Ribosomes are essential components of the protein translation machinery and are composed of more than 80 unique large and small ribosomal proteins. Recent studies show that in addition to their roles in protein translation, ribosomal proteins are also involved in extra-ribosomal functions of DNA repair, apoptosis and cellular homeostasis. Consequently, alterations in the synthesis or functioning of ribosomal proteins can lead to various hematologic disorders. These include congenital anemias such as Diamond Blackfan anemia and Shwachman Diamond syndrome; both of which are associated with mutations in various ribosomal genes. Acquired uniallelic deletion of RPS14 gene has also been shown to lead to the 5q syndrome, a distinct subset of MDS associated with macrocytic anemia. Recent evidence shows that specific ribosomal proteins are overexpressed in liver, colon, prostate and other tumors. Ribosomal protein overexpression can promote tumorigenesis by interactions with the p53 tumor suppressor pathway and also by direct effects on various oncogenes. These data point to a broad role of ribosome protein alterations in hematologic and oncologic diseases. Eukaryotes have 80 S ribosomes, each consisting of a small (40 S) and large (60 S) subunit. The large subunit is composed of a 5 S RNA (120 nucleotides), a 28 S RNA (4700 nucleotides), a 5.8 S subunit (160 nucleotides) and approximately 49 proteins. The 40 S subunit has a 18 S RNA (1900 nucleotide) and approximately 33 proteins.The biogenesis of the ribosome machinery is a highly coordinated process. It is composed of the synthesis and processing of ribosomal RNA , synthesis of ribosomal proteins in the cytoplasm and their import into the nucleus, assembly of rRNA and ribosomal proteins in the nucleoplasm and finally transport of the mature subunits (40 S and 60 S) into the cytoplasm.The basic translation machinery is composed of ribosomal subunits, mRNAs, transfer RNAs (tRNAs), and translational initiation and elongation factors. First, the initiation factors eIF2, eIF3, tRNA and GTP are incorporated into a 40 S ribosomal subunit to form a 43 S complex. Second, eIF4E is recruited into the 43 S complex to form a 48 S complex with mRNA. Finally, a 60 S ribosomal subunit and the 48 S subunit form the final 80 S complex .The relative functional significance of the rRNAs and the ribosomal proteins has been a matter of interest for many years. In the 1970s, it was generally accepted that ribosomal proteins constituted the functionally active part of the ribosome, whereas rRNA was essentially a scaffold that kept the proteins in position for optimal functioning. By the 1980s, the pendulum had swung, with ribosomal proteins thought to be the scaffolding for the rRNA. The reason for this change was the discovery of catalytic RNA and data showing the direct involvement of rRNAs in distinct ribosomal functions, for example, the Shine-Dalgarno interactions between mRNA and rRNA during initiation of bacterial protein synthesis, as well as the predominance of rRNA at the decoding and peptidyl-transferase centre (PTC) of the ribosome. The crystal structures of the ribosome confirmed this predominance of rRNA at these active sites, but also revealed that a number of ribosomal proteins were located in positions of functional importance, for example, RPS12 at the decoding center, RPL11 and RPL10 as components involved in translation factor binding. Over the years, specific functions of many individual ribosomal proteins were identified and currently it is accepted that both proteins and RNA are essential for the optimal functioning of the ribosome..Futhermore, several ribosomal proteins have been found to have extraribosomal functions including DNA repair; autoregulation of ribosomal protein synthesis and translation and regulation of development of malignant transformation mainly through interaction with p53. In E.coli, in addition to these functions, ribosomal proteins have been found to play a role in DNA replication, transcription and RNA processing ,3. The iRibosomopathies are disorders resulting from impaired ribosome biogenesis and function. Diamond-Blackfan anemia and 5q- syndrome are the two clinical syndromes for which there is abundant genetic and experimental evidence that the impairment in erythropoiesis is due to mutations in ribosomal genes. However, there are a few other clinical syndromes in which ribosome dysfunction is thought to play a role and recently, ribosomal dysfunctions have been identified in some malignancies as well.1. Diamond-blackfan anemia (DBA):DBA is a congenital aregenerative anemia accompanied by erythroblastopenia (less than 5% of nucleated cells in the bone marrow are erythroblasts). It is often discovered before the age of two years. There are associated developmental malformations in 40% of the cases. The malformations most often involve the head and neck , limbs , urogenital tract, heart and/or the spinal cord.In 1999, Draptchinskaia reported mutations in the gene encoding ribosomal protein S19 in patients with Diamond-Blackfan anemia . The oth2. 5q- syndrome:5q- syndrome is a disorder resulting from deletion of the long arm of chromosome 5 and is characterized by macrocytic anemia with normal/elevated platelets with hypolobulated micromegakaryocytes. It is found predominantly in females of advanced age. It is now classified as an independent subtype of MDS, with better prognosis and a relatively low rate (10%) of progression to acute myeloid leukemia compared with other subtypes of MDS . In pati3. Treacher collins syndrome (TCS):Also called mandibulofacial dysostosis, TCS is a rare autosomal dominant congenital disorder characterized by craniofacial abnormalities such as micrognathia, malformed or absent external ears, underdeveloped zygoma, downward slanting eyes, conductive hearing loss, colobomata of the lower eye lids, cleft palate, brachycephaly, variable degree of facial nerve involvement . Intelli4. Cartilage hair hypoplasia:CHH is an autosomal recessive disorder that results in short limb dwarfism . It is a5. Shwachman diamond syndrome (SDS):SDS is a rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, skeletal abnormalities and a predisposition to leukemia . It is t6. Dyskeratosis congenita (DKC):DKC is a rare, progressive bone marrow failure syndrome characterized by the triad of reticulated skin hyperpigmentation, nail dystrophy and oral leukoplakia. Early mortality is often associated with bone marrow failure, infections, fatal pulmonary complications or malignancy . In all 7. Common variable immunodeficiency (CVID):CVID is a disorder that involves low levels of most or all of the immunoglobulin classes, a lack of B lymphocytes or plasma cells that are capable of producing antibodies and frequent bacterial infections. A diagnosis of CVID is reserved for those with an undefined B cell dysfunction. About 50% of the patients also have T lymphocyte dysfunction. The recognition of a patient with DBA who subsequently developed CVID and the finding of a mutation in the SBDS gene in another patient with CVID has led to the hypothesis that ribosome biogenesis defects are also responsible for a subset of CVID. A review of the literature shows that patients with ribosomal defects may share abnormalities of T and B cell development with many features of CVID, and which may not be recognized as such by non-immunologists .8. Turner syndrome:Turner syndrome results from a chromosomal abnormality in which all or part of one of the female sex chromosomes is absent. Monosomy X in which an entire X chromosome is missing, is most common. Characteristic physical abnormalities include short stature, webbed neck, broad chest, low hairline, low-set ears and edema. Girls with Turner Syndrome typically experience gonadal dysfunction resulting in amenorrhea and sterility. Other health problems frequently present include congenital heart disease, hypothyroidism, diabetes, vision and hearing problems, autoimmune diseases and developmental delays.Human sex linked genes RPS4X and RPS4Y encode 2 isoforms of ribosomal protein S4. RPS4 is a component of 40 S subunit and haploinsufficiency of RPS4 is hypothesized to lead to some features of Turner’s syndrome such as panhypogammaglobulinemia, low IgM, decreased T and B cell numbers ,46.1. HEPATOCELLULAR CANCER: Increased expression of ribosomal protein S2 was found in mouse hepatocellular carcinoma (HCC) samples and in mouse livers after partial hepatectomy. RPS2 is involved in amino-acyl tRNA binding to ribosome, thus potentially affecting the fidelity of mRNA translation. Increased levels of RPS2 were shown to correlate with increased cell proliferation. However, whether the overexpression of RPS2 is a causative factor or just an associated phenomena in increased cell proliferation is unclear . Similar2. Colorectal cancer: Increased expression of ribosomal protein genes, including S3, S6, S8, S12, L5 has been reported in colorectal cancer (CRC) . Another3. Prostate cancer : The RPL19 gene has been found to be highly overexpressed in prostate cancer cell line. An inverse relationship between RPL19 expression and patient survival has been found, with patients expressing low levels of RPL19 surviving significantly longer. The statistical significance was also comparable to the Gleason score (p < 0.05) suggesting that RPL19 could be used as an independent prognostic marker . FurtherIYSNKLM harbors the 7 amino acid peptide insertion IYSNKLM, allowing it to selectively attack the melanoma cells and spare normal cells. In the same study it was shown that mice with the human melanoma xenograft had better survival and tumor regression with a combination of SLT-1AIYSNKLM and Dacarbazine than either alone [4. Malignant melanoma : A single chain ribosome inactivating protein (scRIP), developed from the cytotoxic A subunit of Shiga- like Toxin 1 (SLT-1A), has been shown to have the ability to kill 7 of 8 melanoma cell lines. The protein called SLT-1Aer alone .5. Metastatic sarcoma : The serine/threonine kinase, mammalian target of rapamycin (mTOR), is a protein kinase of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway thought to have a key role in controlling cancer growth and thus is an important target for cancer therapy. Several inhibitors of mTOR are in clinical trials, including AP23573, which is being tested