Extracellular vesicle-derived TP53BP1, CD34, and PBX1 from human peripheral blood serve as potential biomarkers for the assessment and prediction of vascular aging.

BACKGROUND: Vascular aging is an important pathophysiological basis for the senescence of various organs and systems in the human body, and it is a common pathogenetic trigger for many chronic diseases in the elderly. METHODS: The extracellular vesicles (EVs) from young and aged umbilical vein endothelial cells were isolated and identified by qPCR the differential expression levels of 47 mRNAs of genes closely related to aging in the two groups. RESULTS: There were significant differences in the expression levels of 18 genes (we noted upregulation in PLA2G12A, TP53BP1, CD144, PDE11A, FPGT, SERPINB4, POLD1, and PPFIBP2 and downregulation in ATP2C2, ROBO2, RRM2, GUCY1B1, NAT1-14, VEGFR2, WTAPP1, CD146, DMC1, and GRIK2). Subsequent qPCR identification of the above-mentioned genes in PBMCs and plasma-EVs from the various age groups revealed that the trend in expression levels in peripheral blood plasma-EVs of the different age groups was approximately the same as that in PBMCs. Of these mRNAs, the expression of four genes-PLA2G12A, TP53BP1, OPRL1, and KIAA0895-was commensurate with increasing age. In contradistinction, the expression trend of four genes (CREG1, PBX1, CD34, and SLIT2) was inversely proportional to the increase in age. Finally, by taking their intersection, we determined that the expression of TP53BP1 was upregulated with increasing human age and that CD34 and PBX1 were downregulated with increasing age. CONCLUSION: Our study indicates that human peripheral blood plasma-EV-derived TP53BP1, CD34, and PBX1 potentially comprise a noninvasive biomarker for assessing and predicting vascular aging.

Histochemical assessment on osteoclasts in long bones of toll-like receptor 2 (TLR2) deficient mice.

OBJECTIVE: Toll-like receptor 2 (TLR2), recognizes a wide variety of pathogen-associated molecular patterns such as lipopolysaccharides, peptidoglycans, and lipopeptides, and is generally believed to be present in monocytes, macrophages, dendritic cells, and vascular endothelial cells. However, no histological examination of osteoclasts, which differentiate from precursors common to macrophages/monocytes, has been performed in a non-infected state of TLR2 deficiency. The objective of this study was to examine the histological properties and function of osteoclasts in the long bones of 8-week-old male TLR2 deficient (TLR2-/-) mice to gain insight into TLR2 function in biological circumstances without microbial infection. METHODS: Eight-week-old male wild-type and TLR2-/- mice were fixed with paraformaldehyde solution, and their tibiae and femora were used for micro-CT analysis, immunohistochemistry, transmission electron microscopy, and real-time PCR analysis. RESULTS: TLR2-/- tibiae and femora exhibited increased bone volume of metaphyseal trabeculae and elevated numbers of TRAP-positive osteoclasts. However, the number of multinucleated TRAP-positive osteoclasts was reduced, whereas mononuclear TRAP-positive cells increased, despite the high expression levels of Dc-Stamp and Oc-Stamp. Although TRAP-positive multinucleated and mononuclear osteoclasts showed the immunoreactivity and elevated expression of RANK and siglec-15, they revealed weak cathepsin K-positivity and less incorporation of the mineralized bone matrix, and often missing ruffled borders. It seemed likely that, despite the increased numbers, TLR2-/- osteoclasts reduced cell fusion and bone resorption activity. CONCLUSION: It seems likely that even without bacterial infection, TLR2 might participate in cell fusion and subsequent bone resorption of osteoclasts.

Molecular subtyping based on TRP family and prognostic assessment for TRP-associated lncRNAs in pancreatic adenocarcinoma.

