Sjogren’s syndrome (SS) is a complex heterogeneous autoimmune disease resulting in loss of salivary gland and lacrimal gland function that may include multiple systemic manifestations including lymphoma. Multiple cell types participate in disease pathogenesis. This review discusses evidence for abnormal B cell subpopulations in patients with SS, critical roles of B cells in SS and the status of B cell–directed therapies in the management of patients with SS. Sjogren’s syndrome (SS) is an autoimmune disease defined by the presence of abnormal salivary and lacrimal gland function commonly characterized by the detection of specific autoantibodies, especially anti-nuclear autoantibodies (ANAs) and Rheumatoid Factor (RF) ,2,3. DisIn support of this overall concept are results arising from studies of mouse models for SS. Perhaps some of the most fascinating and original findings came from the Igμ gene knockout mouse, NOD-IgμNULL . The NODDespite the fundamental knowledge that B lymphocytes play an important role in the development of SS, we are still, decades later, trying to define basic relationships between B lymphocytes and SS disease. In this review we discuss the critical roles for B cells in SS, evidence for abnormal B cell subpopulations in patients with SS, and the status of B cell–directed therapies in the treatment of SS.Studies evaluating the subpopulations of B cells in the peripheral blood of patients with SS have measured decreased numbers of CD27+ memory B cells ,12,13,14Various studies with SS patients have identified increased serum levels and local expression by epithelial cells, B cells and T cells of B cell activating factor (BAFF), a growth and differentiating factor felt to be critical for the activation and survival of B cells ,22. SeruOverall, patients with SS have skewed subpopulations of B cells with both increased antigen-selected memory B cells, IgM memory B cells and CD5 positive B cells ,28. The Studies to evaluate the potential roles for B cells in SS have been performed out of necessity in animal models. Various models have been used, including the MRL/lpr mouse, which carries a mutation in the fas gene and reproduces the human lymphoproliferative syndrome; the NOD mouse, which developed spontaneous diabetes along with SS; the B6.NOD-Aec1Aec2 mouse, which carries genetic regions from the NOD mouse that predisposes C57BL/6J mice to develop SS without diabetes; the BAFF transgenic mouse, which develops proliferative glomerulonephritis along with SS; and the IL-14α transgenic mouse, which develops all the features of SS seen in patients in the same relative time frame, including hypergammaglobulinemia, autoantibodies, salivary and lacrimal gland hypo-function, lymphocytic pneumonitis, renal disease and lymphoma ,35,36,37Several interesting common features have been observed in these animal models. First, increased numbers of MZB and MZB precursors have been noted ,39,40. IIn experiments done with NOD mice, salivary gland injury was minimized in NOD mice treated with cobra venom to deactivate complement, suggesting that complement, especially C3, is important in disease pathogenesis . FurtherIn the B6.NOD-Aec1Aec2 mouse, elimination of C3 was again shown to dampen the clinical manifestations of SS . SimilarIn the BAFF transgenic mouse, elimination of MZB cells blocks the development of SS, but fails to alter the proliferative glomerulonephritis, suggesting that the nature by which various organs are injured even in the same autoimmune animal are different . EliminaIn the IL-14α transgenic mouse, several additional observations are of interest. First, eliminating lymphotoxin-alpha (LTα) prevents the development of all the clinical manifestations of SS . MZB spoTaken together, these studies in mice clearly indicate a central role for MZB cells in SS. They focus on local and systemic features of SS, although limited studies have evaluated dacryoadenitis. However, significant work is needed to understand the various roles that MZB cells play in the disease pathophysiology. Likely cytokine production, in particular LTα and possibly type 1 interferon, is important. MZB cells can be activated via Toll-like receptors as part of the innate immune system to produce various cytokines. In addition, MZB cells have been shown to carry antigens to germinal centers and present antigens to other cell types ,60. It iWhile autoantibodies are often considered to be the main inducers of local tissue injury, recent studies have raised doubt that this is true. In SS in particular, the functional relevance of autoantibodies to Ro and La, ubiquitous cellular antigens, is unclear. Particular autoantibodies, such as those directed towards muscarinic receptors type 3, have been shown to induce functional changes in vivo and in vitro ,63, whilThe treatment of SS has suffered from many problems: poor understanding of the pathophysiology of the disorder, variability in the disorder itself among different patients, and, most importantly, a delay in the diagnosis of patients with SS. Patients are often diagnosed at a point when there is little healthy glandular tissue remaining in the salivary and lacrimal glands ,72,73,74There have been three B cell–targeted therapies evaluated in SS: epratuzumab, an anti-CD22 monoclonal antibody, rituximab, an anti-CD20 monoclonal antibody, and Belimumab, an anti-BAFF monoclonal antibody ,76,77.Epratuzumab has been used only in one published, open, labeled study with 16 patients, eight of whom showed some clinical improvement . WhetherSimilarly, the work with Belimumab is very preliminary. An initial study with 30 patients demonstrated that 60% had improvement in fatigue, but no patient demonstrated improvement in either salivary or lacrimal gland flow. Improvement in certain parameters of B cell activation was achieved in some patients ,78,79. FRituximab has received the most attention as a therapeutic for SS. It has a much longer history as a therapeutic modality for B cell lymphomas, such as those associated with SS ,81. SeveWhile these B cell–directed therapies show promise, and certainly require further study, there are potential weaknesses that may prevent any of them from becoming the optimal therapy. First of all, rituximab can eliminate all populations of B cells, even though it tends not to impact memory B cells significantly . BeneficStudies evaluating patients with SS, as well as various animal models for SS, demonstrate abnormal B cell subpopulations, activities and products as central to the pathophysiology of SS as it progresses from a localized disease of the salivary and lacrimal glands initiated by an innate immune response to a systemic disease involving both the innate and adaptive immune system that can undergo lymphomagenesis. MZB cells appear to be key players in this disease, while other B cell subpopulations may play a variety of other roles. Further research is necessary to understand the roles of these critical cells so that more specific and efficacious therapies can be developed.


Neurofibromas are the hallmark lesions in Neurofibromatosis 1 (NF1); these tumors are classified as cutaneous, subcutaneous and plexiform. In contrast to cutaneous and subcutaneous neurofibromas, plexiform neurofibromas can grow quickly and progress to malignancy. Curcumin, a turmeric-derived polyphenol, has been shown to interact with several molecular targets implicated in carcinogenesis. Here, we describe the impact of different dietary patterns, namely Mediterranean diet (MedDiet) compared to the Western diet (WesDiet), both with or without curcumin, on NF1 patients’ health. After six months, patients adopting a traditional MedDiet enriched with 1200 mg curcumin per day (MedDietCurcumin) presented a significant reduction in the number and volume of cutaneous neurofibromas; these results were confirmed in subsequent evaluations. Notably, in one patient, a large cranial plexiform neurofibroma exhibited a reduction in volume (28%) confirmed by Magnetic Resonance Imaging. Conversely, neither unenriched MedDiet nor WesDiet enriched with curcumin exhibited any significant positive effect. We hypothesize that the combination of a polyphenol-rich Mediterranean diet and curcumin was responsible for the beneficial effect observed on NF1. This is, to the best of our knowledge, the first experience with curcumin supplementation in NF1 patients. Our report suggests that an integrated nutritional approach may effectively aid in the management of NF1. Polyphenols are secondary metabolites ubiquitous in the plant kingdom with unique antioxidant properties and a wide spectrum of therapeutic activities. It is well known that the phenolic compounds present mainly in olive oil contribute significantly to the superior health profile observed in Mediterranean populations following the traditional Mediterranean diet (MedDiet). In addition, curcumin, the most bioactive polyphenolic constituent of turmeric and an integral part of the Indian diet, exerts protective effects on a variety of diseases such as cardiac diseases, cancer, diabetes, Alzheimer’s disease, rheumatoid arthritis and psoriasis ,3,4,5,6.However, the mechanisms by which dietary polyphenols can affect human diseases are multiple and complex. Various stages of carcinogenesis may be inhibited by polyphenols in in vitro or in vivo systems. For example, curcumin is able to inhibit diethylnitrosamine-induced hepatocarcinogenesis in mice at a concentration of 0.2% in the diet , but theInterestingly, it has also been reported that polyphenols can affect tumor progression by interfering with the dynamic interactions between several components within the tumor microenvironment and the cancer cells. Indeed, the bi-directional interaction between cancer cells and the surrounding cells increases tumor proliferation, invasion and metastasis, and allows the tumor cells to resist therapeutic insults . CurcumiIn our study, we evaluated the effects of a traditional Mediterranean diet (rich in polyphenol content) compared to a Western dietary “pattern”, both with or without curcumin supplementation, in case series of patients with Neurofibromatosis type 1 (NF1), one of the most common autosomal dominant genetic disorders