on metastatic sarcomas and other tumors. Phosphorylated S6 ribosomal protein is a marker for the activity of mTOR and it was found that the level of phosphorylated S6 ribosomal protein expression was predictive of early tumor response to the mTOR inhibitor. Patients with a high expression of phosphorylated S6 responded better to the mTOR inhibitors, suggesting that this is a promising predictive sarcoma marker for targeted mTOR inhibitor therapy [6. Non hodgkins lymphoma: RPS6 is highly expressed in Diffuse large B cell lymphomas and genetic modulation of RPS6 protein levels with specifically targeted short hairpin RNAs (shRNA) lead to a decrease in the actively proliferating population of cells compared with control shRNA. RPS6 was shown to associate with multiple mRNAs containing a 5’TOP tract, which encode the translation machinery .1. Protein biosynthesis function of ribosomal proteins in cancer: Both tumor suppressors and oncogenes have been shown to modulate the ribosome protein biosynthesis as well as ribosome translation initiation in various models .. MYC, aRps19 mutant mouse model used to study DBA, induction of p53 and p53 target genes was identified with the mutation. These mice had decreased hematocrit and increased MCV and phenocopied the human disease. The anemia observed in these mice was partially abrogated with monoallelic inactivation of p53. Homozygous inactivation of p53 in Rps19 mutant mice fully corrected the hematologic abnormality [p53 mRNA translation. Haploinsufficiency of a subset of r-proteins  in zebrafish is linked to the development of malignant peripheral nerve sheath tumors. Interestingly, these nerve sheath tumors fail to produce p53 protein, despite presence of p53 mRNA [p53 mRNA translation during the DNA damage response by direct interactions with both MDM2 protein and p53 mRNA [2. Interaction of p53 with ribosomal proteins: Likely mechanism involved in some features of ribosomopathies and associations between ribosomal proteins and cancer.A number of events, including haploinsufficiency of some ribosomal proteins, impart instability to ribosomal biogenesis and cause nucleolar stress. In response to this stress, some ribosomal proteins  bind to MDM2 and block MDM2-mediated p53 ubiquitination and degradation, resulting in p53-dependent cell cycle arrest. By doing so, the ribosomal proteins play a crucial role in connecting deregulated cell growth with inhibition of cell division. The ribosomal protein-MDM2-p53 signalling pathway provides a molecular switch that may constitute a surveillance network monitoring the integrity of ribosomal biogenesis . . Interestingly, all these features have been shown to be caused by a deranged RP-MDM2-p53 pathway. Development of therapeutic targets against this pathway could possibly reverse the hematological manifestations. In Cartilage Hair Hypoplasia, Shwachman Diamond syndrome and Dyskeratosis Congenita, although there are significant ribosomal dysfunctions found, there still remains a small doubt whether they are causally associated with the conditions, particularly given the fact that the genes involved have other functions, which could also explain the clinical manifestations.Ribosomal protein disorders in malignancies seem to be more complex. Perturbation in extraribosomal functions of ribosomal proteins seems to be directly involved in tumorigenesis and deranged ribosomal function seems to be the consequence or an associated feature. While some ribosomal proteins have been shown to exert a direct effect on proto-oncogenes and tumorigenesis, others interact directly or indirectly with the p53 tumor suppressor pathway. Further mechanistic studies will reveal distinct sets of ribosomal proteins that are deranged in specific malignancies and will potentially uncover newer therapeutic targets.We have no competing interests.NS wrote the article, RK, TB, SB, YY performed literature search and contributed to the text, AV wrote the article. All authors have read and approved the final manuscript.


Dengue virus (DENV) affects over half the world’s population in 112 countries, and dengue fever (DF) is the second largest arthropod borne infectious global hazard after malaria with complications like Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS), accounting for significant morbidity and mortality world-over. Pakistan is significantly affected with DENV infection and to-date no study identifying risk factors associated with development of severe complications of DF has been done.997 confirmed cases of DF were collected from a tertiary care hospital in Lahore, Pakistan and their clinical and biochemical data were collected. Univariate, multivariate and logistics regression analysis was performed to identify risk factors associated with development of DHF and DSS.Bleeding OR 70.7 (CI 38.4–129.9), deranged liver function test OR 1.9 (CI 0.97–0.99), presence of urinary red blood cells OR 1.4 (95%CI 0.179–0.900) and presence of urinary protein OR 1.1 (95%CI 0.191–0.974) were related to development of DHF and DSS.Severe Dengue, like DHF and DSS can be predicted by the presence of clinical and biochemical factors like signs of bleeding, deranged liver function test, presence of urinary red blood cells and urinary protein; so that the patients at high risk for complication be identified early and started on treatment timely.Predictors of severe dengue are identified in this study but further large scale multi-centered studies are needed for better interpretation. Infection with the Dengue virus (DENV) is increasingly recognized as an important arthropod-borne viral infection infecting about 2.5 billion people worldwide; being endemic to over a 100 countries with over 975 million belonging to tropical and sub-tropical countries in Southeast Asia, the Pacific and the America.7th century in Chinese illustrations but the first detailed description of dengue shock syndrome was accounted by Benjamin Rush in 1780.12Dengue viral infection can either cause dengue fever (DF), dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). The classical dengue fever is mild, febrile illness which usually results after primary infection with dengue virus which is cleared in approximately seven days by a complex immune response.3 and hematocrit value of 50%, or a rise of more than 22% from baseline hematocrit, high viral load, intense activation of the immune system, various host conditions such as extremes of age, genetics, nutritional status, coexisting conditions and WHO recommended clinical criteria of abdominal tenderness, hepatomegaly, lethargy, cold extremity, and bleeding, in addition to Caucasian race, and people with AB blood group.Clinical trials show various factors associated with development of DHF including DENV serotype, of ≤ 75,000/mmTo assess the clinical and biochemical factors affecting the disease severity and mortality among patients with dengue fever in a tertiary care hospital in Pakistan.Severity was defined as presence of complications of dengue fever in the form of Dengue Hemorrhagic Fever or Dengue Shock Syndrome; both also known as Severe Dengue.The study was conducted during the Dengue Fever epidemic in a tertiary care hospital  in Lahore, Pakistan from August 1, 2011 till the September 30, 2011. The inclusion criterion was the presence of dengue fever (DF) which defined as finding three out of ten classical symptoms as described by World Health Organization (WHO) criteria and confirmed on the basis of presence dengue antibodies in a person from an endemic area.15The patients who were found to be negative for DF antibodies by Enzyme-linked immunosorbent assay (ELISA) or who had another cause identified for their symptoms like malaria, sepsis or infection elsewhere were excluded from the study. The data regarding physical characteristics including signs and symptoms, biochemical data, complications in the form of DHF and DSS and outcome in terms of numbers of days admitted and mortality if any, were recorded on a specially designed proforma. Data were then entered and analyzed using SPSS  for windows version 16. Data were tested for normality using Kolmogrov and Smirnove test. Categorical variables were presented as number and percentage while numerical variables were presented as mean (standard deviation) if normally distributed or median (range) if not normally distributed. Univariate and multivariate statistical analysis were undertaken successively.A univariate analysis was carried out using ANOVA test or Kruskal Wallis test for comparison between continuous variables, whereas the χ2 test or Monte Carlo exact test was used to assess differences between Categorical variables. Multivariate analysis was carried out by using Multinomial logistic regression analysis (MLR) where all the significant variables in the univariate analysis were included in multivariate analysis to adjust for confounders. This model was used to assess the association between the predictors  and multinomial outcome of each case as DF, DHF or DSS (3 possible outcomes). DF was considered as the reference group. Categorical independent variables were included as “factors” while quantitative independent variables were included as “covariates”. Odds ratio (OR) with 95% confidence intervals (95%CI) were used to identify the likelihood of categorical variables predicting the outcome. A p-value of 0.05 or less was taken as significant.Of the 1200 patients initially included in this study, 223 were excluded with other causes of febrile illness. Overall 977 were confirmed as dengue illness; including 686 cases with DF (70.2%), 280 cases with DHF (28.7%) and 11 cases with DSS (1.1%).Among 977 confirmed dengue illness, ages ranged from 5 to 87 years with a mean + SD age of 39.9 + 6.7 years and there was no significant difference between different diagnosis  regarding age (see There was no significant difference between DF and complicated cases (DHF and DSS) in presentation with headache, high fever, vomiting, abdominal pain and ascites and pleural effusion (see It was seen that majority of patients with urinary proteins (URP) and Urinary Red blood cells (URBC) (72.7%) develop shock  where all the significant variables in the univariate analysis were included in multivariate analysis to adjust for confounders. This model was used to assess the association between the predictors  