BACKGROUND: Transient receptor potential (TRP) channels have high permeability to Ca2+ ions because they are non-selective ion channels. TRP channels have been implicated in tumor onset and progression, proliferation, and migration in recent years. However, the prognostic value of genes related to TRP and their specific mechanism in pancreatic adenocarcinoma (PAAD) are yet to be understood. METHODS: Public databases such as TCGA and GEO were used to retrieve data on gene expression and clinical information of patients with pancreatic adenocarcinoma for our study. ConsensusClusterPlus package was used for unsupervised clustering analysis. The microenvironment cell population (MCP)-counter approach was employed to measure the immune cells infiltration status. The Pearson correlation was performed to identify TRP-associated lncRNAs. RESULTS: Initially, we separated PAAD patients into three clusters depending on TRP-related genes, and of the three clusters, cluster B showed the least immune cell infiltration, which was correlated with poor prognosis. Moreover, GSVA enrichment analysis further revealed that cluster A was subjected to a considerable enrichment in carcinogenic signaling pathways, whereas cluster C was enriched in immune-related pathways. Then, using TRP-associated lncRNAs as a starting point, we constructed a prognostic risk model for PAAD patients that could efficiently predict their prognosis. Further, GSEA revealed that cancer-related pathways, for instance, the cell cycle, p53 signaling pathway, etc. were considerably enriched in the high-risk group. In addition, we looked into the link between the prognostic model and the immunological microenvironment. Lower cytotoxic lymphocytes, NK cells, CD8 T cells, and endothelial cells infiltration were found to be associated with high risk using the MCP-counter algorithm. The expression of CD274, POLE2, MCM6, and LOXL2 was also found to be higher in the high-risk group. TMB was also considerably greater in high-risk individuals, indicating that immune checkpoint inhibitors (ICIs) therapy may benefit them more. Lastly, qRT-PCR further confirmed the differential expression of these prognostic TRP-associated lncRNAs, indicating that these lncRNAs play an imperative role in PAAD tumorigenesis. CONCLUSION: TRP family genes may represent a new class of candidate molecular markers of the occurrence and progression of PAAD. Risk models based on TRP-associated lncRNAs could provide important new references for immunotargeted therapy of pancreatic adenocarcinoma.

Vasculature-on-chip for Assessment of Bioresorbable Scaffolds and Endothelial Barrier Integrity.

ABSTRACT: Endothelial cells adhere to one another through junctional structures formed by intercellular adhesion molecules. These intercellular proteins regulate barrier function in response to the hemodynamic shear rate and enable the selective passage of solutes and fluids across the endothelium. After endovascular device implantation, the endothelial barrier is compromised and becomes discontinuous, which increases permeability, allowing transmigration of leukocytes and lipoproteins and leading to the accumulation of lipid-laden foamy macrophages in the subendothelial space. Drug-coated bioresorbable vascular scaffold implants have been associated with unexpected thrombotic complications, which were not predicted in animals because of dissimilarities in endothelial regeneration and realignment between animals and humans. The development of a microengineered, microfluidics-based system of patterned channels lined with human endothelial and smooth muscle cells perfused with blood allows for the evaluation of endothelial function and barrier integrity. This review highlights the translational potential of vasculature-on-chip, which recreates the microphysiological milieu to evaluate the impact of drug-eluting bioresorbable vascular scaffolds on endothelial barrier integrity and to characterize polymer biodegradation behavior and drug release kinetic profiles over time.

Altered Cytokine Response of Human Brain Endothelial Cells after Stimulation with Malaria Patient Plasma.

Infections with the deadliest malaria parasite, Plasmodium falciparum, are accompanied by a strong immunological response of the human host. To date, more than 30 cytokines have been detected in elevated levels in plasma of malaria patients compared to healthy controls. Endothelial cells (ECs) are a potential source of these cytokines, but so far it is not known if their cytokine secretion depends on the direct contact of the P. falciparum-infected erythrocytes (IEs) with ECs in terms of cytoadhesion. Culturing ECs with plasma from malaria patients (27 returning travellers) resulted in significantly increased secretion of IL-11, CXCL5, CXCL8, CXCL10, vascular endothelial growth factor (VEGF) and angiopoietin-like protein 4 (ANGPTL4) if compared to matching controls (22 healthy individuals). The accompanying transcriptome study of the ECs identified 43 genes that were significantly increased in expression (≥1.7 fold) after co-incubation with malaria patient plasma, including cxcl5 and angptl4. Further bioinformatic analyses revealed that biological processes such as cell migration, cell proliferation and tube development were particularly affected in these ECs. It can thus be postulated that not only the cytoadhesion of IEs, but also molecules in the plasma of malaria patients exerts an influence on ECs, and that not only the immunological response but also other processes, such as angiogenesis, are altered.