that affects ~1 in 3500 individuals . The NF1Our series included eleven NF1 patients identified on the basis of the National Institutes of Health (NIH) Consensus Conference criteria , clinically followed up at our Division of Neurology, Neurofibromatosis and Rare Diseases Center of the University Hospital, University of Campania Luigi Vanvitelli, Italy . Of thesEight patients were screened for NF1 mutations see . Geneticn = 2) was instructed to follow a Western diet (WesDiet); the second (n = 3) a traditional Mediterranean-style diet (MedDiet); the third (n = 3) a Western diet enriched with curcumin (WesDietCurcumin); and the fourth (n = 3) a traditional Mediterranean-style diet enriched with curcumin (MedDietCurcumin). All the diets were evaluated by a dietitian. In particular, the Western diet included ad libitum the consumption of red and processed meat, refined grains, French fries, sweets and desserts. No total calorie restriction was advised.The NF1 patients were divided into four groups on the basis of their dietary regimen: the first group , the consumption of ≥2 daily servings of vegetables, ≥3 daily servings of fresh fruits, ≥3 weekly servings of legumes, ≥3 weekly servings of fish or seafood, white meats instead of red meats or processed meats , and avoiding the consumption of cream, butter, margarine, pate, duck, carbonated and/or sugared beverages, pastries, industrial bakery products and desserts, French fries or potato chips, and sweets. Curcumin was supplemented at a dose of 1200 mg to the third and fourth groups at lunchtime ,20. Fina® software by Adobe Systems Software, Belfast, Ireland). Physical activity was not specifically advised for all NF1 patients. Compliance with the program was assessed by attendance at the follow-up visits and completion of the diet diaries. The patients were seen at two-monthly intervals over the six months of follow-up, after the introduction of the different diets. A complete physical examination, an evaluation of plasmatic curcumin level, integrated by photographic documentation were performed at each visit. Manual counting one by one of cutaneous neurofibromas was performed on all images  was performed. Head MR was performed using a 1.5 T MRI unit (Philips Gyroscan) including axial Spin-Echo T1, Turbo Spin-Echo (TSE) T2 and FLAIR (slice thickness: 5 mm), coronal FLAIR (slice thickness 3 mm), sagittal TSE T2 (slice thickness: 4 mm) and axial DWI (Diffusion Weighted Imaging). Volumetric analysis was performed using an algorithm for volumetric evaluations available in OsiriX, a software for viewing and processing DICOM images. It was necessary to make a correction of the gap between the slices (intergap correction). The tumor profiles were manually traced “slice by slice”.The patients’ nutritional status was evaluated applying a combination of clinical observation, bioimpedentiometric analysis and anthropometric and biochemical parameters.This study was approved by the Medical Ethics Committee and Safety Board of the University of Campania Luigi Vanvitelli, in accordance with the Declaration of Helsinki on ethical principles for medical research involving human subjects (protocol number 479/13). Written informed consent was obtained from each patient before admission to the study and to allow the initiation of any study-related procedures. In addition, the patients authorized the publication of this manuscript, its accompanying images and other data.v/v), sonicated in a water bath for 15 min and evaporated to dryness at 30 °C under negative pressure in a centrifugal concentrator. This process was repeated for a total of two extractions. The dried extract was reconstituted in methanol and subjected to HPLC analysis.A plasma aliquot was first treated with 10 μL of 6.0 M HCl and then with 10 U of β-glucuronidase type H-1 from Helix pomatia in 0.1 M phosphate buffer (pH 6.86). The resulting mixture was then thoroughly vortexed and incubated at 37 °C for 1 h to hydrolyze the phase-2 conjugates of curcuminoids. After incubation, curcuminoids were extracted with 2 volumes of methanol/chloroform  protected by a guard column  and maintained at 30 °C. A linear elution gradient consisting of a mobile phase A (0.1% acetic acid), B (Acetonitrile), and C (Methanol) was programmed as follows: initially 50% A, 45% B, and 5% C, linearly changed to 30% A, 65% B, and 5% C over 5 min, and then held for 4 min at 30% A, 65% B, and 5% C. The system was then re-equilibrated for 5 min with the initial solvent. The detection wavelength was set at 420 nm. The quantitation of curcuminoids is by peak area ratio  and is based on a standard curve in a plasma or urine matrix, generated by using an external standard to spike plasma or urine. A linear curve is generated from a single analysis of six different standard concentrations. System control and data acquisition were performed using the ChemStation software .The HPLC-UV procedure was conducted according to Heath et al.  with somp < 0.05, using GraphPad Prism Version 5.04 software .Statistical analysis was conducted using a one-way ANOVA test, with the significant differences determined at Patients 1, 2, 6, 7 and 8 did not adhere to the MedDiet and did not significantly modify their dietary habits, which were consistent with a WesDiet; among them, Patient 6, 7 and 8 agreed to introduce curcumin to their diet (WesDietCurcumin). Patients 3, 4 and 5 followed a MedDiet. Patients 9, 10 and 11 agreed to follow a MedDietCurcumin. As shown in Clinical follow-up integrated by self-assessment of outcome in the first (WesDiet—patients 1 and 2), second  and third group  did not detect significant phenotypic variations in the number, size and color of cutaneous neurofibromas or in other signs and symptoms of disease, clinically detectable, at the different time points, compared to baseline. Patient 9 , a 54-yePatient 10, , a 27-yePatient 11 , a 50-yeFrom a metabolic point of view, as shown in All patients following a Mediterranean diet with curcumin supplementation  or without curcumin  presented a general improvement in their metabolic status, with a BMI reduction and an increase in body hydration, due to the greater intake of water; in all of these patients, the percentage of fat-free mass (FFM), and basal metabolism (BMR) increased. Phase angle (PA) improved in the Mediterranean and MedDietCurcumin patients, with the exception of patient 11, for whom it remained stable. Na/K exchange remained essentially unchanged in all of these patients.No patient reported any side effects of curcumin consumptionThe curcumin supplemented dietary regimen was established on the basis of previous clinical studies for inflammatory conditions, where active dosages of around 1–2 g/day of curcuminoid were used ,20. For The association between NF1 and malignant tumors , leukemia and rhabdomyosarcoma) has been largely described . The mutHere we report the beneficial effects of a traditional Mediterranean Diet (MedDiet) enriched with curcumin (MedDietCurcumin) on the number and size of neurofibromas in all NF1 patients consuming this diet (patients 9–11). Indeed, besides the improvement in the general metabolic status, and particularly of the lipid profile, we observed a marked reduction in the number (ranging from 30 to 51%) and volume of neurofibromas after six months of MedDietCurcumin. Notably, the large cranial plexiform neurofibroma in patient 11 exhibited a marked volume reduction (28.2%), as shown by a conventional imaging method. In contrast, we observed no significant effect on the pattern of neurofibromas in the patients who did not follow MedDietCurcumin.Epidemiological evidence and many case-control studies suggest that a Mediterranean diet plays a pivotal role in lowering the risk of several chronic diseases, including cardiovascular disease, neurodegenerative disease, diabetes, and cancer. A high intake of olive oil is considered a hallmark of the traditional Mediterranean diet. In this study, we used extra-virgin olive oil (EVO—the juice of the olive obtained solely by pressing and consumed without any further refining process) having >2000 mg/kg total phenol concentration. It has been reported that EVO consumption increases the monounsaturated fatty acid content in phospholipids and cholesterol esters by modifying the fatty acid composition of the plasma membrane, which influences the association of G proteins and PKCa with the lipid bilayer in elderly persons with type 2 diabetes . The autIn particular, curcumin has been extensively studied for its role in Ras oncogenic signaling pathways, notably by abolishing the RAS-ERK signaling mechanism . DependiUnfortunately, while curcumin is one of the most used nutraceuticals, its utility as a therapeutic agent is limited by its poor water solubility, short biological half-life, and low bioavailability after oral administration in certain tissues. Therefore, various approaches including the use of adjuvants, liposomes, nanoparticles, phospholipid complexes and reformulation with various oils have been tried . InteresNF1 presents a unique situation in which the existence of widespread neurofibromas, and their tendency to recur, calls for more effective and sustainable medical intervention than the simple surgical removal of individual lesions. The results presented in this study are of particular interest as they are the first clinical demonstration of the therapeutic activity on NF1 of curcumin in combination with a dietary approach rich in polyphenols.As shown by our data, the plasma level of curcumin increased in patients following MedDietCurcumin; this suggests that curcuminoid bioavailability is positively influenced by polyphenol-rich foods in the Mediterranean diet. However, this synergism between curcumin and polyphenols needs to be confirmed on a larger cohort of NF1 patients.Further studies are necessary to elucidate the molecular mechanisms by which MedDietCurcumin is able to reduce both the number and volume of neurofibromas. In addition, clinical trials involving new nanoformulations of curcumin conjugated with long-term observations may provide further data on the potential therapeutic role of curcumin in NF1 as well as in other inherited or sporadic cancer syndromes.


