years) who were carriers of rs5918ITGB3 polymorphism had an early onset of DVT (34.5 vs 39.4 years, χ2 = 7.027, P = .008) as analyzed by Kaplan-Meier and a higher risk of the recurrent event (χ2 = 3.405, odds ratio = 2.581, P = .044). The period before recurrent venous thromboembolism event was related to smoking status and BMI in young female who were carriers of rs5918 polymorphism but not in the males. CONCLUSIONS: Carriage of genetic variant rs5918(C) polymorphism in ITGB3 gene in women contributes to higher risk of single and recurrent DVT events at younger age.

PlA2 Polymorphism in Glycoprotein IIb/IIIa Modulates the Morphology and Nanomechanics of Platelets.

Glycoprotein IIb/IIIa (GPIIb/IIIa) is the most abundant platelet surface receptor for fibrinogen and von Willebrand factor. Polymorphism PlA1/A2 in the gene of GPIIb/IIIa is among the risk factors for the development of arterial and venous thrombosis. The aim of this study is to evaluate the effect of the carriage of PlA1/A2 on the size, topographic features, and membrane stiffness of platelets from healthy controls and patients with deep venous thrombosis (DVT). Atomic force microscopy (AFM) imaging and nanoindentation (force-distance curves) were applied to investigate the morphological and nanomechanical properties (Young's modulus) of platelets immobilized on glass surface. The surface roughness ( Ra) and height ( h) of platelets from patients with DVT, carriers of mutant allele PlA2 ( Ra = 30.2 ± 6 nm; h = 766 ± 182 nm) and noncarriers ( Ra = 28.6 ± 6 nm; h = 865 ± 290 nm), were lower than those of healthy carriers of allele PlA2 ( Ra = 48.1 ± 12 nm; h = 1072 ± 338 nm) and healthy noncarriers ( Ra = 49.7 ± 14 nm; h = 1021 ± 433 nm), respectively. Platelets isolated from patients with DVT, both carriers and noncarriers, exhibit much higher degree of stiffness at the stage of spreading ( E = 327 ± 85 kPa and 341 ± 102 kPa, respectively) compared to healthy noncarriers ( E = 198 ± 50 kPa). In addition, more pronounced level of platelet activation was found in polymorphism carriers. In conclusion, the carriage of PlA2 allele modulates the activation state, morphology, and membrane elasticity of platelets.

Costs associated with implementation of computer-assisted clinical decision support system for antenatal and delivery care: case study of Kassena-Nankana district of northern Ghana.

OBJECTIVE: This study analyzed cost of implementing computer-assisted Clinical Decision Support System (CDSS) in selected health care centres in Ghana. METHODS: A descriptive cross sectional study was conducted in the Kassena-Nankana district (KND). CDSS was deployed in selected health centres in KND as an intervention to manage patients attending antenatal clinics and the labour ward. The CDSS users were mainly nurses who were trained. Activities and associated costs involved in the implementation of CDSS (pre-intervention and intervention) were collected for the period between 2009-2013 from the provider perspective. The ingredients approach was used for the cost analysis. Costs were grouped into personnel, trainings, overheads (recurrent costs) and equipment costs (capital cost). We calculated cost without annualizing capital cost to represent financial cost and cost with annualizing capital costs to represent economic cost. RESULTS: Twenty-two trained CDSS users (at least 2 users per health centre) participated in the study. Between April 2012 and March 2013, users managed 5,595 antenatal clients and 872 labour clients using the CDSS. We observed a decrease in the proportion of complications during delivery (pre-intervention 10.74% versus post-intervention 9.64%) and a reduction in the number of maternal deaths (pre-intervention 4 deaths versus post-intervention 1 death). The overall financial cost of CDSS implementation was US$23,316, approximately US$1,060 per CDSS user trained. Of the total cost of implementation, 48% (US$11,272) was pre-intervention cost and intervention cost was 52% (US$12,044). Equipment costs accounted for the largest proportion of financial cost: 34% (US$7,917). When economic cost was considered, total cost of implementation was US$17,128-lower than the financial cost by 26.5%. CONCLUSIONS: The study provides useful information in the implementation of CDSS at health facilities to enhance health workers' adherence to practice guidelines and taking accurate decisions to improve maternal health care.

Plasma cyclic 3',5'-adenosine monophosphate in patients with elevated pulmonary pressure due to left-to-right shunt.

BACKGROUND: Increased pulmonary blood flow in patients with left-to-right shunt has been shown to be associated with alterations in prostacyclin-synthesis. There are limited data with respect to the plasma cAMP in patients with elevated pulmonary artery pressure due to left-to-right shunt. We hypothesized that plasma cAMP might be influenced by pulmonary blood flow or pressure in patients with left-to-right shunt. METHODS: Plasma cAMP from venous blood was measured in 122 healthy controls aged 8.3 (0.2 - 14.9) years (median [range]) and in 130 patients with left-to-right shunt aged 1.4 (0.1 - 19.1) years by radioimmunoassay. RESULTS: Plasma cAMP in controls and in patients was similar and decreased with age. Healthy infants (n = 42) showed higher plasma cAMP (46 [27-112] nmol/l) than children > 6 years of age (n = 40, 39 [19-73] nmol/l; P < 0.001). CONCLUSIONS: These values should be taken into consideration when reporting plasma values for cAMP in patients with congenital heart disease. The values for healthy children obtained in this study should allow a better interpretation of plasma cAMP levels in various disease conditions such as chronic renal failure, liver chirrhosis, hyperthyreosis, or children with intellectual disability.