Flow Cytometric Assessment of Endothelial and Platelet Microparticles in Patients With Atrial Fibrillation Treated With Dabigatran.

The prothrombotic state in patients with atrial fibrillation (AF) is related to endothelial injury, the activation of platelets and the coagulation cascade. We evaluated the levels of platelet- (CD42b) and endothelial-derived (CD144) microparticles in the plasma patients with non-valvular AF treated with dabigatran at the time of expected minimum and maximum drug plasma concentrations. Following that, we determined the peak dabigatran plasma concentration (cpeak ). CD42b increased after taking dabigatran (median [IQR] 36.7 [29.4-53.3] vs. 45.6 [32.3-59.5] cells/µL; p = 0.025). The concentration of dabigatran correlated negatively with the post-dabigatran change in CD42b (ΔCD42b, r = -0.47, p = 0.021). In the multivariate model, the independent predictors of ΔCD42b were: cpeak (HR -0.55; with a 95% confidence interval, CI [-0.93, -0.16]; p = 0.007), coronary artery disease (CAD) (HR -0.41; 95% CI [-0.79, -0.02]; p = 0.037) and peripheral artery disease (PAD) (HR 0.42; 95% CI [0.07, 0.74]; p = 0.019). CD144 did not increase after dabigatran administration. These data suggest that low concentrations of dabigatran may be associated with platelet activation. PAD and CAD have distinct effects on CD42b levels during dabigatran treatment.

Quantitative assessment and clinical relevance of VEGFRs-positive tumor cells in refractory brain tumors.

Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)1 and 2 signaling is a potent activator of tumor angiogenesis. Although the expressions of VEGFR1 and VEGFR2 were initially thought to be limited to the endothelial cells, it is now known that both the receptors are expressed in tumor cells. This is the first study wherein VEGFRs-positive tumor cells are quantitatively evaluated for brain tumors with upregulated VEGF/VEGFR signaling. The percentage of VEGFRs-positive tumor cells was quantitatively evaluated in various brain tumors (10 glioblastomas, 22 neurofibromatosis type 2 [NF2]-related schwannomas, 21 sporadic schwannomas, 27 chordomas, 36 meningiomas, 29 hemangioblastomas, 11 hemangiopericytoma, and 13 ependymomas) using immunohistochemistry. VEGF-A expression was also analyzed using quantitative real-time polymerase chain reaction. Double immunofluorescence staining using anti-PDGFR-ß and anti-CD34 antibody, microvessel density, and vessel diameter were analyzed to evaluate the vascular characteristics. Chordomas demonstrated an extremely higher percentage of VEGFR1 and VEGFR2-positive tumor cells than other tumors. In contrast, meningiomas and hemangiopericytomas showed few VEGFRs-positive tumor cells. The percentage of positive tumor cells in chordomas, hemangioblastomas, and NF2 schwannomas was associated with clinical courses, such as shorter progression free survival, and growth speed. Glioblastomas and NF2 schwannomas showed larger tumor vessels without pericyte coverage. The present study is the first to quantitatively analyze VEGFR1- and VEGFR2- positive tumor cells in various types of refractory brain tumors. This novel parameter significantly correlated with the progressive clinical courses.

Glomerular Endothelial Cells: Assessment of Barrier Properties In Vitro.

Endothelial cells form the inner lining of all blood vessels and play a vital role in regulating vascular permeability. This applies to the circulation in general and also to specific capillary beds including the renal glomerular capillaries. Endothelial dysfunction, including increased permeability, is a key component of diabetes-induced organ damage. Endothelial cells together with their glycocalyx, grown on porous membranes, provide an excellent model to study endothelial permeability properties. Here we describe the measurement of two characteristics of glomerular endothelial cell (GEnC) monolayers: electrical resistance and macromolecular passage. Trans-endothelial electrical resistance provides a measure of small-pore pathways across the endothelium and provides an index of monolayer confluence and cell-cell junction integrity. Measurement of macromolecular passage provides an index of large-pore pathways and use of labeled albumin provides direct relevance to the clinically important parameter of albuminuria. The combination of the two approaches provides a fantastic tool to elucidate endothelial barrier function in vitro including in response to cytokines, pathological stimuli, and potential therapeutic agents.