and multinomial outcome of each case as DF, DHF or DSS (3 possible outcomes). DF was considered as the reference group. Categorical independent variables were included as “factors” while quantitative independent variables were included as “covariates”. The interpretation of MRL results was as following:The relationship between the dependent and independent variables was evaluated; the correlation was based on the statistical significance of the chi-square model. In this analysis, the distribution reveals that the probability of the chi-square model (54.3) was 0.003; that suggests a statistically significant relationship between the independent variable and the dependent variable, being the level of significance <0.05 (i.e. p<0.05).Once the relationship is established, the next important thing to do is to test the strength of multinomial logistic regression relationship.Using Cox & Snell R Square and the Nagelkerke R square value, they provide an indication of the amount of variation in the dependent variable, these are described as pseudo R square, this model revealed that the values are 0.181 and 0.322 respectively, suggesting that between 18.1% percent and 32.2% percent of the variability is explained by this set of variables used in the model.2 + 0.2872 + 0.0112 = 0.575). The proportional by chance accuracy criteria however was 71.9% (1.25 × 57.5% = 71.9%). The classification accuracy rate was 86.1% which was greater than the proportional by chance accuracy criteria of 71.9%, suggesting that the model was useful.The proportional by chance accuracy rate was computed by calculating the proportion of cases for each group based on the number of cases in each group and then squaring and summing the proportion of cases in each group  antibody positive for West Nile, Japanese encephalitis and DENV-2 Flaviviruses, which rapidly increased from July to October in patients ranging from 6 to 20 year age.Recent studies have shown that in addition to recommended WHO criteria, AB blood type, Caucasian race; new clinical and biochemical markers like clinical bleeding, high serum urea, low serum protein, low lymphocyte proportion, viral load assessment, viral serotype testing, cytokine, elastase, hyaluronan, soluble thrombomodulin, and nitric oxide level, and circulating endothelial cell detection test are associated with development of complications like DHF and DSS.Dengue virus circulates in Pakistan throughout the year with a peak incidence in the post monsoon period which is made worse by floods and as there is no licensed vaccine or antiviral compounds available against dengue virus, prevention and early identification remains the keystone of treatment. Recently there have been studies on ovicidal and adulticidal activities of plant leaf extracts against mosquito vector which appears to show promise in the future for widespread use as an ideal eco-friendly insecticide for the control of mosquitoes that spread malaria, yellow fever, filariasis and other arboviruses.23We need to identify patients at risk of developing severe complications and treat them promptly. Until we come up with an antiviral compounds that can target all four serotypes of dengue with same efficiency, recognizing factors that can help predict well in advance the severe form of DF, like DHF and DSS, could focus treatment in these group of patients. Our study showed increased liver function tests in serum, OR 1.9 (95%CI 0.97–0.99); presence of URBC, OR 1.4 (95%CI 0.179–0.900); and presence of URP, OR 1.1 (95%CI 0.191–0.974) can help predict patients at high risk for developing severe DF, both DHF and DSS. The most significant factor in identifying severe DF is the development bleeding  in patients with DF which had OR 70.7 (95%CI 38.4–129.9), making them susceptible to develop DHF; while the risk for progressing to DSS in them is even more, OR 608.2 (95%CI 33.9–108.9). More studies that can help add these factors as part of the warning signs for prediction of severe DF.Dengue infection has emerged as a major health concern in Southeast Asia especially Pakistan and till a specific vaccine or an antiviral agent become available, we have to rely on early identification of risk factors associated with developing complications of DF; just like the warning features specified by WHO as well as the ones identified in this study. Our study attempts to identify clinical and biochemical factors like signs of bleeding, deranged liver function test, presence of urinary red blood cells and urinary protein as possible predictors of severe DF so that a proactive approach is taken to treat these patients. However more large scale multi-centered trials are needed to confirm the findings in this study and to may be add these predictors later-on as a part of the warning signs for development of severe DF.


Background. Alterations in thyroid hormones regulation and metabolism are frequently observed in patients with cirrhosis. Aims. To assess alterations in thyroid volume (TV), haemodynamics, and hormones in patients with cirrhosis and their relation to hepatic arterial haemodynamics, and disease severity. Methods. Forty cirrhotic patients were compared to 30 healthy subjects regarding TV, free triiodiothyronine (fT3), free tetraiodothyronine (fT4), thyroid stimulating hormone (TSH), and pulsatility and resistance indices in the inferior thyroid and hepatic arteries. Results. TV (P = 0.042), thyroid volume standard deviation score , Inferior Thyroid Artery Pulsatility Index , Inferior Thyroid Artery Resistance Index , Hepatic Artery Pulsatility Index  and Hepatic Artery Resistance Index  were higher among cases being highest in Child-C patients. FT3 was lower in patients than controls (P = 0.001) and correlated negatively with ITAPI  and ITARI . ITAPI and ITARI correlated directly with HAPI and HARI . Conclusions. Thyroid is involved in the haemodynamic alterations of cirrhosis. Routine study of thyroid by Doppler and assessment of thyroid functions should be performed in patients with cirrhosis to offer proper treatment if needed. Liver cirrhosis is an irreversible alteration of the liver architecture which occurs in response to chronic liver injury from a variety of causes including toxins, chronic viral infection, cholestasis, and metabolic disorders. It consists of diffuse fibrosis of hepatic parenchyma resulting in nodule formation . Cirrhosis is characterized by complex changes in the systemic and splanchnic haemodynamics . Within 3 concentration probably due to impaired conversion of T4 to T3 in the liver. In severe cases, low T4 concentrations are also observed [ Alterations in thyroid hormone regulation and metabolism are frequently observed in patients with liver cirrhosis with decreased serum Tobserved .3 decreases systemic vascular resistance by dilating resistance arterioles of the peripheral circulation through a direct effect on vascular smooth muscle cells. Therefore, alterations in T3 may participate in the haemodynamic alterations of cirrhosis [ Thyroid hormones affect the vascular system, Tirrhosis . In medical application, ultrasonic Doppler equipment can be employed for detection and evaluation of the characteristics of blood flow in arteries and veins with the ultrasound beam directed toward the vessel of interest. The sources of echo signals are the red blood cells flowing in the vessel . With this background, we were stimulated to assess thyroid haemodynamic and hormonal alterations in patients with liver cirrhosis and their relation to hepatic arterial haemodynamics and disease severity. This cross-sectional case-control study was conducted over a period of 1 year from the beginning of August 2008 to the end of July 2009. It included 40 Egyptian patients with liver cirrhosis, regularly attending the Pediatric Hepatology Clinic, Children's Hospital, Ain Shams University, Cairo, Egypt. They were 22 males and 18 females with a mean age of 7.8 ± 5.5 years (range: 0.5–16 years). The diagnosis of cirrhosis was based on liver histology and clinical examination together with presence of portal hypertension . The etiology of cirrhosis was posthepatitic in 5 patients (2 posthepatitis B and 3 posthepatitis C), glycogen storage disease in 8 patients, biliary atresia in 7 patients, Niemann Pick disease in 6 patients, autoimmune hepatitis in 4 patients, Budd Chiari syndrome in 6 patients, and Wilson's disease in 4 patients. Patients were classified according to Child-Turcotte- Pugh classification  which us Group A included 15 patients  with a mean age of 6.6 ± 4.5 years (range: 1–14 years), group B included 15 patients  with a mean age of 5.9 ± 6.2 years (range: 0.5–15 years), and group C included 10 patients  with a mean age of 7.2 ± 3.0 years (range: 1–16 years).n = 12) and nonascitic groups (n = 28) based on clinical examination and ultrasound assessment [ Patients were also classified into ascitic  whose mean age was 7.2 ± 2.0 years ranging between 1–15 years. Children with primary thyroid disorders or positive family history of thyroid disorders or history of intake of thyroid modifying drugs in the past 3 months or those who had abnormal thyroid functions or living in areas known to be iodine deficient were excluded from the study. An informed written consent was signed up by the parents or caregivers of all studied subjects before enrollment in the study. This study was approved by the Bioethical Research Committee, Faculty of Medicine, Ain Shams University hospitals, Cairo, Egypt. Full medical history especially age, sex, residence, history of drug intake, and positive family history of thyroid disorders.Examination for signs of hypothyroidism and neck examination to detect presence of goiter if present.3, fT4, and TSH with the Immulite 2000 Analyzer using chemiluminescent immunometric assay [Thyroid function tests, including serum fTic assay .Liver function tests: which included Alanine amino transferase , Aspartate amino transferase , serum bilirubin , total proteins (gm/dL), and albumin (gm/dL) using Automated assay, Synchron clinical system, Beckman.Doppler ultrasound: using a color Doppler of Power Vision SSA-380A system  with muti-Hertz (3 to 5 MHz) convex and sector-pulsed probes. This was carried out on:Thyroid gland: TV in mL was calculated by ellipsoid method (length × width × thickness × 0.523) . The totLiver: Sonographic examination