Despite the successful clinical application of titanium (Ti) as a biomaterial, the exact cellular and molecular mechanisms responsible for Ti osseointegration remains unclear, especially because of the limited methodological tools available in this field.In this study, we present a microscopic and molecular characterization of an oral implant osseointegration model using C57Bl/6 mice.Forty-eight male wild-type mice received a Ti implant on the edentulous alveolar crest and the peri-implant sites were evaluated through microscopic  and molecular  analysis in different points in time after surgery .The early stages of osseointegration were marked by an increased expression of growth factors and MSC markers. Subsequently, a provisional granulation tissue was formed, with high expression of VEGFb and earlier osteogenic markers . The immune/inflammatory phase was evidenced by an increased density of inflammatory cells, and high expression of cytokines  chemokines  and chemokine receptors (CCR2 and CCR5). Also, iNOS expression remained low, while ARG1 was upregulated, indicating predominance of a M2-type response. At later points in time, the bone matrix density and volume were increased, in agreement with a high expression of Col1a1 and Col21a2. The remodelling process was marked by peaks of MMPs, RANKL and OPG expression at 14 days, and an increased density of osteoclasts. At 21 days, intimate Ti/bone contact was observed, with expression of final osteoblast differentiation markers , as well as red spectrum collagen fibers.This study demonstrated a unique molecular view of oral osseointegration kinetics in C57Bl/6 mice, evidencing potential elements responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel microscopic analysis. No antibiotics and anti-inflammatory drugs were administered to the animals after implantation surgery and there was no evidence of weight loss, infection and persistent inflammation in the surgical sites. This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of HealthForty-eight male wild-type mice (C57Bl/6)  were obtained from the animal facilities of FOB/USP. Thirty-six animals were used for microscopic analysis  and twelve animals were used for molecular assays, distributed along 4 experimental periods: 3, 7, 14 and 21 days after surgical procedure. Throughout all experimental periods of this study, the mice were provided sterile water In an attempt to employ a titanium screw comparable to the one clinically used in Dentistry, a screw with Ø 0.6 mm, titanium-6 aluminum-4 vanadium alloy  and machined titanium surface was used in this study, as previously described in the oral osseointegration model in CD1 mice®, Agribrands Brasil LTDA, Paulínia, SP, Brazil) and 160 mg/kg of xylazine chloride  in a 1:1 proportion, which was determined according to the animal’s weight. Subsequently, the mice were placed on a surgical table with a mouth retractor, as previously described in other Dentistry mice models,®, British Columbia, CA, USA). A small detachment of the mucoperiosteum was made and the subjacent bone was drilled using a pilot drill with Ø 0.50 mm . The pilot hole was performed using a surgical motor , with 600 rpm speed and 35 N to kilogram force, under continuous irrigation with cold saline solution, in order to avoid heating and subsequent bone necrosis. The Ti-implant was screwed down in the implant bed using a Castro Viejo Micro Needle Holder   . All surThirty-six mouse maxillae containing the Ti-implants were scanned with the Skyscan 1176 System  at 80 kV, 300 μA, 180 degrees of rotation and 1-degree exposure range. The images were captured with a 12.45 μm pixel size resolution. The projection images were reconstructed using the NRecon software  with 35% of Beam Hardening Correction and subsequently aligned using the Dataviewer 1.4.4.0 software  to standardize the position of all specimens for subsequent quantitative evaluation . The thrper HE section, comprising the region adjacent to the thread spaces, were captured using a 100× immersion objective. A grid image was superimposed over each histological field, with 10 parallel lines and 100 points in a quadrangular area, by using the Image J software . Briefly, the points were counted coinciding with the following parameters of the osseointegration process: blood clot, inflammatory cells, other elements (empty spaces left by the implant’s space), blood vessels, fibroblasts, collagen fibers, osteoblasts, osteoclasts, and new bone matrix. The results were presented as the mean area density for each structure considered in each examined group.The same mice maxillae used for microCT scanning were processed for histological analysis. Forty semi-serial sections were cut with 4 µm thickness, of which nine serial sections considering the central region of bone for implant contact were chosen for histomorphometry and stained for hematoxylin and eosin (H&E) staining. The analyses were performed by a single calibrated investigator with a binocular microscope  using a 100x immersion objective. Six histological fields For birefringence analysis, 4 sections with 5 µm thickness histological slides considering the central region of the bone for implant contact were used for picrosirius red staining and birefringence analysis. As previously described®, Thermofisher, Waltham, MA, USA) until the RealTime PCR array reactions. The RealTimePCR array reactions were performed as previously described,,The samples containing only the region of the implant bed were resected and stored in a RNA Stabilization Solution  and Mann-Whitney test were used. The statistical significance of the experiment involving the PCR Array was evaluated through the Mann-Whitney test, and the values were tested for correction of Benjamini and HochbergDifferences between data sets were statistically analyzed through One-Way Analysis of Variance (ANOVA) followed by Bonferroni’s multiple comparison Our focus is this study was to address a pre-clinic murine model of oral osseointegration, previously developed in CD1 miceSEM micrographs demonstrated uniform unidirectional threads, with no deposits and no particular features or deformation and characteristics of a clearly machined surface topography, such as small irregularities. In the composition characterization, Ti screw alloy had a mass with 75.35% of Ti, 14.66 % of V, 5% of N and 4.20% of Al. Other evaluated chemical elements were found in minor concentration, less than 1% .For developing the surgical protocol, the Ti-screw was implanted in the edentulous space between the maxillary right first molar and the incisor. After a day of surgery, the animals were able to eat crumbled food and were acting normally, with no signs of distress. All animals had complete oral mucosal healing by day 7, as clinically demonstrated in Subsequently, we evaluated the sites of Ti-implantation through microtomographic qualitative and quantitative analyses of mineralized bone matrix . The thrConsidering the histological analysis, the panoramic transversal image of mouse maxillae demonstrated that the Ti-screw was projected through the palatal bone into the olfactory epithelium of the maxillary sinus  from the new bone matrix and initial granulation tissue. A negligible quantity of collagen fibers was found after 3 to 7 days around the Ti threads, emitting birefringence in the green spectrum (i.e. immature and thinner fibers) . From 7 th day, followed by a gradual decrease in their expression in subsequent experimental periods  to late experimental periods (14 and 21 days) in the oral cavity of C57Bl/6 mice .per se, as has been frequently used in experimental studies using craniofacial,While the C57Bl/6 strain was used in a previous study as recipients/hosts of Ti devices in the oral cavityThe surgical procedures used in this study were performed following the same principles and procedures used in Dentistry, to avoid lack of primary stability and overheating. Of all titanium implants with adequate primary stability, 77.78% achieved osseointegration, demonstrated through µCT and histological data  and 4, win vitro studies,Initially, our histological characterization demonstrated that blood is the first biological element in contact with the Ti surface, evidenced by the formation of a highly organized clot in contact with the Ti threads and native bone after 3 days  and B´ a,,,Concurrently with the early upregulation of the MSCs markers, a provisional extracellular matrix is formed and gradually evolves into a highly vascularized granulation tissue  and 5, w,Also in these earlier stages of osseointegration, the immune/inflammatory response is triggered at the Ti/host interface, which integrates the key molecular events for determining the success or failure of osseointegration,Indeed, in addition to the classical role of macrophages on debris clearance after injury, these cells are key regulators of inflammatory and regenerative processes, by releasing different mediators in response to the state of polarization towards the M1 (inflammatory) or M2 (reparative) phenotype, and orchestrating the outcomes of inflammation and bone healing,,Following the resolution of inflammation , while tst day, with an average 42.12±3.01% mineralized bone matrix (BV/TV) detected around the Ti threads via microCT analysis analysis , and alsanalysis , possiblIn summary, this study originally demonstrated a unique molecular view of the kinetics of osseointegration, evidencing elements that could be responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel histological, birefringence and μCT analysis . Conside


Aspergillus versicolor Tiraboschi was often encountered on building partitions (frequency 4: frequently). The ability to synthesize the carcinogenic sterigmatocystin (ST) means that it poses a risk to humans and animals. Biotoxicological tests of biomasses of A. versicolor were conducted in the Microbiological and Toxicological Laboratory, using the planarians Dugesia tigrina (Girard). The obtained results of the tests covered a broad range of toxicity levels of isolated strains: from weakly toxic (100–1000 mg·L−3) to potently toxic (1–10 mg·L−3). The high-performance liquid chromatography (HPLC) physicochemical method confirmed the ability of A. versicolor strains to synthesize sterigmatocystin. All of the samples of the air-dry biomasses of the fungi contained ST in the range between 0.03 and 534.38 mg·kg−1. In the bio-safety level (BSL) classification A. versicolor belongs to category 1. Additionally, A. versicolor is an allergenic mould. In an area representative of a moderate climate zone (Lubuskie Province in Poland), mycological tests in over 270 flats demonstrated the occurrence of 82 species of moulds.  Aspergillus versicolor and Stachybotrys chartarum [S. chartarum  and A. versicolor (sterigmatocystin) may be present in the majority of samples coming from the building materials of damp flats or from samples of settled dust. The problem with the occurrence of moulds in buildings is not only the biodeterioration of technical materials, but also the deterioration of the sanitary condition of the indoor air ,2. This hartarum ,8,9,10. hartarum  showed tAspergillus versicolor is a saprotrophic fungus, which is widespread in nature. It is often present in soil , on many rotting food products , and on cereals. It produces proteolytic enzymes and often appears on fats and their derivatives, as well as on bread. It also tolerates a very broad pH range, including an alkaline environment. The optimum temperature for its growth amounts to 20 °C–30 °C . Conidia germinate at temperatures between 12 °C–37 °C [A. versicolor are easily inhalable [°C–37 °C ,14. The °C–37 °C . Spores nhalable ,17. TheiAspergillus versicolor is a primary coloniser capable of growing at aw > 0.65–0.70 [.65–0.70 ,18,19. T.65–0.70 ,23,24,25.65–0.70 , and its.65–0.70 ,26.Aspergillus versicolor synthesises important toxic metabolites: 5–methoxysterigmatocystin and dihydroxy–sterigmatocystin, 5,6-dimethoxysterigmatocystin (with cytotoxic properties), averufin, deoxyaverufin, cyclopiazonic acid, nidulotoxin, versicolorin A, B, and C , versiconol (with low toxicity), and sterigmatocystin (ST)  [−1 of body weight, and kidney damage at higher doses. Although sterigmatocystin may initiate liver cancer in rats and cause the development of tumours at the site of its administration, its carcinogenic effect is ten times smaller than that of AFB1. The toxic effect is also smaller [A. versicolor consistently produces carcinogenic sterigmatocystin on the majority of laboratory media. It synthesises substantial quantities of ST and 5 methoxy-sterigmatocystin on building materials [A. versicolor from the air inside the rooms proved that these strains produced ST in laboratory cultures, and the extracts of the cultures demonstrated cytotoxicity. It was determined that conidia of two strains of A. versicolor comprised 0.375 and 1.060 pg (10−12 g) of ST in the spore [Artemia salina) are 5.95 mg·L−3 and 0.54 mg·L−3 [−1) amounts to: ducklings, 1.0; albino rats, 166 ; albino