A phenomenological model for cell and nucleus deformation during cancer metastasis.

Cell migration plays an essential role in cancer metastasis. In cancer invasion through confined spaces, cells must undergo extensive deformation, which is a capability related to their metastatic potentials. Here, we simulate the deformation of the cell and nucleus during invasion through a dense, physiological microenvironment by developing a phenomenological computational model. In our work, cells are attracted by a generic emitting source (e.g., a chemokine or stiffness signal), which is treated by using Green's Fundamental solutions. We use an IMEX integration method where the linear parts and the nonlinear parts are treated by using an Euler backward scheme and an Euler forward method, respectively. We develop the numerical model for an obstacle-induced deformation in 2D or/and 3D. Considering the uncertainty in cell mobility, stochastic processes are incorporated and uncertainties in the input variables are evaluated using Monte Carlo simulations. This quantitative study aims at estimating the likelihood for invasion and the length of the time interval in which the cell invades the tissue through an obstacle. Subsequently, the two-dimensional cell deformation model is applied to simplified cancer metastasis processes to serve as a model for in vivo or in vitro biomedical experiments.

Primary Human Lung Alveolus-on-a-chip Model of Intravascular Thrombosis for Assessment of Therapeutics.

Pulmonary thrombosis is a significant cause of patient mortality; however, there are no effective in vitro models of thrombi formation in human lung microvessels that could also assess therapeutics and toxicology of antithrombotic drugs. Here, we show that a microfluidic lung alveolus-on-a-chip lined by human primary alveolar epithelium interfaced with endothelium and cultured under flowing whole blood can be used to perform quantitative analysis of organ-level contributions to inflammation-induced thrombosis. This microfluidic chip recapitulates in vivo responses, including platelet-endothelial dynamics and revealed that lipopolysaccharide (LPS) endotoxin indirectly stimulates intravascular thrombosis by activating the alveolar epithelium, rather than acting directly on endothelium. This model is also used to analyze inhibition of endothelial activation and thrombosis due to a protease activated receptor-1 (PAR-1) antagonist, demonstrating its ability to dissect complex responses and identify antithrombotic therapeutics. Thus, this methodology offers a new approach to study human pathophysiology of pulmonary thrombosis and advance drug development.

An intra-body molecular communication networks framework for continuous health monitoring and diagnosis.

Intra-body communication networks are designed to interconnect nano- or micro-sized sensors located inside the body for health monitoring and drug delivery. The most promising solutions are made of implanted nanosensors to timely monitor the body for the presence of specific diseases and pronounce a diagnosis without the intervention of a physician. In this manner, several deadly health conditions such as heart attacks are avoided through the early in vivo detection of their biomarkers. In reality, nanosensors are challenged by the individual specificities, molecular noise, limited durability, and low energy resources. In this paper, a framework is proposed for estimating and detecting diseases and localizing the nanosensors. This framework is based on molecular communication, a novel communication paradigm where information is conveyed through molecules. Through the case study of the shedding of endothelial cells as an early biomarker for heart attack, the intra-body molecular communication networks framework is shown to resolve major issues with in vivo nanosensors and lay the foundations of low-complexity biomedical signal processing algorithms for continuous disease monitoring and diagnosis.

Assessment of endothelial cell density and corneal thickness in corneal grafts an average of 5 years after penetrating keratoplasty.

BACKGROUND: Corneal transparency is a useful indicator for corneal function. Our aim was to investigate central corneal endothelial cells and corneal thickness in transplanted corneas at an average of 5.4 years after penetrating keratoplasty PATIENTS AND METHODS: The study involved 68 perforated keratoplasty patients with at least a 1 year follow-up. Post-operatively, the central corneal endothelial layer was observed using a contact specular microscope. Central endothelial cell density, corneal thickness and the coefficient of variation of endothelial size were statistically analysed. RESULTS: The post-operative follow-up time was ranging from 12 months to 23 years. Endothelial cell density (ECD) was 1,501 ± 249 cell/mm(2). The average cell size was 673.6 ± 98.3 µm(2), and the coefficient of variation of cell size was 0.61 ± 0.11. No difference in ECD was detected between diagnostic groups. Corneal thickness was 0.56 ± 0.06 mm. Correlation between ECD and post-operative time was not significant (r = 0.02; p = 0.85). CONCLUSION: Our study concluded that ECD showed a higher rate of decrease after penetrating keratoplasty with no relation to pre-operative diagnosis.