was carried out 8 hours after the last meal. Color Doppler allowed identification of hepatic artery which was evaluated by an intercostal approach via demonstration of right and left portal veins under a 60 degree angle. In some subjects, hepatic artery traces were seen at least 2 cm away from right to left portal vein bifurkation, therefore, measurements were done from there. Doppler system was placed inside this vessel at an angle of less than 60 degree between the vessel and ultrasound beam and the blood flow velocity waveform was recorded.In both inferior thyroid and hepatic arteries, resistance and pulsatility indices were calculated according to the following formulae [All children were subjected to the followingformulae :(1)Resice versa . A valuece versa .t-test of 2 independent samples was used to compare 2 quantitative groups for parametric data while Mann-Whitney test (z-test) was used to compare 2 quantitative groups for nonparametric data. Pearson correlation coefficient (r-test) was used to relate different variables to each other. A value of P < 0.05 was considered significant. The data were statistically analyzed using SPSS statistical package version 10 . Description of quantitative variables was in the form of mean ± SD, median, and range, while that of qualitative variables was given as frequency and percentage. Student 3 was significantly lower among patients than controls with a nonsignificant difference regarding fT4 and TSH (P > 0.05). None of our cases had signs of hypothyroidism nor visible or palpable goiter on clinical examination. TV and its SDS and thyroid and hepatic haemodynamic parameters were significantly higher, while fT and TSH . In addi and TSH . Moreove3 going from group A to C . On the other hand, fT4 and TSH levels did not significantly differ between the 3 groups. Both ITAPI and ITARI increased progressively from group A to C . Also, both HAPI and HARI increased progressively from group A to C . Thus, all thyroid and hepatic haemodynamic parameters increased with increased severity of liver disease. In addition, the frequency of increased TV, ITAPI and ITARI among cirrhotic patients increased progressively going from group A to C  and ITARI  while fT4 and TSH did not correlate significantly with each of ITAPI and ITARI (P > 0.05). Moreover, there were significant positive correlations between fT3 and each of serum albumin  and total proteins  and significant negative correlations between fT3 and each of ALT  and AST . Also, there were significant positive correlations between thyroid and hepatic arterial indices [ITAPI, and HAPI  [3 syndrome” with increased rT3 and decreased T3: T4 ratio [4 to T3 and rT3 to T2 could take part in the modification in circulating hormone levels described in cirrhosis [3 levels have been demonstrated in cirrhotic patients with a high degree of liver damage [3, was found suggesting a role of hyperglucagonemia in the pathogenesis of low T3 values in these patients [3 with normal total T4 and TSH concentration in the absence of clinical hypothyroidism have been frequently reported in patients with nonthyroidal illnesses. Moreover, an inverse correlation between serum T3 concentration and the severity of liver dysfunction was reported [3 and each of ALT and AST. Also, progressive decrease in T3 level in chronic liver disease has been described as indicative of a poor prognosis [3 syndrome in such patients as indicated by the positive correlation between fT3 and each of serum albumin and total proteins in our study. On the other hand, in 2001, Desi and his coworkers [3 in patients with liver cirrhosis in relation to controls, which is against our results. This difference might be caused by the fact that the patients included in their study may have had an underlying thyroidal illness which was not excluded by the authors. Also, Gardner and his associates [3 between cirrhotic patients and controls. This difference might be caused by the fact that the patients included in their study had less severe liver failure than those included in our study. In the current study, fT studies , 17. TheT3 (rT3) . In cirrT4 ratio . Severalirrhosis . High ser damage . Moreover damage . A signipatients . Thyroidreported , which wrognosis . Moreoveoworkers  found thsociates  found a 4 and TSH did not differ between patients and controls which was also confirmed by other authors. [4 level in patients with liver cirrhosis whose cause was acute hepatitis but there was a nonsignificant difference in serum-free T4 and TSH in patients with liver cirrhosis due to other causes when compared to controls. They explained this by the fact that increased T4 might be attributed to the viral infection that could cause thyroiditis and raised fT4. In our series, fTauthors. , 17, 27.authors.  found an3 levels and thyroid Doppler indices suggest that thyroid hormones changes could directly participate in haemodynamic alterations in cirrhotic patients suggesting their role in pathophysiology [3, decrease systemic vascular resistance by dilating resistance arterioles of the peripheral circulation through a direct effect on vascular smooth muscles. This vasodilating effect is attenuated by endothelial denudation, cyclooxygenase inhibition, and NOS (nitric oxide synthetase) inhibition that occur in cirrhosis. The latter hypothesis confirms the involvement of low fT3 in thyroidal arteries vasoconstriction in cirrhotic patients [Both ITAPI and ITARI were significantly higher in patients than controls with a progressive increase in the frequency of their rise going from group A to C, that is, with increased severity of liver disease. The reason for the increase in ITAPI and ITARI in cirrhotic patients is the vasoconstriction in thyroid circulation which is a part of the same haemodynamic picture of cirrhotic portal hypertension, secondary to increased vasoconstricting hormones. Also, the inverse correlation between fTysiology . Thyroidpatients . Moreovepatients  found th Moreover, in our patients, Doppler indices in inferior thyroid artery correlated directly with hepatic arterial indices which was confirmed by other authors . This su In addition, our series revealed higher HAPI and HARI in patients than controls. Schenk and his collegues  explaine On the other hand, Doppler haemodynamic parameters and thyroid function tests did not significantly differ according to the etiology of liver disease meaning that the different pathogenetic mechanisms of liver cirrhosis have no influence on the hepatic/thyroid gland hemodynamic interaction.3 may play a role in the pathophysiology. Therefore, thyroid functions should be performed in patients with liver cirrhosis to detect the early changes in thyroid hormones and to offer proper treatment if needed. Also, study of the thyroid gland by Doppler could be useful in patients with liver cirrhosis to assess severity of liver cirrhosis in addition to Child-Turcotte-Pugh classification in order to predict its prognosis.In conclusion, the thyroid gland is involved, primarily and secondarily, in the haemodynamic alterations of cirrhosis; a reduction in vasodilator fT


Statement of Problem. The chemical or topographic modification of the dental implant surface can affect bone healing, promote accelerated osteogenesis, and increase bone-implant contact and bonding strength. Objective. In this work, the effects of dental implant surface treatment and fibronectin adsorption on the adhesion of osteoblasts were analyzed. Materials and Methods. Two titanium dental implants (Porous-acid etching and PorousNano-acid etching followed by fluoride ion modification) were characterized by high-resolution scanning electron microscopy, atomic force microscopy, and X-ray diffraction before and after the incorporation of human plasma fibronectin (FN). The objective was to investigate the biofunctionalization of these surfaces and examine their effects on the interaction with osteoblastic cells. Results. The evaluation techniques used showed that the Porous and PorousNano implants have similar microstructural characteristics. Spectrophotometry demonstrated similar levels of fibronectin adsorption on both surfaces (80%). The association indexes of osteoblastic cells in FN-treated samples were significantly higher than those in samples without FN. The radioactivity values associated with the same samples, expressed as counts per minute (cpm), suggested that FN incorporation is an important determinant of the in vitro cytocompatibility of the surfaces. Conclusion. The preparation of bioactive titanium surfaces via fluoride and FN retention proved to be a useful treatment to optimize and to accelerate the osseointegration process for dental implants. The phenomenon of endosseous implant osseointegration, conceptualized by Branemark as the “direct, structural and functional link between the living and orderly bone and the surface of an implant subjected to functional loads” , is fundThis paper shows that the bone-implant interface strength is greater in dental implants with rough surfaces than in those with smooth surfaces , 6. TreaAppropriate changes in dental implant surface roughness can produce better anchoring strength and mechanical locking in the early stages of osseointegration , 6. Morein vitro tests have shown that titanium fluoride treatments have a greater capacity for the nucleation of phosphate crystals than sandblasted Ti implants. In vivo fluoride ion-modified implants have generally proven superior to sandblasted surfaces in terms of osseointegration, ultimately increasing the removal torques.Johansson et al.  observedFibronectin is a major extracellular matrix protein that is known to promote cell attachment and spreading, differentiation, and phagocytosis. It is a dimeric glycoprotein found in all vertebrates in two basic forms: soluble (plasma and other fluids) and insoluble (extracellular matrix of various tissues). It has a molecular weight between 440 and 500 kDa. Disulfide bridges link one subunit to another via sites near the carboxy termini of each subunit. The fibronectin protein has folds that lead to structural remodeling and various conformations according to the medium –12.Fibronectin (FN) functions in cell adhesion, migration, survival, proliferation, and differentiation as well as tissue organization. The FN molecule can interact with other biomolecules, such as collagen, proteoglycan, heparin, hyaluronic acid, fibrin/fibrinogen, plasmin, gangliosides, complement components, and also integral proteins of cell plasma membrane-integrins, as well as with itself .Menezes  conducteα5β1 (FNIII7–10) increase osteoblastic differentiation and optimize tissue formation and functional integration compared with untreated surfaces or surfaces containing only the RGD sequence.Petrie et al.  conducteThe purpose of this study was to evaluate the effect of the fluoride treatment of cp titanium samples on the adhesion and proliferation of osteoblastic cells on surfaces with and without fibronectin coating. 