rats, 60 ; albino rats, 120 ; albino rats, 65 ; male monkeys, 32 (i.p.) [enicity) ,28,29,30 smaller . A. versaterials . The teshe spore . In labohe spore . Other s4 mg·L−3 , respect2 (i.p.) .A. versicolor include 2-ethyl-1-hexanol, 1-octen-3-ol, 3-octanone, 2-heptanone, 2-pentanone, 2-hexanone, and 2-methylfuran. Isolates produce pronounced earthy volatiles [Volatile metabolites (MVOCs) produced by olatiles . Aspergillus versicolor, and also the results of biotests from extracts of A. versicolor biomasses), which indirectly confirm the health risk for people living in mouldy premises. The research was conducted in the area of the Lubuskie province in Poland. The area is representative of many buildings erected within the moderate climate zone. Collection of the big number of results  allowed the establishment of the frequency of occurrence of the respective taxons on building partitions, as well as the indication of 10 species which colonise walls most frequently. The results, after taking into consideration the potential risks gathered on the basis of many different published references, constituted the grounds for the selection of the species (out of 10 species on the list) for mycotoxicological tests (high-performance liquid chromatography (HPLC) and biotests). Aspergillus versicolor became our choice as it is often present in residential housing and consequently synthesises sterigmatocystin, which is a carcinogenic substance from category 2B  [The paper presents the results of research conducted for many years (more than 20 years), with regards to the occurrence of moulds in residential housing, as well as the assessment of potential risks  and real risks  ), on theA. versicolor obtained in laboratory cultures and coming from buildings were subjected to toxicity tests. The biotoxicological testing of methanol extracts from mould biomasses was carried out using Dugesia tigrina (Girard). Forty-five mould samples were analysed for the presence of sterigmatocystin (ST) in the Department of Pharmacology and Toxicology of the National Veterinary Research Institute in Puławy, using HPLC. Mycological and mycotoxicological tests have been carried out in the Microbiological and Toxicological Laboratory at the Department of Applied Ecology, Institute of Environmental Engineering, University of Zielona Góra (Poland). Forty-five air-dry biomasses of Moulds were collected in over 270 buildings from the inner surfaces of building partitions with visible mould. The partitions were made of plasterboard, bricks, and breeze blocks, and the finishing materials included plaster, acrylic paint, wallpaper, and others. The samples were collected directly at the site of occurrence and transferred onto Petri dishes, which contained such media as malt extract agar (MEA), Merck and Czapek–Dox (Cz), and Merck. Mycological analysis was performed in accordance with the methodology established by CBS  . The dirAcari) were taken directly from the place where moulds were present onto surfaces with two media (Cz and MEA), using Petri dishes. Mites were mounted in a Hoyer medium on slides, and the species and life stages were determined with the aid of an Axioscope 2 Zeiss compound microscope . Solarz [Samples of mites  before their filtration through Whatman 4 blotting paper placed on a filter flask. In the applied procedure, 1 mL of extract is obtained from 10.0 mg of sample (mould plus medium). In order to carry out 45 toxicity analyses of  Piontek ,38,39. OA. versicolor isolated from flats was investigated. For all of them, toxicological tests using Dugesia tigrina were carried out. The obtained results were the basis for the calculations of a lethal concentration LC 50, (mg·L−3) for test organisms. The method of the cultivation of the planarians for toxicological tests was developed by Piontek [3 were poured into beakers with capacities of 50 cm3. 10 cut specimens were introduced into each beaker [2 test was used in the calculations. The tested distributions were considered sufficiently convergent with the normal distribution, if the likelihood in the χ2 test was higher than 0.7 [The toxicity of 45 strains of  Piontek . The toxh beaker ,50,51,52than 0.7 . The obtthan 0.7  . The determination of the content of sterigmatocystin in fodder and mycelium was conducted by means of high-performance liquid chromatography  [Forty-five air-dry biomasses of 1.04.28) . A detai1.04.28) ,39.The toxicity of ST was evaluated based on the classification established by Chełkowski ,33, Tab. In the mycological tests conducted in over 270 flats, 82 species of moulds  were isoAcari), belonging to Tyrophagus putrescentiae was found [The presence of two or more species of moulds in one sample was confirmed in reference to all of the tested samples. As well as the moulds, the presence of numerous bacteria and allergenic mites . Two species were present frequently: Ulocladium chartarum and Aspergillus versicolor . The following species of moulds were present with the highest frequency: The specification of the obtained results of physicochemical and biotoxicological tests is presented in A. versicolor were weakly toxic , 26 strains (58%) belonged to Class III (medium toxic), for which the LC 50 ranged between 10 and 100 mg·L−3, and six strains (13%) were potently toxic (Class II). At the National Veterinary Research Institute in Puławy, the analysis was performed based on 45 biomasses of A. versicolor , which classifies these strains as very potently toxic, 51% of biomasses were potently toxic (the quantity of ST ranged between 13.6 and 99.7 mg·kg−1). 20% of biomasses were weakly toxic, whereas four biomasses of A. versicolor with the quantity of sterigmatocystin below 1 mg·kg−1 were non-toxic (n.t.).As the obtained results of the biotoxicological tests indicate, 30% of the strains of rsicolor . The quaolor  on building materials ,27,31,32The risk of the release of mycotoxins may occur when spores and fragments of moulds, as well as particles containing mycotoxins such as dust or mouldy substances, are inhaled and swallowed. Therefore, the most important issue is to determine which, as well as how often and how many, mycotoxins are present naturally in mouldy building partitions in residential housing . The toxA. versicolor is the main producer of carcinogenic sterigmatocystin with nephrotoxic, hepatocarcinogenic and immune-suppressive properties. The tests performed in the Microbiological and Toxicological Laboratory confirm that A. versicolor is a species of mould which may constitute a risk to residents of infected rooms. It is often present on the building partitions of buildings located in the areas representative of a moderate climate (D. tigrina demonstrated the occurrence of potently toxic strains (13%), which means that the biological methods are applicable for the evaluation of the mycotoxic risk in buildings (−1 of it (highly toxic strains according to Chełkowski) justifies the need to conduct research on this species. Chemical analysis unfriendly to the environment could be replaced by biological one. Physicochemical analysis (HPLC) should be conducted in the case of a high toxicity level of the sample.  climate . The toxuildings . The varA. versicolor. The majority of the strains (98%) of A. versicolor isolated from the dust was able to synthesise ST in vitro [Penicillium chrysogenum, and among Aspergillus, Aspergillus versicolor [Tests of dust from damp residential premises conducted in Germany demonstrated the occurrence of the species of mould called in vitro . Mycologrsicolor . This faOn the basis of the mycological tests performed, it is possible, based on literature on the subject, to determine the potential hazard for humans inside the infected rooms. In 1996, a list was drawn up under the auspices of the European Confederation of Medical Mycology, which specified the classification of biosafety levels (BSL) for the respective species of fungi (BSL 1/3) .According to this list, it is possible to specify species of moulds occurring in residential housing, which are pathogenic for humans and animals and which were most frequently present on building partitions in the Lubuskie province .A. versicolor, are classified into the BSL1 category. This means that infections which may be caused by these species are most frequently benign, superficial, and non-invasive. In the rooms subjected to the tests, the most threatening pathogens were not present on the walls (BSL 3).The isolated moulds, including Aspergillus, Cladosporium, and Penicillium present in damp buildings may cause asthma and/or rhinitis among atopic residents. It has been proved that raw extracts including A. versicolor may cause the release of histamines without the mediation of IgE in human cells of the mucous membranes from lungs in vitro [d-glucans), microbial volatile organic compounds (MVOC) and mycotoxins, as well as other organisms which occur together with the moulds, such as mites (Acari) [Aspergillus and Penicillium [Moulds are the cause of many allergic diseases ,17,58,59in vitro . Allergi (Acari) ,61,62. Ticillium ,17.Aspergillus versicolor may cause allergic diseases. Among 82 species of moulds occurring in residential housing in the Lubuskie province, the other allergenic fungi include: Penicillium chrysogenum, Aspergillus niger, Cladosporium herbarum, Alternaria alternata, and Aspergillus flavus. The moulds which may occur with lower frequency in buildings, but still may cause allergic diseases, include Penicillium glabrum, Aspergillus fumigatus, and Aureobasidium pullulans (present paper).At present, the time of exposure and the concentration of mycotoxins which may have a negative impact on the human health of residents is not known . HoweverDugesia tigrina Girard instead of the costly physicochemical analysis by means of HPLC, which is additionally not friendly to the natural environment. This was confirmed by the results of the analyses carried out by means of HPLC. Strains of A. versicolor, which have the biological abilities to synthesise sterigmatocystin, occur on building partitions. However the number of highly mycotoxigenic strains in laboratory conditions is small. All of the research results presented in the paper are innovative as, owing to consequent complementation , they led to valuable conclusions. The paper includes quotations from our own 17 research publications which were continued until our own two tables presented in this paper were drawn up . The conducted toxicological tests proved that Aspergillus versicolor is the most dangerous species for humans and animals in infected rooms owing to the synthesis of toxic and carcinogenic sterigmatocystin ST  in various quantities. All of the samples of air-dry mould biomass with the laboratory medium contained sterigmatocystin. A. versicolor is an allergenic species which may cause benign fungal infections. Biotoxicological tests of extracts from the biomasses of A. versicolor are necessary for the evaluation of the mycotoxic risk in buildings.The mycological tests demonstrated the frequent occurrence of


Dietary intake was measured using 24-h dietary recalls. Baseline characteristics were collected. Associations between BCAA intake and demographic, socioeconomic and lifestyle factors were determined using linear regression. Results: Total BCAA intake was 217.14 mg/kg·day . BCAA intake was negatively associated with female sex in adolescents and adult groups, with no white race in adolescents, and with former smoker status in adults. Conversely, BCAA was positively associated with household per capita income in adolescents and adults. No associations were observed in the older adults group. Main food contributors to BCAA were unprocessed red meat, unprocessed poultry, bread and toast, beans and rice. Conclusions: Adolescents and adults were the most vulnerable to having their BCCA intake influenced by demographic, socioeconomic and lifestyle factors.Background: Identifying which risk groups have a higher intake of branched chain amino acids (BCAA) is important for the planning of public policies. This study was undertaken to investigate BCAA consumption, the foods contributing to that consumption and their association with demographic, socioeconomic and lifestyle factors. Methods: Data from the Health Survey of São Paulo, a cross-sectional population-based survey ( The branched-chain amino acids (BCAA) leucine (Leu), isoleucine (Ile) and valine  are an important class of essential amino acids. These amino acids play an important role in the human body for protein synthesis, production of energy and synthesis of many neurotransmitters ,2. MoreoSeveral studies have shown that elevated levels of BCAA in blood, including Leu, Ile and Val, may be related to the risk of developing type 2 diabetes, insulin resistance and cardiovascular diseases ,5,6,7. TStudies evaluating the dietary intake of BCAA are scarce. Moreover, despite the literature demonstrating the importance of identifying the relationship between dietary intake and socioeconomic status for advances of public health , there iTo our knowledge, there have been no population-based studies that examined the association of dietary BCAA intake with demographic, socioeconomic and lifestyle factors in Brazil. Therefore, the aim of this study was to investigate BCAA consumption and their association with demographic, socioeconomic and lifestyle factors, and to identify the main foods contributing to BCAA intake.This study was approved by the Ethics Committee of the Public Health School of the University of São Paulo . All participants provided written informed consent before all procedures.Data were drawn from the 2008 Health Survey of São Paulo . This is a cross-sectional population-based survey that used a complex, stratified, multistage probability sample of individuals living in permanent households from urban areas of Sao Paulo, in southeastern Brazil. In this survey, health, nutritional information and lifestyle conditions were collected.The sample was obtained in two stages. As the primary sampling units, 70 census tracts were randomly selected from all urban census tracts in the city of São Paulo. In the second stage, 16,607 households were randomly selected within these census tracts and stratified. Eight domains were fixed according to age and sex: infants ; children ; male adolescents (12–19 years); female adolescents (12–19 years); male adults (20–59 years); female adults (20–59 years); male older adults (60 years or more) and female older adults (60 years or more). To preserve the representativeness of each domain and to correct differences in the relative participation of the age groups in the population of the city of Sao Paulo, different sampling fractions were applied. The sample size was calculated to estimate proportions of 0.5 with a sample error of 0.07 at a 5% significance level and design effect of 1.5.n = 560), adults (n = 585) and older adults (n = 517) of both sexes, who answered a structured questionnaire and at least one 24-h dietary recall (24HR). The survey response rate was 77%. The findings presented here are based on the complete case analysis.This ISA-Capital study included 2691 individuals aged 12 years and older for both sexes . In the present study, we included only data obtained from adolescents . The household head’s level of education was measured in years of study. Household per capita income was calculated by adding the monetary income reported by all family members and dividing by the number of family members . The smoking status was obtained from questions about current or former smoking habits and the number of cigarettes smoked per day were classified as never, current and former smoker status. The intake of alcoholic beverages was obtained by asking questions about the amount, frequency and preference of the consumption of alcohol, and the individuals were classified as consumer or non consumer of alcoholic beverages [Age was calculated by the difference between the interview date and the date of birth. Self-reported race was classified as everages .insufficiently active or sufficiently active .Level of physical activity was collected by the long version of the International Physical Activity Questionnaire . Leisure2)), from self-reported weight and height and classified according to the cut-off for adolescents  [2; healthy weight, BMI 18.5–24.9 kg/m2; overweight, BMI 25.0–29.9 kg/m2; obese, BMI ≥ 30 kg/m2) [2; healthy weight, BMI 23.0–27.9 kg/m2; overweight, BMI 28–29.9 kg/m2; obese, BMI ≥ 30 kg/m2) [Body mass index (BMI) was calculated using the Quetelet equation (BMI = weight (kg)/height (mcentile) , adults 0 kg/m2)  and olde0 kg/m2) . This in0 kg/m2)  in a preDietary intake was assessed based on two 24HRs. Both were collected by trained interviewers on non-consecutive days, during all seasons of the year and days of the week. The first 24HR was collected in the household and the second was collected by telephone. These 24HRs were collected using, respectively, the Multiple-Pass Method and the Automated Multiple Pass Method, which have been described in detail by Guenther et al. and Blanton et al. ,18. The The prevalence of inadequate dietary intake of BCAA was determined using the estimated average requirement (EAR) cut-off point approach, a simplification of the probability method, which estimates the proportion of individuals with usual intakes below the EAR (median requirement).Food contributors to BCAA intake were investigated using the method of considering the sampling design, proposed by Block et al. . This meThe misreporting percentage was estimated using the equation: Energy intake—EER (estimated energy requirements)/EER∙100 . The equThe characteristics of subjects were presented as medians and interquartile range (IQR) for continuous variables, and percentages for categorical variables. The total BCAA was defined as the sum of the energy-adjusted dietary Leu, Ile and Val. The usual BCAA intake was estimated using the Multiple Source Method (MSM) . This wep-values < 0.05 as statistically significant.All analyses considered the complexity of the sample design and were performed with STATA version 13.0 , considering n = 1662) was predominantly composed of the female sex (56.8%), the adults age group (35.2%), never smoked status (69.5%), self-reported white skin colour (58.0%) and non-consumer of alcohol beverages (58.3%). The median of the household head’s education was eight years (IQR 4–11) and household per capita income was US$275.0 (IQR 164.0–476.0). Excess body weight was observed in 35.7% of the individuals and 84.7% had insufficient leisure time physical activity.The sample  and the total BCAA with socioeconomic, demographic and lifestyle conditions are shown in The main food contributors to Leu, Ile, Val, and total BCAA intakes were unprocessed red meat, unprocessed poultry, bread and toast, and beans and rice .In this study, we observed consistent associations of BCAA consumption, including Leu, Ile and Val, with socioeconomic, demographic and lifestyle factors in adolescents and adults. Compared to dietary reference intake (DRI), residents of São Paulo appear to have a low prevalence of inadequacy. It is important to emphasize that some authors have raised questions about the DRI for BCAA intake and suggested higher values of requirement. Riazi et al. , for exaIn adults, protein requirement estimations has been described in the literature and depend on one of two main approaches, namely, the factorial method and nitrogen balance. The BCAA-requirement estimation methods have limitations and are inconclusive. The DRI community uses the average of the values found by the methods . BesidesRegardless of age, women had a lower association with the consumption of these amino acids when compared with men. Micha et al.  observedFormer smoker status was associated with higher intake of individual and total BCAA in adults. Smoking cessation appears to be associated with being overweight and having a higher intake of energy , cholesterol, saturated fatty acids, and alcohol . In addiThe results obtained in the present study pointed to the association between self-reported race, household per capita income, and higher intake of all these amino acids. This association corroborates with the results of Carvalho et al. , which sOur findings demonstrate that unprocessed red meat is the main source of dietary Leu, Ile, Val and total BCAA intake for all age groups, constituting 22% of food intake. Regarding the BCAA intake, our results were similar to the US adult population, in that the major food contributors were red meat (~37%), milk (~12%) and fish (~8%) . HoweverIn older adults, no significant results were found. This age group generally consumes less protein and some of the major dangers of this is the loss of muscle mass, strength and sarcopenia, which is a function that progressively occurs with aging. Several studies have identified animal protein, which contains essential amino acids, as a key nutrient for muscle health in older adults. These populations are less responsive to the anabolic stimulus of low doses of amino acid intake compared to younger individuals . TherefoIn a recent population-based cohort study, Zheng et al.  showed tOur study has limitations. First, this is a cross-sectional study, consequently, it is impossible to determine causality between the factors evaluated and BCAA intake. In addition, the dietary assessment used was the 24HR, which is a method that has a source of errors such as recollection of memory, omissions and possible errors in portion size estimation. However, we sought to minimize the influence of the potentially confounding variables. We measured the database from two 24HRs, through the Multiple Pass Method and finally, we used the MSM to estimate usual intake.Individuals with a higher household per capita income, of the male sex, white race and those who were former smokers are the groups that are most vulnerable to higher Leu, Ile, Val, and total BCAA intake. These characteristics may be used to better target the planning of public health policies.


At 8 weeks after surgery, the NBV increased in all four groups. However, larger NBVs were observed in the BG and DHT + BG groups, and a significant difference was no longer observed between the two groups. Histologic analysis demonstrated that the graft materials sustained the center of the defect in the BG and DHT + BG groups, which was shown in histomorphometric analysis as well. These results suggest that DHT membrane is a safe biomaterial with adequate tissue integration, and has a positive effect on new bone formation. Moreover, the best effects were achieved when DHT was used in conjunction with BG materials.In this study, the bone regeneration efficacy of dehydrothermally (DHT) cross-linked collagen membrane with or without a bone graft (BG) material was evaluated in a critical-sized rat model. An 8-mm-diameter defect was created in the calvaria of 40 rats, which were randomized into four groups: (1) control; (2) DHT; (3) BG; and, (4) DHT + BG. Evaluations were made at 2 and 8 weeks after surgery using micro-computed tomographic (micro-CT), histological, and histomorphometric analyses. Micro-CT analysis showed an increase in the new bone volume (NBV) of the BG and DHT + BG groups at 2 weeks after surgery, representing a significant difference ( The presence of sufficient bone volume is a prerequisite for the predictable osseointegration of a dental implant. However, alveolar ridge resorption over time, combined with tooth loss and the presence of anatomical structures such as the maxillary sinus, nasal cavity, and inferior alveolar nerve all limit the amount of available bone for implant placement ,2. ThereGuided bone regeneration (GBR) is a well-established and widely used technique that promotes new bone formation using a barrier membrane to exclude epithelial and connective tissue proliferation within the defect ,4. The bSince collagen is the main component of periodontal tissues, collagen-based materials are representative degradable membranes with many advantageous properties. These include minimal inflammation rates, low immunogenicity and cytotoxicity, hemostasis, and the ease of manipulation during surgical procedures . CollageSeveral investigations have been performed in a rat calvarial defect model to evaluate the effects of various materials on new bone formation. This model is established by forming a bone defect with the diameter of 8 mm in the center of the parietal bone of a rat calvarium using a trephine bur. This defect is regarded as a critical-sized defect, as it does not spontaneously heal during the lifetime of the animal . This moIt is essential for collagen membranes utilized in GBR to maintain physical integrity long enough to allow bone regeneration. However, there are few available membranes that can completely satisfy all requirements. Therefore, the purpose of the present study was to evaluate bone regeneration efficacy in a critical-sized rat calvarial defect model, using DHT cross-linked collagen membrane with or without BG material.The post-operative soft tissue healing was uneventful, with no complications  or other inflammatory reactions observed in any of the rats. At 2 and 8 weeks post-surgery, surgical sites showed the evidence of incision and sutures. After harvesting of the surgical sites, the bone defect areas were clearly surrounded by periosteum and dura mater under visual inspection.The results of micro-CT measurements are summarized in p = 0.031; DHT + BG, p = 0.002). While that of the DHT + BG group was significantly greater than those of the DHT and BG groups . Both BG and DHT + BG groups showed significantly higher NBV , BVF , and BMD . Moreover, the NBV and BVF values were significantly higher in the DHT + BG group than in the BG group .At 2 weeks after surgery, there was no statistically significant difference between the control and DHT groups in terms of total augmented volume (TV), new bone volume (NBV), bone volume fraction (BVF), and bone mineral density (BMD). In contrast to this, TV was significantly