[Effect of posterior neodymium:YAG capsulotomy. Safety evaluation of macular foveal thickness, intraocular pressure and endothelial cell loss in pseudophakic patients with posterior capsule opacification]. / Efecto de la capsulotomía posterior con láser neodymium:YAG. Valoración de seguridad en el grosor foveal, la presión intraocular y el recuento de células endoteliales en pacientes pseudofáquicos con opacidad de cápsula posterior.

OBJECTIVE: To determine the effect of posterior capsulotomy on macular thickness, intraocular pressure and endothelial cell loss in pseudophakic patients with posterior capsule opacification using the other eye of every patient as a control. METHODS: An observational prospective study was conducted on 31 pseudophakic patients with posterior capsular opacification in one eye, using the other eye as a control. Patients did not suffer any other ocular pathology. All patients were treated by posterior capsular opacification with Nd:YAG capsulotomy, and followed up for a three-month period. The ocular examination included, best corrected visual acuity (BCVA), intraocular pressure (IOP), macular optical coherence tomography (OCT), and endothelial cell assessment (including densitometry, cell size and coefficient of variation, hexagonal cell percentage and pachymetry), which were determined in both eyes before treatment, and one week, one month and 3 months after capsulotomy. RESULTS: Generalized estimating equations (GEE) were used to assess the capsulotomy effect adjusted by corresponding baseline measurements and time. No association was found between capsulotomy and IOP (P=.597), macular thickness (P=.085) or ECA (densitometry (P=.422), average size of cells (P=.299), variation coefficient (P=.495), hexagonal cell percent (P=.093) and corneal pachymetry (P=.423). A significant increase of 0.15 Snellen units in BCVA was found during the 3-month follow-up period (P<.001). CONCLUSION: This study shows that after Nd:YAG capsulotomy, BCVA improves significantly without any IOP, OCT or ECA changes during the three-month follow-up. Nd:YAG capsulotomy is a safe procedure in pseudophakic patients without any other ocular pathology.

Assessment of inflammatory infiltration and angiogenesis in the thrombus and the wall of abdominal aortic aneurysms on the basis of histological parameters and computed tomography angiography study.

The proliferation of vessels within the aneurysm's wall and the intraluminal thrombus of abdominal aortic aneurysm (AAA) may be the main factor responsible for progression and rupture of AAA. The aim of this study was to compare the parameters of the thrombus (size, density, contrast enhancement) measured by computed tomography (CT) with histological assessment of thrombi removed during surgery. 29 patients with AAA were examined with angio-CT. Post-surgery histopathological evaluation of AAA was performed. Slides were stained with markers of B- (CD20) and T-lymphocytes (CD3), and markers of endothelial cells (CD34). Thrombi were enhanced after contrast media administration in angio-CT (p = 0.002). There was a statistically significant correlation between contrast enhancement and the presence of B lymphocytes. Intensity of endothelial cell marker expression significantly correlated with the presence of inflammatory T- and B-cells. No statistical significant correlation was found between contrast enhancement of the thrombus and markers of endothelial cells. The accumulation of inflammatory cells in the wall of AAA thrombus results in the formation of new vessels which participates to the instability of the thrombus and AAA wall. Assessment of the inflammation and neovascularization in the wall and thrombus of the AAA might be an important factor in monitoring the progression and the risk of aneurysm's rupture.