3, H2SO4, and HCl (surface treatment similar to Porous implants available from Conexão Sistemas de Prótese);Porous: samples treated in acidic solutions containing HNO PorousNano: treatment similar to Group 1 followed by fluoride ion modification by immersion for one hour in a solution containing fluorine ions; Porous-FN: treatment similar to Group 1 with FN incorporation; PorousNano-FN: treatment similar to Group 2 with FN incorporation.Implants and discs of grade 4 machined cp Ti were provided by Conexão Sistemas de Prótese . Samples were submitted to surface treatment and divided into four groups:After treatments, samples from the Porous and PorousNano groups were washed with distilled water and absolute alcohol, dried in oven at 70°C for two hours, and packed and sterilized by gamma irradiation (25 kGy).To characterize the surface morphology and identify differences in samples submitted to treatments with acids and/or fluorides, the samples were characterized by a high-resolution scanning electron microscopy . The results were complemented by analysis with an MFP-3D atomic force microscope  operating in contact at room temperature mode. The cantilevers used were V shaped, NP-S model  with an 0.08 N/m spring constant, and calibrated using the thermal noise method. To reduce damage to samples and reduce noise, images were acquired using low-frequency scanning (1.0 Hz) with 256 × 256 pixel resolution. Image processing was performed in the program IGOR PRO  using a MFP-3D platform developed by Asylum Research.α = 1,542 Å) with an RU 200B model Rigaku generator and 0.02° step/minute.An X-ray diffractometer was used to identify crystalline phases on discs. X-ray diffraction for the analysis of thin films (grazing incidence technique) was conducted at 40 kV and 30 mA. A copper anode was used ) was diluted to 10 g/mL, pH 4.5 in previously filtered 20 mM sodium acetate  buffer solution. NaCl was added to the solution to maintain the medium's ionic strength between 0.145 and 0.150 mol·dmμg/mL) controls were performed. The wavelength used was 550 nm (protein reading). Samples from the Porous and PorousNano groups were coated with fibronectin at room temperature for 2 hours. Substrates with FN were washed with PBS  to remove nonadsorbed molecules. Then, the adsorbed molecules were detached using 0.1% trypsin and PBS. One to two minutes later, the excess was removed, and the resulting solution was collected and analyzed with a Spectrum 22PC spectrophotometer to quantify the adsorbed molecules. Spectrophotometry was also used to determine the FN's absorbance on both surfaces (protein concentration in solutions that absorb radiation). Negative (PBS) and positive  buffered with 10 mM HEPES (Sigma-Aldrich) and 14.3 mM NaHCO3 (Reagen). The pH of the medium was adjusted to 7.2. Cultures were incubated at 37°C in 5% CO2 atmosphere. The enzymatic cell detachment technique was used to transpose cells from the stock culture flask to substrates for the adhesion assay. Confluent cultures were treated with 0.2% trypsin  and 0.02% EDTA (Sigma-Aldrich) in saline solution  for 5 minutes at 37°C. Then, the detached cells were collected, and the proteolytic action of trypsin was inhibited by adding fetal calf serum to the solution. The suspension was then centrifuged at 1500 rpm at 22°C, and the pelleted cells were resuspended in culture medium without fetal calf serum. The cell concentration/density of the suspension was estimated by counting in a hematimetric Neubauer chamber.Cells were maintained in polystyrene bottles containing DMEM (Dulbecco's Modified Eagle Medium) culture medium with low glucose, 10% fetal bovine serum , and 1% essential amino acids solution  ascorbic acid  to surfaces were washed with PBS and fixed using glutaraldehyde (2.5% in PBS). Glutaraldehyde was used as fixative to avoid damaging cell integrity . This procedure was adopted because the use of formaldehyde  as a fixative profoundly deformed the cells. After fixation, cells were trypsinized and counted in a hematimetric Neubauer chamber. After the cell concentration of the suspension was measured in a hematimetric chamber, 103H]-thymidine (1143 cpm). After allowing incorporation for a period of 12 hours, the confluent cells were again detached and washed in DMEM without serum, and a liquid scintillator  was used to evaluate the radioactivity associated with cells. The resulting values were expressed as counts per minute (cpm). These cells, incorporating [3H]-thymidine, were associated with different surfaces  for a period of 3 hours, and counts were carried out after 1, 2, and 3 hours. This cell behavior evaluation method allows accurate reproduction, favoring the future applicability of FN incorporation onto surfaces of dental implants.Human osteoblastic cells (HOBs) were cultivated to evaluate cell adhesion and proliferation by liquid scintillation counting. Cells from confluent HOB cultures were detached with trypsin, washed, and counted in a hematimetric chamber. Then, the culture was resuspended in DMEM containing serum and -thymidine for 12 hours, the radioactivity associated with osteoblast cells was evaluated. Subsequently, 1.8 × 106 cells/mL, corresponding to 1,100 cpm, were delivered to Porous, Porous-FN, PorousNano, and PorousNano-FN surfaces. The results are shown in After the incorporation of [After one hour of interaction, 70% of cells (0.751 cpm) were associated with the PorousNano surface. This number is most likely low because some cells died or were not associated with the sample at the beginning of the process. The number of associated cells increased with interaction time, reaching 0.864 cpm after three hours; that is, there was a 15% increase in the amount of associated cells due to proliferation and cell division.Only 64% of the cells interacting with the Porous surface (0.687 cpm) remained associated after one hour, but this number increased approximately 32% after 3 hours of interaction, reaching 0.905 cpm.On the Porous-FN surface, 90% of cells (0.976 cpm) were associated after 1 hour of interaction. The number of attached cells increased 9% after three hours, reaching 1.064 cpm. For the PorousNano-FN surface, 92% of cells (0.986 cpm) were associated after one hour of interaction, and this number increased by 11.5% over three hours, reaching 1.100 cpm. Ra values than acid-treated surfaces without fluoride. Images obtained by atomic force microscopy  show thaAs measured based on the images obtained through AFM, the roughness of the PorousNano surface sample was lower than that of the Porous surface, demonstrating that treatment with fluoride reduced the summit height, most likely due to the reaction of titanium oxide with fluoride ions. This ultrastructural aspect of the summits contributes to the more homogeneous roughness pattern of the PorousNano surface, in addition to the presence of smoother areas and larger microcavities.The presence of only one crystalline phase of titanium was revealed by X-ray diffraction of the Porous and PorousNano samples. It is likely that the immersion in a solution containing fluoride ions adds only a small amount of this element to the titanium surface and that this trace amount of fluoride cannot be detected by the XRD technique for the analysis of thin films (grazing incidence technique).Approximately 80% of the FN allowed to interact with Porous and PorousNano surfaces was adsorbed (2.473 AU). This result demonstrates that the chemical treatment with acids (Porous) and chemical treatment with acids followed by immersion in solution containing fluoride ions (PorousNano) did not affect the incorporation of biomolecule; that is, the presence of the fluoride ion did not influence the protein adsorption. Dos Santos et al.  observed5 cells/mL) than the Porous surface  after one hour of interaction. Because 106 cells/mL were taken to interact with surfaces, approximately 8% adhered to the Porous sample, while 23% were associated with the PorousNano sample. These indices suggest that the surface subjected to chemical treatment followed by immersion in a solution containing fluoride ions favors the adhesion of most cells during the initial interaction period. As mentioned earlier, the association indices of the Porous and PorousNano surfaces without fibronectin were considered null for evaluations of the influence of protein on cell behavior. Thus, the number of cells associated with the PorousNano with FN surface increased 57.4% compared with the same surface without the biological variable. For the Porous with FN surface, the increase in cell adhesion was 44.7% compared to the same area without the protein. These indices show that the protein variable is responsible for the significant increase in the number of cells attached to the surfaces, confirming the results of Ku et al. [2, cell adhesion was initiated after 3 hours and had significantly lower cell numbers for all measurement points compared with FN. The present work showed the same results.Cell counting in a hematimetric chamber is a sensitive and accurate technique for the evaluation of cell adhesion to titanium surfaces. In this study, the PorousNano surface showed a stronger association with osteoblastic cells  were allowed to interact with Porous, Porous-FN, PorousNano, and PorousNano-FN titanium surfaces for three hours. After one hour of interaction, 92% of the cells were associated with the PorousNano-FN surface, and 90% of cells were associated with the Porous-FN surface, while 70% and 64% of cells were associated with the PorousNano and Porous surfaces (without FN), respectively. These results confirm that the protein coating accelerated the adsorption of cells during the initial interaction period (adaptation period). This can be explained by the fact that when fibronectin is allowed to interact with titanium samples under ideal conditions of pH such that its cryptic sites are exposed, the fibronectin signals to osteoblasts to activate the cell cycle and initiate the secretion of ECM proteins. A total of 1.8 × 10The Porous-FN and PorousNano-FN surfaces showed similar behavior during the three-hour interaction, both during the initial adherence of cells (approximately 90% for both surfaces) and in their proliferation. The cell number increased by 14% for the sample PorousNano-FN and 12% for Porous-FN in the first 3 hours of interaction . Ku et ain vitro, in addition to offering greater osseointegration resistance in vivo. Although previous studies have used different methodologies for fluoride treatment, their results also suggest that the presence of fluoride ions on titanium surfaces facilitates various osseointegration processes. Analysis of the experimental cell adhesion and proliferation data presented in In samples without FN, this study showed that within one-to-three hours of interaction, the number of cells attached to the PorousNano surface increased by 32%, while the number of cells attached to the Porous surface increased by 15%. This difference shows that the surface that received acid treatment followed by immersion in a solution containing fluoride ions (Nano) showed accelerated cell division and proliferation compared to the Porous surface. the surfaces of titanium samples treated with fluoride ions (PorousNano) retained the basic microstructural characteristics of surfaces not treated with fluoride (Porous),the Porous and PorousNano surfaces incorporated similar levels of FN (approximately 80%) over the time tested (3 hours), demonstrating that the presence of fluoride ions did not influence protein adsorption,in vitro cytocompatibility of surfaces,the association indices of HOB cells to the four tested surfaces suggest that FN incorporation is critical for the FN-treated samples showed significantly higher percentages of associated cells during the initial period of one hour, confirming that FN  had a greater effect on the adhesion and proliferation of cells than the fluoride treatment of titanium surfaces used in this study.Based on the experimental results, it can be concluded that


Pogosta disease is a mosquito-borne viral disease in Finland, which is clinically manifested by rash and arthritis; larger outbreaks occur in 7-year intervals. The causative agent of the disease has been suspected of being closely related to Sindbis virus (SINV). We isolated SINV from five patients with acute Pogosta disease during an outbreak in fall 2002 in Finland. One virus strain was recovered from a whole blood sample and four other strains from skin lesions. The etiology of Pogosta disease was confirmed by these first Finnish SINV strains, which also represent the first human SINV isolates from Europe. Phylogenetic analysis indicates that the Finnish SINV strains are closely related to the viral agents isolated from mosquitoes and that cause clinically similar diseases in nearby geographic areas. Alphavirus in the family Togaviridae, was first isolated in 1952 in the Nile River delta in Egypt from a pool of Culex pipiens and Cx. univittatus mosquitoes (Sindbis virus (SINV), a member of the Western equine encephalomyelitis virus complex of the genus Culex and Culiseta, from which SINV has previously been isolated in Sweden  as anticoagulant and skin lesion biopsy specimens were collected, if possible. The skin biopsy specimens were taken from one papulopustule with a punch or a surgical knife into a dry tube, usually by a local physician. The samples were kept at +4°C, and transported to the local central hospital usually within the next 24 hours to be frozen at –70°C. All samples were kept frozen until they were processed at the department of Virology, University of Helsinki. Informed consent was obtained from all patients. The study was carried out under the permission of the coordinating ethical committee of the Hospital District of Helsinki and Uusimaa.A 35-year-old man was most probably exposed to SINV in August 2002, in Ilomantsi, a province of North Karelia. Typical Pogosta disease symptoms began with polyarthritis, first affecting the fingers, wrists, and the left shoulder, then extending to the left elbow, and finally both knees and the right ankle. Joint symptoms were often aggravated by exertion. At the acute phase, the patient also had fatigue, nausea, muscle pain, headache, low fever, and itchy rash throughout the body. A skin biopsy was taken from one papulopustule . Basic blood parameters at the acute phase were within normal range, including blood cell counts, hemoglobin, platelets, and the erythrocyte count indices. The joint symptoms lasted for 3 to 4 months. Besides these symptoms, no other residual symptoms were encountered.All patients were screened for SINV immunoglobulin (Ig) M and IgG with enzyme immunoassay (EIA) and for total antibodies with the hemagglutination inhibition test (HI). EIA was performed with purified SINV antigen, directly coated on microtiter well plates  was apparent, immunofluorescence assay (IFA) was performed, and the cells were passaged.The frozen skin samples were cut into small pieces, then homogenized in a mortar, and suspended in 100–150 μL Dulbecco’s minimal essential medium plus 0.2% bovine serum albumin. A total of 50–100 μL of this suspension diluted in 500 μL of culture medium, containing minimal essential medium and 2% fetal calf serum with a mixture of glutamine, ampicillin, and penicillin, was added to confluent Vero cells in 25-cmSeveral measures were undertaken to avoid laboratory contamination. SINV was not handled in the two laboratories where the samples were prepared and the cells were cultured, and separate sets of instruments were always used. Samples were handled and cells cultured in separate laboratories. Cross-contamination was avoided by culturing in cell culture flasks instead of culture plates. At least two flasks of noninfected Vero cells were cultured in the same incubator every time and went through the same procedures as the potentially infected cells.An IFA was developed to confirm SINV infection of the cells. Cells were harvested in 600 μL phosphate-buffered saline (PBS), washed, and centrifuged at 1,800 rpm for 3 min five times and dried on a slide. For immunofluorescence staining the slides were fixed for 7 min in ice-cold acetone. A pool of 10 SINV IgG–positive serum samples were diluted 1:20 in PBS, added to slides, and incubated in a moist chamber at 37°C for 30 min. The slides were washed three times in PBS and once in water, then incubated at 37°C for 30 min with fluorescein isothiocyanate–conjugated F(ab')2 goat anti-human IgG diluted 1:100 in PBS. After another wash, the slides were dried, mounted, and screened with a fluorescence microscope.tttagcggatcggacaattc-3′ and 5′-gcggtgacgaactcagtag-3′. The RT reaction was carried out as follows: 10 μL of RNA dissolved in water was mixed with 2 μL of each primer (10 pmol/μL), 2 μL of M-MuLV reverse transcriptase , 5 μL of 5 x RT-buffer (MBI Fermentas), 2 μL of dNTP-mix , and 2 μL of Ribonuclease inhibitor . The mixture was incubated at 37°C for 90 min.Viral RNA was extracted from culture supernatant with TriPure Isolation Reagent  by following the manufacturer’s instructions. The primers used in RT-PCR were 5′-For the PCR 7 μL of cDNA were incubated at 95°C for 5 min, cooled immediately on ice, and mixed with 2 μL of each primer (10 pmol/μL), 10 μL of 10 x Taq Extender PCR Buffer , 8 μL of dNTP mix , 1 μL of Taq DNA Polymerase (recombinant) , 1 μL of Taq Extender PCR Additive (Stratagene), and 69 μL of water. The reactions were amplified through 35 cycles using a DNA thermal cycler with the following steps: denaturation at 95°C for 45 s, annealing at 55°C for 2 min, and elongation at 72°C for 3 min, followed by a final extension at 72°C for 10 min.Escherichia coli cells on bacterial plates containing x-gal  and IPTG  for blue-white screening. The plasmid DNA was isolated with QIAprep Miniprep kit (Qiagen). and restriction analysis was performed. Vector-based primers M13 Reverse and T7 were used for automatic sequencing with ABI PRISM .The PCR amplicons were purified with QIAquick gel extraction kit . The amplicons were cloned with TOPO TA Cloning Kit for Sequencing , following the manufacturer’s instructions, and transformed into TOP10 chemically competent http://www.psc.edu/biomed/genedoc/) editor program. Sequence was confirmed from at least three different clones. PHYLIP program package  was used to create 5,000 bootstrap replicates on the sequence data (SEQBOOT). Distance matrices were calculated with DNADIST program with Kimura’s two-parameter model of substitutions and analyzed by Neighbor-joining tree-fitting algorithm with NEIGHBOR program. The bootstrap support values were calculated with CONSENSE program.Sequences were aligned with Clustal W1.75 program  were collected from patients with acute SINV infection and used in this study for isolation attempts.A physician examined all patients at the acute phase. To determine the most likely place and time of exposure to SINV, background information about the physical location of the patients before the onset of symptoms was also collected by using questionnaires. The exact incubation period in Pogosta disease is unknown, and determining the time and place of exposure was difficult.Virus isolation was successful from 4 of 23 skin samples and 1 of 73 whole blood samples  Figure . All isoThe median and average number of days of rash for patients with the positive skin samples before sampling were 1.5 and 2.5, respectively, and SINV IgM antibodies were not detectable (IgM status for patient 4 at the time of sampling was not available) . The medAedes mosquitoes in 1983 in central Russian Karelia, approximately 200 km north of Ilomantsi, Finland , Ilomantsi-2002B (AY532326), Ilomantsi-2002C (AY532324), Kiihtelysvaara-2002 (AY532325), and Johannes-2002 (AY532323). In addition, we sequenced from this region the LEIV-9298-strain (AY532321), isolated from This study describes the first human SINV isolates from Europe, one strain from blood and four from skin lesions. One of the strains (Johannes-2002) is apparently Russian, since