increased in both groups receiving BG when compared with that in the control group . Additionally, the TV value in the DHT + BG group was also significantly higher than those in the DHT and BG groups . Similar to the results obtained at 2 weeks, both BG and DHT + BG groups showed significantly higher NBV values , BVF , and BMD  values. However, no statistically significant difference was observed in NBV, BVF, and BMD values between the BG and DHT + BG groups.At 8 weeks after surgery, all values were shown to have increased, and no statistically significant difference was observed between the control group and DHT group in the four variables. In contrast, the BG and DHT + BG groups showed significantly higher TV values when compared with that in the control group .Comparisons within the same experimental groups showed only statistically significant differences in NBV and BVF values in the BG and DHT + BG groups with time  and DHT group . However, a significant difference was obtained between two BG groups (p = 0.009). At 2 weeks of healing, no significant differences in TA and NB were observed between the control and DHT groups. In contrast, these values were significantly higher in the BG and DHT + BG groups when compared with those determined in the control group  and NB (p = 0.001 and p < 0.001) values in the BG and DHT + BG groups, respectively, were significantly higher when compared with those determined in the control group. The TA values in two BG groups were shown to be significantly different (p = 0.009). The NB values were significantly higher in the BG (both p < 0.001) and DHT + BG (p = 0.001 and 0.003) groups, when compared with those in the control and DHT groups, respectively. TA values were significantly higher in both BG and DHT + BG groups compared with that of determined in the DHT group .At 8 weeks after surgery, no statistically significant differences were observed in TA and NB values between the control and DHT groups. However, the TA . The amount of graft materials, including the bone material and membrane, decreased in all three experimental groups between 2 and 8 weeks after surgery.In the BG and DHT + BG groups, significant differences in the NB values were detected between 2-week and 8-week healing periods (Proliferating cells were highlighted using the proliferating cell nuclear antigen (PCNA) antibody by immunohistochemistry both in the control and the experimental group. We found that the control group mainly showed negative patterns for PCNA expression a,b. By cBiologically, an optimal barrier membrane must induce hemostasis, be integrated by the host tissues, maintain chemotaxis for periodontal ligament fibroblasts, and possess low cytotoxicity. Moreover, the membrane should be able to exclude unwanted cells to protect the wound area and prevent infection ,4,11. ThGood biocompatibility and tissue integration are crucial elements for the reduction of the inflammatory response and membrane exposure . These pVarious graft materials have been developed and used to obtain favorable outcomes in periodontal and implant surgeries, including autogenous bones, allografts, xenografts, and bone substitutes . Freeze-The aim of this study was to evaluate new bone formation using DHT cross-linked collagen membrane with or without bone materials. Some studies reported unfavorable mechanical strength and inadequate barrier function as the major disadvantages of the use of collagen membrane ,29. ApprPrevious studies reported that a cross-linked collagen membrane promotes new bone formation in bone defects. Park et al.  histologAccording to the results of micro-CT analysis, TV values in the regions of interest increased in a time-dependent manner in all groups. Significant differences in the NBV, BVF, and BMD values, which are the key indices of bone regeneration, were observed between the BG and DHT + BG groups and the control group. Additionally, significant differences were observed between the BG and DHT + BG groups. Based on these results, DHT cross-linked collagen membrane represents a safe biomaterial and has a positive effect on new bone formation, showing good potential to serve as a barrier membrane in the GBR procedure. Given the short evaluation period and the limitations of this study, further investigations are necessary to confirm the effectiveness of the DHT membrane for the GBR in clinical practice.The DHT collagen membrane was obtained from the native porcine dermis with additional physical cross-linking, and was mainly composed of type I collagen. The experimental membrane was processed by mechanical cleaning, chemical treatment, lyophilization, and compression. This was followed by the cross-linking using DHT in a vacuum at 110 °C and sterilization. Various DHT membrane properties were investigated, including morphology, enzyme resistance, and mechanical properties. The surface of the DHT membrane had macro-micro interconnective porous structure to permit cellular invasion.FDBA was used as the graft material in this study. The allograft , a particle-type graft material, was composed of a cortical and cancellous powder with a weight ratio of 80/20. The sizes of the particle and pore were 0.2–1.0 mm and 250 µm, respectively.A total of 40 albino rats of the Wistar strain  weighing between 350 g and 370 g were used in the present study. All rats were housed individually in standard cages under specific pathogen-free conditions, and fed a standard laboratory diet and water. The animals were allowed to acclimate to the new environment for 7 days before performing the surgery. The animal selection, management, preparation, and surgical protocol were evaluated and approved by the institutional Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea .A critical-sized defect of 8 mm in diameter was created in the parietal bone of each rat calvarium. A total of 40 defects were formed. Ten rats were allocated to each of the following groups, which were split into two subgroups for the examination at 2 and 8 weeks after surgery: (1) sham surgery control group, in which the bone discs were removed and the surgical defects were not filled with any membrane or material; (2) DHT membrane group, in which the surgical defects were covered by DHT collagen membranes; (3) BG group, in which the surgical defects were filled with FDBA; and, (4) DHT + BG group, in which the surgical defects were filled with FDBA and covered with DHT collagen membranes.2 and then anesthetized by an intraperitoneal injection of 15 mg/kg zoletile  and 10 mg/kg rompun . Under local anesthesia with 2% lidocaine hydrochloride containing 1:100,000 epinephrine, after disinfection with povidone iodine , a middle skin incision was made on the skull, and a full-thickness flap was reflected. Under copious saline irrigation, a standardized, round transosseous defect of 8 mm in diameter was created in the center of the calvaria with a trephine bur. After removal of the trephined calvarial disc, four groups of ten animals each received the DHT collagen membrane, BG, BG with collagen membrane, or sham-surgery control procedure. For the groups with the collagen membrane, it was cut to a size of 10 × 10 mm2 squares to cover the outer surface of the bony defect, and then placed over the defect. For the groups involving BG, a sufficient amount of graft material was applied to completely fill the defect by applying a gentle pressure using a surgical instrument. After obtaining adequate hemostasis, the periosteum and skin were repositioned and sutured. Post-operatively, to minimize post-operative pain and prevent infection, all rats were injected subcutaneously with antibiotics  and analgesics .Initially, the animals were sedated in a chamber with 4% isoflurane  in 100% O2 chamber. Subsequently, block sections of the rat calvaria were harvested and fixed in a 10% neutral buffered formalin solution.After healing periods of 2 and 8 weeks, five rats in each group were euthanized in a COAll samples were scanned using high-resolution micro-CT system  at a pixel size of 14.91 µm. Prior to scanning the samples, calibration was performed using water, air, and synthetic bone samples. The digital images were obtained under a source voltage of 130 kV and a current of 60 µA. The scanned images were then reconstructed using CT-analyzer software . The regions of interest of each sample were determined in 3D images for the analysis of TV, NBV, BVF , and BMD.After obtaining micro-CT scans, block sections of the experimental sites were fixed in a 10% neutral-buffered formalin solution for 10 days. The fixed specimens were decalcified in 5% formic acid for 14 days and embedded in paraffin. Serial sections of 5 µm were cut through the central portion of each experimental site. Only the central sections were chosen and stained with hematoxylin-eosin for histological and histomorphometric analysis.An experienced researcher, blinded to the specific experimental conditions, performed the microscopic examination and histomorphometric analysis. Digital images of histologic slides were obtained using a binocular microscope , coupled with a color camera , and saved as digital files. Histometric measurements in the defects were made using automated image analysis software  at 12.5× magnification. The following parameters were measured: (1) TA, including newly formed bone, connective tissue, remaining membrane, and grafted materials within the defect; (2) NB, representing the area of newly formed bone within the defect; (3) remaining membrane area (RMA), representing the area of residual membrane within the defect; and (4) residual materials (RM), grafted bone materials within the defect.Activity of cell proliferation in new bone area was identified using immunohistochemical analysis with an anti-PCNA monoclonal antibody. Endogenous peroxidase activity was inactivated with 1% hydrogen peroxide solution for 30 min. The sections were blocked with PBS containing 5% bovine serum albumin at room temperature for 10 min and reacted with the primary antibody  at room temperature for 1 h. After washing in tris-buffered saline, the sections were reacted with secondary antibody  at room temperature for 30 min. Then, the bound antibodies were visualized with avidin-biotin DAB system , and counter-stained with hematoxylin. The PCNA-positive cells were observed under optical microscopy .p-value less than 0.05 was considered statistically significant.The statistical analysis was performed using a commercially available software program . The bone defect of each rat was regarded as a statistical unit. Data obtained in each group are expressed as mean values and standard deviations. Kruskal–Wallis one-way analysis of variance based on ranks and the post-hoc Mann–Whitney U test were used to assess the differences among groups at each time point. The Wilcoxon signed rank test was used to compare the data obtained in the same group between two healing periods. A


Globally, road traffic injury (RTI) causes 1.3 million deaths annually. Almost 90% of all RTI deaths occur in low- and middle-income countries. RTI is one of the leading causes of death in Bangladesh; the World Health Organization estimated that it kills over 21,000 people in the country annually. This study describes the current magnitude and risk factors of RTI for different age groups in rural Bangladesh. A household census was carried out in 51 unions of seven sub-districts situated in the north and central part of Bangladesh between June and November 2013, covering 1.2 million individuals. Trained data collectors collected information on fatal and nonfatal RTI events through face-to-face interviews using a set of structured pre-tested questionnaires. The recall periods for fatal and non-fatal RTI were one year and six months, respectively. The mortality and morbidity rates due to RTI were 6.8/100,000 population/year and 889/100,000 populations/six months, respectively. RTI mortality and morbidity rates were significantly higher among males compared to females. Deaths and morbidities due to RTI were highest among those in the 25–64 years age group. A higher proportion of morbidity occurred among vehicle passengers (34%) and pedestrians (18%), and more than one-third of the RTI mortality occurred among pedestrians. Twenty percent of all nonfatal RTIs were classified as severe injuries. RTI is a major public health issue in rural Bangladesh. Immediate attention is needed to reduce preventable deaths and morbidities in rural Bangladesh. There is a growing consensus in the international health community that road traffic injury (RTI) is a leading cause of death, illness, and disability throughout the world ,2,3. AccDue to rapid motorization and urbanization in Bangladesh, RTIs are on the rise as in other LMICs, and RTI also represents a leading cause of injury deaths ,11,12,13Bangladesh has had a gradual shift from infectious disease to non-communicable disease and injuries in the past couple of years ,18,19,20“Saving of Lives from Drowning (SoLiD)”, an implementation research study, was