Immunomorphological assessment of regional lymph nodes for predicting metastases in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma is the most common neoplasm and comprises of approximately 80% of the cancers occurring in the oral cavity. The role of the host response to this neoplasm has been recognized, and for many years the regional lymph node in tumor-bearing hosts has been considered as an anatomic barrier to the systematic dissemination of tumor cells. Morphological evaluation of the regional nodes has aided in understanding the immune response. AIM: The current study was carried out to observe the morphological changes occurring in the regional lymph nodes and to evaluate whether these features could be helpful in assessing the immunological status of the patient, and thereby, the prognosis of the patient. MATERIALS AND METHODS: The study was based on lymph nodes from 63 patients with oral squamous cell carcinoma, who underwent radical neck dissection or modified neck dissection. In the lymph node, four morphological patterns were observed that included lymphocyte predominance, germinal center predominance, mixed pattern (sinus Histiocytosis), and an unstimulated pattern. The cases were then divided into four groups according to the predominant immunoreactivity pattern based on the World Health Organization (WHO) standardized system for reporting human lymph node morphology. RESULTS: Revealed that risk of metastases to cervical lymph nodes in patients with lymphocyte predominance was less (28.6%) when compared to the high risk of metastases with germinal center predominance (68%), and these results were statistically significant (P < 0.05). Patients with a mixed pattern showed less risk of metastases (45.4%), while those with an unstimulated pattern had increased risk of metastases (66.6%), but the results were not statistically significant. It was also found that in the positive nodes, germinal center hyperplasia (50.2%) was the predominant pattern. CONCLUSION: The present study revealed that patients with lymphocyte predominance had less risk of metastases and patients with germinal center predominance had a high risk of metastases to the lymph node.

Biophysical assessment of single cell cytotoxicity: diesel exhaust particle-treated human aortic endothelial cells.

Exposure to diesel exhaust particles (DEPs), a major source of traffic-related air pollution, has become a serious health concern due to its adverse influences on human health including cardiovascular and respiratory disorders. To elucidate the relationship between biophysical properties (cell topography, cytoskeleton organizations, and cell mechanics) and functions of endothelial cells exposed to DEPs, atomic force microscope (AFM) was applied to analyze the toxic effects of DEPs on a model cell line from human aortic endothelial cells (HAECs). Fluorescence microscopy and flow cytometry were also applied to further explore DEP-induced cytotoxicity in HAECs. Results revealed that DEPs could negatively impair cell viability and alter membrane nanostructures and cytoskeleton components in a dosage- and a time-dependent manner; and analyses suggested that DEPs-induced hyperpolarization in HAECs appeared in a time-dependent manner, implying DEP treatment would lead to vasodilation, which could be supported by down-regulation of cell biophysical properties (e.g., cell elasticity). These findings are consistent with the conclusion that DEP exposure triggers important biochemical and biophysical changes that would negatively impact the pathological development of cardiovascular diseases. For example, DEP intervention would be one cause of vasodilation, which will expand understanding of biophysical aspects associated with DEP cytotoxicity in HAECs.

Comparative assessment of transient exposure of paclitaxel or zotarolimus on in vitro vascular cell death, proliferation, migration, and proinflammatory biomarker expression.

Both paclitaxel and zotarolimus are currently employed in vascular interventional therapies, such as drug-eluting stents, and are under investigation for use in other novel drug-device combination products. Paclitaxel is a microtubule-stabilizing compound with potent antiproliferative properties and antimigration effects, whereas zotarolimus is a potent mammalian target of rapamycin inhibitor with antiproliferative and antiinflammatory properties. This study was intended to compare paclitaxel and zotarolimus for intravascular applications in which drug exposure time may be reduced, such as in drug-coated balloons. These applications are generally aimed at reducing neointimal hyperplasia by limiting smooth muscle cell (SMC) proliferation and inflammatory cell recruitment, while minimally interfering with vessel reendothelialization after balloon denudation. In the cellular models described in this study, transient exposure of zotarolimus resulted in the sustained inhibition of SMC proliferation and reduced endothelial cell (EC) proinflammatory cytokine expression, while not affecting EC migration and viability. Transient exposure of paclitaxel inhibited SMC proliferation, EC migration, and overall cell viability, with no effect on expression of the proinflammatory biomarkers studied.

Assessment of silk fibroin for the repair of buccal mucosa in a rat model.