the Finnish patient most likely was exposed to SINV in Russian Karelia. The four other strains represent the first SINV isolates from Finland. Phylogenetic analysis of the strains shows a close relationship to Swedish and Russian SINV strains, isolated approximately 20 years ago from mosquitoes.The possibility of laboratory contamination was minimized by various measures as described in Methods. Only a few samples were prepared at the same time; each new virus strain was isolated from separate set of samples. No CPE was apparent in the negative control cells at any stage and they were all negative in immunofluorescence staining as well.SINV could be recovered from one blood sample of 73. This sample was positive by nested RT-PCR method as well; full characterization of clinical and laboratory data will be presented later . As now proven, SINV is present in the blood during acute infection. The most viremic window appears to be very narrow and the level of viremia can vary considerably between persons. These presumptions make future laboratory diagnostics based on viral detection challenging, and serology likely will remains the method of choice for diagnosis.In skin tissue, the viral persistence seems to last for several days, if not weeks. The Johannes-2002 strain was isolated from a biopsy specimen taken 7 days after the onset of rash. SINV could be recovered from 17% of the skin biopsy samples. Whether the virus persists in synovial fluid and whether this could explain the prolonged joint symptoms in a substantial proportion of Pogosta disease patients  remain to be determined. Some patients have had borderline results in IgM serologic testing even months or years after the onset of disease , which suggests that local ecologic and environmental factors are involved in the epidemiology of Pogosta disease in Finland. Data are not available about whether the epidemics in northern Karelia were accompanied by similar epidemics on the Russian side of the Finnish-Russian border, although one of our patients  was most probably exposed while traveling in Russian Karelia. Ecologic circumstances likely alter the viral cycle in nature, and the prevalence of mosquitoes carrying SINV varies. However, differences in the pathogenicity of viral strains cannot be excluded. In Australia, for instance, SINV is the most common arbovirus isolated from mosquitoes (Phylogenetic analysis of the Finnish SINV strains for a 1.2-kb genome segment indicates that Finnish SINV strains are closely related to each other and to the Swedish and Russian strains, isolated 2 decades ago . Within South African SINV strains have been shown to not vary substantially from Swedish strains . Indicat


Background: Inhalation is one of the main means of human exposure to polycyclic aromatic hydrocarbons (PAHs) because of their ubiquitous presence in the atmosphere. However, most studies have considered only PAHs found in the particle phase and have omitted the contribution of the gas-phase PAHs to the risk.Objective: We estimated the lifetime lung cancer risk from PAH exposure by inhalation in people living next to the largest chemical site in Southern Europe and the Mediterranean area.a]pyrene (BaP) equivalents (BaP-eq) for individual PAHs and applied the World Health Organization unit risk (UR) for BaP (UR = 8.7 × 10–5) to estimate lifetime cancer risks due to PAH exposures.Methods: We determined 18 PAHs in the atmospheric gas and particle phase. We monitored the PAHs for 1 year in three locations near the chemical site in different seasons. We used toxic equivalence factors to calculate benzo[–4.Results: We observed some spatial and seasonal variability in PAH concentrations. The contribution of gas-phase PAHs to the total BaP-eq value was between 34% and 86%. The total estimated average lifetime lung cancer risk due to PAH exposure in the study area was 1.2 × 10–3 that is considered an indication of definite risk according to similar risk studies. The results also showed that risk may be underestimated if the contributions of gas-phase PAHs are not considered.Conclusions: The estimated risk was higher than values recommended by the World Health Organization and U.S. Environmental Protection Agency but lower than the threshold value of 10 Polycyclic aromatic hydrocarbons (PAHs) are a large group of organic contaminants, characterized by the presence of at least two fused aromatic rings. These compounds result from pyrolytic processes and the incomplete combustion of organic matter at high temperatures, and anthropogenic activities are the main source of PAHs in the environment. Because of their adverse effects on human health, their persistence in environmental matrices, and their reactivity and ability to transform into more active species, PAHs have been classified as priority pollutants by both the U.S. Environmental Protection Agency (EPA 1994) and the a]pyrene (BaP) has been the most extensively studied PAH and is the usual marker for carcinogenic levels of PAHs in environmental studies. However, uncertainties about the suitability of BaP as a cancer risk indicator have also been discussed , as probably carcinogenic to humans (group 2A); and other PAHs, such as naphthalene (Nap), benzo[a]anthracene (BaA), chrysene (Chr), benzo[b]fluoranthene (BbF), benzo[j]fluoranthene (BjF), and indenopyrene (Ind) as possibly carcinogenic to humans (group 2B). PAHs can also metabolize and become reactive electrophilic intermediates that can form DNA adducts, which may induce mutations and ultimately tumors (IARC 2010). Some PAHs, such as fluoranthene (FluT), which IARC classified as weak carcinogens (group 3), have mutagenic characteristics and may therefore play an important role in carcinogenesis  1998] and occupational studies in humans  demonstriscussed . The Intogenesis .Studies of other outcomes have suggested effects of PAHs on the development of arteriosclerosis , reproduIngestion is quantitatively the main route for PAH human exposure. However, inhalation is also a significant route because of the ubiquitous presence of these compounds in the atmosphere , in both the gas and particle phases. We monitored three locations near the two industrial complexes in the region from June 2008 to June 2009. We evaluated the distribution of PAHs in the gas and particle phase, their seasonal and spatial distributions, and the contribution of individual PAHs to estimated risks.Few studies related to industrial atmospheres consider the contribution of both the atmospheric gas phase and the particle phase to risk . To our Study area. The largest chemical site in Southern Europe and the Mediterranean area is located in the Tarragona region (a region . Most ofSample collection. We collected samples using a TE-1000 polyurethane foam (PUF) high-volume air sampler , which simultaneously collects total suspended particles and airborne pollutant vapors, meeting the specifications of U.S. EPA method TO-13 (U.S. EPA 1999). We took samples for 24 hr at a flow rate of approximately 0.2 m3/min on quartz microfiber filters  for trapping particulate matter and PUF cartridges  for gas-phase pollutants. Before the sampling step, we conditioned the QFFs at 400°C for at least 6 hr, and the PUFs using dichloromethane (DCM) Soxhlet extraction for 24 hr and then dried them in a vacuum desiccator. After the conditioning step, we individually wrapped the QFFs and PUFs in aluminum foil, protected them in a sealable plastic bag, and kept them at –18°C before and after the sampling, until analysis. We analyzed the samples within 2 weeks of collection.We conducted the sampling campaign over 1 year between June 2008 and June 2009. During this period, we took a total of 153 samples of matched air and particulate matter in three weekly samples over approximately 4 months in each location . We collected samples during two phases in each location, including 2 months during a high-temperature period and 2 months during a low-temperature period, to determine whether temperature may influence PAH values in the atmosphere.Chemical standards and reagents. We measured 18 target PAHs and used five deuterated PAHs as internal standards. Standards for Nap, acenaphthylene (AcPy), acenaphthene (AcP), fluorene (Flu), phenanthrene (PA), anthracene (Ant), FluT, pyrene (Pyr), BaA, Chr, BbF, benzo[k]fluoranthene (BkF), BaP, and DahA were supplied by Sigma-Aldrich . Standards for BjF, benzo[e]pyrene (BeP), Ind, benzoperylene (BghiP), and the deuterated PAHs  were supplied by Supelco . Dimethylformamide (DMF) was provided by Merck . The purity of all standards was > 96%.DCM with purity > 99.9%  was used to extract the analytes and prepare the standard dilutions. Helium and nitrogen gas (99.999% purity) were used for the chromatographic analysis and extractions, respectively , and Hyflo Super Cel diatomaceous earth (Sigma-Aldrich) was used to fill the QFF extraction cells of the pressurized liquid extraction (PLE) equipment. Diatomaceous earth was conditioned at 400°C for 6 hr and then kept in a desiccator.Sample extraction. We extracted PAHs by PLE using Accelerate Solvent Extraction ASE 200 equipment  and DCM as solvent. For the pressurized solvent extraction of the PUFs, we cut them in two pieces using acetone-rinsed scissors and then placed them in 33-mL extraction cells. Extraction of the PUFs consisted of one cycle that began with a preheating step of 5 min and a static time of 5 min with a temperature of 100°C and a pressure of 1,500 psi. Flush volume was 50%, and purge time was 120 sec. We cut the QFFs into small pieces, mixed them with diatomaceous earth, and placed them in an 11-mL cell. Extraction of QFFs was performed in two cycles under the same conditions as the PUF extraction. The PLE extraction conditions of PUFs and QFFs were optimized in a previous study , using a capillary column Zebron ZB-5 . The gas chromatography–mass spectrometry (GC-MS) analysis was performed by injecting 1 μL extract at 270°C, in splitless mode, at a helium constant flow of 1 mL/min. The oven temperature program started at 100°C, held for 4 min, was raised to 290°C at a rate of 6°C/min, and held there for 10 min.m/z interval from 35 to 280, operating at electron impact energy of 70 eV. Qualitative identification of target PAHs was based on the match of the retention times and the ion ratios of the target quantifier and qualifier