conducted between 2013 and 2015 in Bangladesh. In this program, a baseline census was conducted between June–November 2013 in 51 unions of seven sub-districts . The sevTrained data collectors gathered required information by face-to-face interviews with the heads of households or any household member over 18 years of age who were the most knowledgeable about the household. A repeat visit was made to those households if there was no adult member present or if the respondent was physically or mentally unable to participate in the interview. If an additional second or third visit was unsuccessful, the household was excluded from the census. A set of seven pre-tested structured questionnaires was used to collect relevant information. Data was collected in two stages; the first round collected information on socioeconomic and demographic factors , household environment, child and birth history, and health-seeking behavior to understand the household and family status. The survey also collected information on fatal and non-fatal injuries. If a specific injury mortality or morbidity was identified in first round, detailed information was collected regarding the underlying injury mechanisms in the second round. In this study, injury was defined as any external harm resulting from an assault, burn, fall, animal bite, transportation of goods and persons, cuts, poisoning, blunt objects, operating machinery, suffocation, or (near) drowning resulting in the loss of one or more days of normal daily activities, work, or school, and its methodology is described elsewhere . This paTo ensure the data quality, trained supervisors observed 10% of the conducted interviews and checked 10% of the collected data. They also re-interviewed 2% of the visited households. Field-level research officers also re-checked all data for inconsistencies. If any discrepancy was found, the household was revisited to collect correct information.A data entry program using SQL Server 2008 was developed and collected data were entered, and then transferred to SPSS version 21 for analysis. All related information of fatal and non-fatal RTIs were retrieved from the primary database for analysis and were de-identified. To analyze the characteristics of RTIs, standard descriptive statistics were used. Description of population by fatal and non-fatal RTIs, sex, age, level of education, socioeconomic status (SES), and sub-district, as well as the place, time, and prior activities of fatal and non-fatal RTIs was provided with proportion. Nonfatal injury events were classified into low, medium, and highly severe injuries based on an index that summarizes indicators such as the number of days an individual required assistance, the number of days lost at work or school, post injury immobility, anatomic and physiologic profile of an injury, post injury hospitalization, surgical treatment, and post injury disability for all events . Fatal ROdds of fatal and nonfatal RTI outcomes given independent variables such as age, sex, SES, education, and occupation were assessed using logistic regressions. Results from both bivariate and multivariate logistic regressions are presented. Age was treated as a categorical variable (comprising eight groups: <5 (reference group), 5–9, 10–14, 15–17, 18–24, 25–64, 65+ years). Sex was considered as a binary predictor . Educational (A level and above as a reference group), occupation (agriculture as a reference group), and SES (from lowest to highest) were categorical variables.Ethical clearance was obtained from the Institutional Review Boards of the Johns Hopkins Bloomberg School of Public Health , International Centre for Diarrheal Disease Research, Bangladesh and the Center for Injury Prevention Research, Bangladesh. For inclusion in the study, informed consent was given by all respondent before they participated.Around 1.2 million people from seven selected sub-districts were covered in the census, and the proportion of the population in each sub-district varied depending upon the number of unions covered for the census. In the census, the proportion of males (48.5%) and females (51.5%) were almost equal. Among the total sample, 39.1% were children (<18 years). A total of 6303 deaths (preceding year) and 119,669 non-fatal injury events (preceding six months) were identified during the census . A totalRTI deaths comprised about 1.3% of the deaths that occurred due to any cause in the surveyed population over a recall period of one year. The mortality rate due to RTI was 6.8 (95% CI 55–85) per 100,000 populations per year. The mortality rate was the highest in Raiganj  followed closely by Manohardi .Across gender profiles, RTI deaths were significantly more in males than females, the mortality rates being 9.2 deaths (95% CI 6.9–12.01) per 100,000 in males compared to 4.7 deaths (95% CI 3.2–6.7) per 100,000 in females. The count of deaths due to RTI was highest in the 25–64 years age group; the unadjusted mortality rate was, however, highest among the elderly age group  . IndividThe morbidity rate due to RTI was 889 injuries (95% CI 866–900) per 100,000 population per six months. The RTI morbidity rate was highest in Raiganj , followed by Manohardi . However, rates of non–fatal RTIs where highest  in the highest SES quintile .Transport workers had the highest rates for both RTI mortality  and morbidity  among all occupations . When coAround 40.0% of road traffic injuries occurred while an individual was on his way to work, and 21.5% occurred among individuals who were wandering on the streets. One-fifth of the victims were engaged in driving .The RTI injury severity index showed that 50% of RTI cases had low severity. Almost 20% of cases had been severely injured in a road traffic crash. The highest proportion of high injury severity was found among passengers (37.7%), followed by pedestrians (22.4%) .The survey findings revealed that 81.4% of motorcyclist RTI victims did not use safety devices .Most of RTIs happened earlier in the day, between 9:00 a.m. and 12 noon . Most ofp = 0.000) when compared with females. The risk of a non-fatal road traffic crash increased significantly with increasing age. Individuals aged 15 to 24 years were at the highest risk. Transport workers, such as those driving rickshaws and buses, were 6 times likelier to be in a non-fatal RTI  than agricultural workers. Education level was not seen to be associated with non-fatal RTI risk. Increasing socioeconomic status was significantly associated with increasing risk of non-fatal RTI  at risk than agricultural workers to die in a road traffic accident .The incidence of RTI fatalities was found to be 6.8 deaths (95% CI 55–85) per 100,000 population and non-fatal RTIs were calculated as 889.0 injuries (95% CI 866–900) per 100,000 population. Although RTIs occur in all age groups, the highest rate of fatality  was observed among the older age group (65+ years) followed by 15–17 years , and the highest rate of morbidity was found in the group aged in 24–64 years . These findings were consistent with other studies from the developing world such as India, Pakistan, Nepal, Vietnam, and Ghana, where RTI was found to be the leading killer and the productive age group was found to be the most likely victim ,30,31,32Male preponderance in fatal and non-fatal RTIs is concurrent with other studies from Bangladesh and the surrounding countries ,37,38,39The study findings noticed that no education and lower socioeconomic conditions put individuals at higher risk for road traffic injury deaths. Other studies conducted in LMICs and HICs found similar patterns ,41,42,43In this study, mortality and morbidity were highest among transport workers, such as rickshaw pullers. It has been reported in different studies that more RTIs were seen among students and laborers ,44,45,46In the present study, the highest number of crashes happened between morning and noon (9 a.m. to 12 p.m.). This pattern is similar to other studies from Bangladesh and the surrounding regions, such as India, where the highest incidence of RTIs occurred in the same time frame ,16. ThesThe strength of this study is that data was collected from a large sample size covering all individuals in the survey area. The information was collected through two-stage verbal interviews by trained data collectors and all data was cross-checked. However, data was collected mostly from rural areas of Bangladesh; therefore, the findings may not be generalizable to urban areas of Bangladesh and these sample households were not nationally representative . HoweverAdditionally, the study did not collect data on other risk factors such as exposure time, kilometers driven, and knowledge of safety practices. Furthermore, not all respondents were victims or eye witnesses, which gives some limitation to data accuracy.Other established common risk factors such as lack of awareness, lack of engineering modification, and travelling on overcrowded or poorly maintained vehicles could not be captured in the context of this study.The magnitude of fatal and non-fatal RTIs is remarkably high in rural communities of Bangladesh, and the working age group and male population are more at risk. Being a pedestrian or a student were also identified as risk factors for both fatal and non-fatal RTIs. Lower socioeconomic condition and no education were the important risk factors for fatal and non-fatal RTIs.There is obviously a need for targeted and directed intervention approaches to reduce road traffic injuries. Some examples of interventions include road safety education programs for road users, safe child pedestrian programs through school education, community awareness programs, first responder training at the community level, the implementation of safety measures for non-motorized vehicles, and engineering modifications for speed calming. Such approaches should be directed towards vulnerable road users ,19,46,51


Transcatheter arterial chemoembolization (TACE) is the mainstay of treatment for unresectable hepatocellular carcinoma (HCC). The efficacy of conventional TACE (cTACE) in liver metastases is not satisfactory, which might be due to the fact of embolic material used. Recently, as a new type of drug-loading microsphere, HepaSphere has been introduced in China. In this study, there were total 30 patients  with liver cancer underwent embolization with Hepasphere microsphere. And a total of 44 TACE procedures were performed using 50-100μm HepaSphere. There were 16 patients diagnosed as HCC and 14 patients as liver metastases. The follow up period ranged from 3 to 15 months (median 10 months). Response rates were calculated on intention-to-treat basis. One month after TACE, total objective response was 63.3% and disease control rate was 86.7%. No severe complication  was observed. In conclusion, chemoembolization with Hepasphere microsphere may be a safe and possibly effective palliative treatment for patients with liver cancer. Transarterial chemoembolization (TACE) is a well-recommended treatment for unresectable intermediate stage hepatocellular carcinoma (HCC) patients –3. UnforHepaSphere microspheres  are soft, with a high conformability, and can be loaded with doxorubicin. They have been used for number of years, but have just recently been introduced in China. These small, nonabsorbable, doxorubicin-loaded microspheres can be more distally embolized, releasing the chemotherapeutic agent in a controlled and prolonged manner into the tumor, with lower systemic toxicity. Initial clinical results have been published, and show a good tumor response and safety profile –6. Thus The technical success was 100%, with a total of 44 HepaSphere procedures being performed in the 30 patients. There were 16 patients with HCCs and 14 patients with liver metastases, with the following primary tumor sites: colorectal, bile duct, neuroendocrine, gallbladder, pancreatic, lung, and gastric. The overall median follow-up period was six months, ranging from three to 15 months.The tumor response was evaluated using CT/MRI according to the mRECIST criteria . FollowiGrade 2 toxicities (NCI-CTC) occurred in 43.3% (n=13), and grade 3 toxicities occurred in 26.7% (n=8) of all of the courses in terms of the liver function post-TACE  incomplete lipiodol deposition in the tumor, leading to a poorer tumor response , and (2)When compared with lipiodol, which is commonly used in cTACE, HepaSphere microspheres have a superior safety profile and could carry a larger amount of the chemotherapeutic drug that can be released in a slow and controlled manner. The doxorubicin is released by the microspheres for a period of one month after the embolization . This coSince the drug-eluting TACE procedure does not require the use of lipiodol, the evaluation of the tumor is not hindered by lipiodol retention, and it can be performed properly using Modified Response Evaluation Criteria In Solid Tumors (mRECIST) criteria . With reSeveral previous studies have shown good tumor responses in HepaSphere HCC treatment. In one study conducted by Malagari et al. in 2014 , the objSorafenib is recommended for advanced stage HCC treatment. In a Phase II trial of sorafenib conducted by Pawlik, combined with concurrent transarterial chemoembolization and drug-eluting beads for HCC treatment, the results showed a good disease control rate of 95%. This suggests that combining cTACE and sorafenib could lead to better disease control with an acceptable complication rate . Most liOverall, drug-eluting TACE using HepaSphere microspheres is safe and well tolerated. The liver enzymes were slightly increased after embolization in all of the patients, but returned to normal 3-4 days later, a pattern that was also observed in other drug-eluting embolic studies , 24–26. The limitations of our study were that it was a single center study with a small patient number and a short follow-up period, with diverse patient characteristics and tumor backgrounds. Further investigations are required to evaluate the long term efficacy in a larger cohort or a randomized control trial. Despite these limitations, the use of HepaSphere microspheres in liver cancer management may be a safe, well-tolerated, and effective treatment modality, which has been shown to have a good objective response and disease control rate in both hypervascular and hypovascular tumors.Thirty patients from the First Affiliated Hospital at the Zhejiang University School of Medicine in Zhejiang, China, from February 2014 to September 2015, were included in our study. There were 18 males, 12 females, and the median age was 53.5 years old. All of the patients were treated with doxorubicin-loaded HepaSphere microspheres. Each of the HCC patients was diagnosed clinically and pathologically , and allLetters of consent were obtained from all of the patients, and the experimental protocols were approved by the local ethics committee . The procedure was carried out in accordance with the approved guidelines, and informed consent was obtained from all of the subjects. All of the data was anonymized and de-identified prior to the analysis.A patient was included in this study if they: (1) had an unresectable HCC or liver metastasis, were not a candidate for locoregional tumor ablation, were refractory or intolerant of systemic chemotherapy, or had recurrent HCC; (2) were Child-Pugh status A or B, or Eastern Cooperative Oncology Group (ECOG) 0 to 2; and (3) did not have a tumor thrombosis in the main portal vein.A patient was excluded if they: (1) were Child-Pugh status C, (2) had uncorrectable impaired clotting, (3) had a tumor thrombosis in the main portal vein or portal hypertension, (4) had an infectious disease (e.g. liver abscess), (5) had a life expectancy of less than 3 months, or (6) had an aneurysm in the hepatic artery or portal vein.HepaSphere microspheres are loadable microspheres with a dry caliber of 50-100 μm, which expands to 200-400 μm after doxorubicin loading. The drug loading method was conducted as suggested by the manufacturer. The doxorubicin solution was prepared by adding 4 ml of normal saline into each 10 mg vial of doxorubicin powder, creating a concentration of 2.5 mg/ml. Five 20 ml vials of doxorubicin were prepared for each patient. Next, 10 ml of the doxorubicin solution was added into a 50-100 μm HepaSphere vial, mixed gently 5-10 times, and allowed to sit for 10 minutes. After 10 minutes, the remaining doxorubicin solution was loaded into the HepaSphere vial and allowed to stand for 60 minutes. An hour later, all of the supernatant was extracted, and 20 ml of nonionic contrast medium was added into a syringe containing the doxorubicin-loaded HepaSphere microspheres, and mixed gently until homogeneity was reached.The interventional procedure was done by the experienced interventional radiologists at our hospital. Hepatic angiography was performed to identify the feeding artery and collaterals. In the case of a hypovascular tumor, angiography at the superior mesenteric artery, inferior mesenteric artery, and left gastric artery had to be conducted.The TACE procedures followed the classical steps, and were performed superselectively in the tumor-feeding arteries. After the identification of the feeding arteries, a microcatheter was advanced as distally as possible. Then, the HepaSphere microspheres were injected slowly, at a rate of 1 ml/min, until stasis was observed. The angiography was repeated to ensure the devascularization of the feeding arteries had occurred. In those cases in which a tumor stain was observed, the HepaSphere microspheres were injected until the tumor stain disappeared. Normally, one vial of HepaSphere was required, with a maximum dose of two vials. Embosphere microspheres  were injected into the feeding arteries when the tumor stain was still observed after two HepaSphere vials were used.The routine postembolization medication included the following: 100 mg of tramadol was only used in case of pain, 40 mg of esomeprazole was given intravenously every 12 h, 5 mg of ondansetron was given intravenously every 12 h, and 1500 mg cefuroxime sodium was given intravenously every 12 h for 3 days after embolization. Intravenous drops of 80 mg of a glycyrrhizin compound were given daily, with 1000 mg of ademetionine to protect the liver function. Finally, 200 mg of celecoxib was used if the temperature was higher than 38.5°C.CT/MRI images were assessed one month after the procedure using the modified Response Evaluation Criteria in Solid Tumors (mRECIST) . LaboratP value <0.05.The data processing and analyses were performed using SPSS 19.0 . Chi-squared and t-tests were used when appropriate, and statistical significance was defined as a


To assess the prevalence of child underweight, overweight, and obesity in a Malaysian population according to 3 international references because classification of anthropometric status may differ according to the reference used to express body mass index (BMI).We assessed data from 6414 children aged 6-18 years, collected by the South East Asia Community Observatory. Child underweight, overweight, and obesity were expressed according to 3 internationally used BMI references: World Health Organization 2007, International Obesity Task Force 2012, and Centers for Disease Control and Prevention 2000. We assessed agreement in classification of anthropometric status among the references using Cohen's kappa statistic and estimated underweight, overweight, and obesity prevalence according to each reference using mixed effects Poisson regression.There was poor to moderate agreement between references when classifying underweight, but generally good agreement when classifying overweight and obesity. Underweight, overweight, and obesity prevalence estimates generated using the 3 references were notably inconsistent. Overweight and obesity prevalence estimates were higher using the World Health Organization reference vs the other 2, and underweight prevalence was up to 8.5% higher and obesity prevalence was about 4% lower when using the International Obesity Task Force reference.The choice of reference to express BMI may influence conclusions about child anthropometric status and malnutrition prevalence. This has implications regarding strategies for clinical management and public health interventions. In many middle income populations, including those in Asia, the prevalence of child overweight and obesity is increasing even though the burden of child underweight remains high.Although the World Health Organization (WHO) 2006 standards are widely accepted as ideal for assessing anthropometric status among children aged 0-5 years,We used data from a population-wide health survey conducted from 2013 to 2014 by the South East Asia Community Observatory (SEACO), a health and demographic surveillance system in southern peninsular Malaysia.All data for the SEACO health survey were collected on encrypted tablets. For this study, we used information collected from children aged 6-18 years on height and weight, sex, age, and ethnicity. Height and weight measurements were taken by trained staff following standardized procedures in accordance with the WHO STEPwise approach to Surveillance manual,Children's anthropometric status  was expressed using the Zanthro package in Stata  according to 3 BMI references, described below.The WHO 2007 reference, for children aged 5-19 years, is based upon information on 22 917 children from 3 national surveys conducted in the US from 1963 to 1974, with additional data from the WHO Multicenter Growth Reference Study (1997-2003). Underweight, overweight, and obese are defined as BMI-for-age less than 2 SDs below the mean, greater than 1 SD above the mean, and greater than 2 SDs above the mean respectively.27The CDC 2000 reference, for children aged 2-20 years, is based on the US surveys described above, with additional data from further national surveys conducted up until 1994 . Underweight, overweight, and obesity are defined as BMI-for-age less than the fifth percentile, greater than or equal to the 85th percentile and greater than or equal to the 95th percentile, respectively. To note, the CDC terms the latter 2 cut-offs as “at risk of overweight” and “overweight” respectively.282 at 18 years of age for underweight, overweight, and obesity, respectively. Thus, underweight is defined as BMI-for-age less than the 15.5th percentile in boys and the 16.5th percentile in girls, overweight as BMI-for-age greater than or equal to the 90.5th percentile in boys and the 89.3rd percentile in girls, and obesity as BMI-for-age greater than or equal to the 98.9th percentile in boys and the 98.6th percentile in girls.The IOTF 2012 reference was constructed from national surveys undertaken from 1963 to 1993, in 192 727 children aged 2-18 years from 6 countries . The reference provides percentile cut-offs corresponding to a BMI of 18.5, 25, and 30 kg/mTo allow for comparability across references, we used data on children aged 6-18 years. We initially used the Cohen kappa statistic to assess the overall agreement between the 3 references when classifying underweight, overweight, or obesity. Kappa <0.6 was defined as poor agreement, 0.6 to <0.8 as moderate agreement, 0.8 to <0.9 as good agreement and ≥0.9 as excellent agreement.Analyses were conducted using Stata 13 and 14 .The analyses included 6414 children aged 6-18 years, approximately one-half of whom were boys. The majority of children were of Malay ethnicity, followed by Chinese and Indian ethnicity. There were no differences by sex in the distribution of children across categories of ethnicity and age .Table IDwww.jpeds.com). There was poor to moderate agreement between the 3 references when classifying child underweight, with poorest agreement between the WHO and IOTF references. Between the WHO and IOTF references, agreement was particularly poor among girls, children aged 15-18 years, and those of other ethnicity. When classifying child overweight, there was good to excellent agreement between the references, especially between the IOTF and CDC references. Agreement was similar when examining child obesity, particularly between the WHO and CDC references, overall and across population subgroups. Agreement between the other references was more variable across subgroups . Furthermore, the WHO and CDC references are based exclusively on data from children in the US, with no other populations represented.50In light of such limitations, multiple studies, including 1 from Malaysia, have reported the construction of anthropometric references based upon data collected from local populations.53Regardless, consensus on a single international reference is required to allow for accurate comparison of child malnutrition burden across studies worldwide.Importantly, the clinical diagnosis of malnutrition in a child should take into account other relevant factors additional to anthropometry, such as body composition, other signs of clinical undernutrition, or potential genetic syndromes.Despite standardization and training of staff in this study, the potential for measurement error remains because instruments were not calibrated during the survey. This may have some implications in terms of misclassification of anthropometry but is not expected to affect the relative differences and measures of agreement between references. Moreover, although patterns observed here were similar to previous evidence,To conclude, we observed notable differences between 3 international BMI references in the classification of child underweight, overweight, and obesity, and subsequent estimates of prevalence. Our results indicate the need for the universal use of at least 1 reference to ensure comparability across populations, and for further longitudinal studies to assess the comparative ability of both international and local references to predict the future risk of cardiometabolic and other outcomes. Clearer consensus on the use of anthropometric references to measure malnutrition will be important to inform and guide global initiatives which drive national policy, particularly in low- and middle-income countries.