This study evaluated the effectiveness of silk fibroin materials for wound repair confined to the buccal mucosa in a rat model by assessing several key clinical parameters and the associated local and systemic immune response. Ninety male SD rats were subjected to microscopic oral surgery to establish a full thickness wound on the buccal mucosa. Rats were randomly divided into three groups based on the treatments received: group A, covered with polyporous silk fibroin scaffold; group B, repaired with crosslinking silk fibroin film; and group C, control. Visual observation of the wounds suggests that wound shrinkage 5 days after the operation was significantly lower in both silk fibroin repaired groups (A and B) than that in the controls. The distribution of inflammatory neutrophils in group A was significantly lower than those in the control group throughout the entire study. The percentage of fibroblasts and capillary endothelia (CD34(+)), and the subgroups of peripheral lymphocytes (CD3(+), CD4(+), CD8(+)) were similar amongst the groups. The results revealed that placement of silk fibroin in an oral buccal defect can reduce the degree of wound shrinkage and enhance the growth of mucosal epithelial cells without any local or systemic immunological incompatibility.

Power Doppler sonography, a non-invasive method of assessment of the synovial inflammation in patients with early rheumatoid arthritis.

OBJECTIVE: To detect synovitis is important in both the diagnosis and outcome assessment of early rheumatoid arthritis. This study was meant to assess the validity and reproducibility of ultrasonography (US) as a mean of detection for the knee synovitis, by comparing US findings with clinical examination and histopathological findings in synovial membrane. METHODS: The study group included 65 patients with early rheumatoid arthritis - below 12 months from the debut, naive for DMARDs, in whom demographic data - gender, age, disease duration, the number of tender and swollen joints, HAQ score (Health Assessment Questionnaire) and serum samples for CRP, RF, anti-CCP2 antibodies (ELISA, QUANTA LiteTM, CCP IgG, INOVA Diagnostics Inc, USA), VEGF (ELISA, VEGF2, DRG International, IRC, USA) determination were recorded. Disease Activity Score for 28 joints (DAS28) was calculated. PDS signal was scored from 0 to 3 according to the overall expression of PDS findings at the knees. A sample of synovial tissue was obtained in 35 patients during the arthroscopy, and the vascularisation of the synovial tissue was evaluated by immunohistochemistry and was analyzed qualitatively by a pathologist who was unaware of the PDS findings. Written, informed consent was obtained from each patient before entering the study. They all had active synovitis of the knee, ultrasonographically confirmed, with the identification of the target biopsy sites. The study was approved by the Ethics Committee of the University of Medicine and Pharmacy of Craiova. RESULTS: Angiogenesis was evaluated and quantified by immunohistochemical evaluation of synovial VEGF, one of the most specific endothelial growing factors, that proved to correlate significantly with the serum levels of VEGF, DAS28 as well as with the power Doppler sonography (PDS) score. CONCLUSIONS: The statistical analysis of the data showed that PDS could be used as non-invasive marker with predictive value regarding synovial inflammation and disease progression in early forms of the disease as well as a useful method in the assessment of the therapeutic response.

Minimally invasive assessment of tumor angiogenesis by fine needle aspiration and flow cytometry.

The development of a new, less invasive, and more rapidly implemented method of quantifying endothelial cell density in tumors could facilitate experimental and clinical studies of angiogenesis. Therefore, we evaluated the utility of tumor fine needle aspiration (FNA) coupled with flow cytometry for assessment of tumor angiogenesis. Samples were obtained from cutaneous tumors of mice using FNA, then immunostained and assessed by flow cytometry to determine the number of CD31(+) endothelial cells. Results of the FNA/flow cytometry technique were compared with quantification of tumor microvessel density using immunohistochemistry. The ability of the FNA/cytometry technique to quantify the effects of anti-angiogenic therapy and to monitor changes in tumor angiogenesis over time in individual tumors was also determined. We found that endothelial cell percentages determined in tumor tissue aspirates by flow cytometry correlated well with the percentages of endothelial cells determined in whole tumor digests by flow cytometry and with tumor microvessel density measurements by immunohistochemistry. Moreover, we found that repeated FNA sampling of tumors did not induce endothelial cell changes. Interestingly, by employing repeated FNA sampling of the same tumors we were able to observe a sudden and marked decline in tumor angiogenesis triggered when tumors reached a certain size. Thus, we conclude that the FNA/flow cytometry technique is an efficient, reproducible, and relatively non-invasive method of rapidly assessing tumor angiogenesis, which could be readily applied to evaluation of tumor angiogenesis in clinical settings in humans.