ions. We quantified the isomers BjF and BbF together because they coelute and exhibit similar mass spectra.The GC-MS interface was set at 280°C. The mass spectrometer acquired data in scan mode with an 3, limits of detection (LODs) ranged from 0.002 ng/m3 to 0.033 ng/m3, and limits of quantification (LOQs) from 0.033 ng/m3 to 0.167 ng/m3. Repeatability and reproducibility were below the 10% relative standard deviation (RSD) (n = 5) for all target compounds. For further information about the validation parameters of the analytical method, see Supplemental Material, We used the internal standard calibration method for GC-MS quantification. The standards, diluted in DCM, ranged between 0.01 and 50 mg/L with an internal standard constant concentration of 1 mg/L. We prepared all the standards on the day of use and kept them refrigerated. For an average air preconcentration of 300 mQuality assurance. Because of the ubiquitous presence of PAHs in the atmosphere, precautions to avoid sample contamination were taken throughout the entire process. In addition to the precautions mentioned previously, we followed U.S. EPA method TO-13 (U.S. EPA 1999) guidelines. Furthermore, components of the extraction cells and collecting vials were ultrasound-cleaned with isopropanol followed by DCM and dried inside a fume cupboard. All glassware, including GC syringes, was rinsed three times with isopropanol and three times with DCM before use. Field blanks, process blanks, and solvent blanks were also performed according to section 14 of method TO-13. Taking these precautions into account, no blank showed any detectable signals for the target PAHs.Toxic equivalency factors: BaP equivalency. For risk assessment calculations, we ranked the 18 target PAHs according to their cancer potency relative to BaP. Most of the TEFs used in this study were proposed by We calculated BaP equivalents (BaP-eq) by multiplying each individual PAH concentration with its corresponding TEF and determined the concentration of total PAH expressed as BaP-eq. Following U.S. EPA criteria (U.S. EPA 2000) for risk assessment calculations, we substituted half the LOD for measured values < LODs and half the LOQ value for measured values < LOQs.Lifetime lung cancer risk of PAHs in the atmosphere defined by the WHO unit risk. The estimated lifetime lung cancer risk from PAHs in the atmosphere based on the WHO unit risk (UR) (3 BaP (UR = 8.7 × 10–5) over a lifetime of 70 years.isk (UR)  is 8.7 cThe risk of developing lung cancer can thus be calculated as3) × UR. [1]Lifetime lung cancer risk =  BaP-eq  × lifetime lung cancer risk ÷ 70 (years of exposure). We defined the exposed population for this study  based on the proximity to the industrial complexes and the prevailing winds.PAH gas and particle phase values. PAHs in air can be present either in the gas phase or in the particulate phase. The phase distribution of the PAHs depends on the vapor pressure of the compound, the atmospheric temperature, the PAH concentration, the affinity of the PAH for the atmospheric suspended particles (determined by the partitioning coefficients Koc), and the nature and concentrations of the particles .3 (nitrate) radicals increase with higher temperatures. However, other factors, such as routine plant maintenance and seasonal holidays, may also influence seasonal variation in PAH distributions.Although low temperatures facilitate the condensation of volatile and semivolatile PAHs onto preexisting particles, we found that PAH concentrations in the gas phase increased more than those in the particle phase when temperatures decreased . This trIn general, the highest total PAH values were associated with the gas phase in both sampling periods, especially in winter . This trEuropean legislation only regulates some heavy PAHs  in the particulate phase . Based oSpatial distribution of PAHs. The volatile PAHs  were most abundant at all three sampling sites, especially in the gas phase, and they made a large contribution to the total PAH values . Because site 1 is a rural area, the PAH values primarily may reflect activities in the North Industrial Complex, particularly refining, which can be an important source of particulate phase bounded PAHs.3 in the high-temperature season and 67.2 ng/m3 in the low-temperature season, whereas site 2 presented the lowest average values in both phases  and in winter (1.992 ng/m3).It is worth noting that BaP-eq values for the particle phase were higher than those for the gas phase in winter in site 1 but comparable between the particle and gas phases in summer at this site, and comparable between the two phases during both seasons at site 2 . For sitLifetime lung cancer risk of 18 PAHs in the atmosphere.–4  for the summer period and 1.7 × 10–4 for the winter period. In the 2008 sampling periods, site 2 and site 3 had estimated lifetime lung cancer risks of 1.5 × 10–4 and 1.4 × 10–4, corresponding to a summer and autumn season, respectively, that are consistent with estimates for site 1. However, in the 2009 sampling periods the estimated risks for site 2 and site 3 were 2.2 × 10–5 (winter–spring) and 2.9 × 10–5 (spring), respectively, which were much lower than those estimated for 2008. These differences could reflect external factors, such as the international economic crisis that affected many industries located at the chemical site and probably caused reductions in production or shutdowns of some chemical plants (AEQT 2009), thus changing emissions.Concerning the individual toxicity of the target PAHs, the compounds that contributed most to the total estimated risk were BaP (19–45%), DahA (24–67%), and FluT . The contribution of FluT that we found mainly in the gas phase and that had high values in site 1 in winter and in site 3 in autumn was also noteworthy. Although FluT is classified as a weak carcinogen, it is mutagenic and therefore has a high TEF (0.05). BbF, BjF, BkF, Chr, and Ind also contributed to the overall risk with maximum individual contributions between 4% and 7%. Although PA had the highest average concentration of the individual PAHs at the three sampling sites, it made a small contribution to the overall risk because of its low TEF.–4  for the study area as a whole. This represents the estimated risk above the baseline lifetime lung cancer risk in the absence of atmospheric PAH exposure. Taking into account the calculated average lifetime risk and assuming a homogeneous exposure of the inhabitants  around the chemical site, 0.3 annual cases of lung cancer can be attributed to this PAH exposure. Furthermore, in the absence of major indoor PAH sources, such as tobacco smoking, cooking fumes, and residential heating, outdoor PAHs can also substantially contribute to indoor PAH levels , and higher than the 10–6 risk that is the U.S. EPA guideline .In this study, we determined 18 PAHs in 153 matched samples of atmospheric air and particulate matter from three sampling locations around a chemical site. PAH values were generally higher in low-temperature periods than in high-temperature periods, with higher individual PAH concentrations observed in the gas phase, especially for the most volatile PAHs.Risk calculations showed that the contribution of the gas-phase PAHs to the total risk should be taken into account, as well as the contribution of volatile PAHs, such as FluT, and of the heaviest PAHs, such as BaP or DahA, especially during high-temperature periods.–3 considered a definite risk according to criteria used in similar risk assessment studies. Despite uncertainties associated with the sampling methodology and QRA calculations, our findings suggest that it would be prudent to take PAH concentrations in both gas and particle phases into account in future health-risk legislation. The real risk values may otherwise be underestimated.The average estimated lifetime lung cancer risk for our study area was higher than the WHO and the U.S. EPA recommended values but lower than the threshold value of 10(164 KB) PDFClick here for additional data file.


Objective Vitamin D deficiency is an important health problem in both developed and developing countries. Recent reports on the extraskeletal effects of vitamin D have led to increased interest in prevalence studies on states of deficiency/insufficiency of vitamin D. The aim of this study was to determine the frequency of vitamin D deficiency and insufficiency in children and adolescents residing in Ankara, Turkey and to investigate the factors associated with low vitamin D status. Methods: A total of 440 children and adolescents aged between 0 and 16 years were enrolled in this study. The subjects were divided into three groups according to their vitamin D status  and also according to their age . Results: Overall, 40% of the subjects were found to have 25 hydroxy vitamin D [25(OH)D] levels of less than 20 ng/mL. The levels indicated a deficiency state in 110 subjects (25%) and insufficiency - in 66 (15%). The rate of vitamin D deficiency was higher in girls, regardless of age. 25(OH)D levels correlated negatively with age , body mass index (BMI)  and intact parathyroid hormone (iPTH) level . A positive correlation was observed between 25(OH)D and serum ferritin levels . Conclusions: The high frequency of vitamin D deficiency in childhood  indicates a need for supplementation and nutritional support. Conflict of interest:None declared. Vitamin D deficiency is an important public health problem in both developed and developing countries, with a reported worldwide prevalence of 30-80% in children and adults ,2,3. ThePrevalence studies on vitamin D deficiency and/or insufficiency in the Turkish population, especially among prepubertal children over 3 years of age, are relatively scarce . The aimThis study was undertaken by the Fatih University Pediatrics Department and conducted in the University hospital, situated in Ankara, Turkey. We retrospectively reviewed the records of a total of 440 children and adolescents aged between 0 and 16 years who had presented to the outpatient clinic between January 2008 and January 2010. All patients were subjected to a careful physical examination. Weights were measured using a calibrated digital scale. Height measurements were done in triplicate to the nearest millimeter using a calibrated stadiometer. Body mass index (BMI) were calculated according to the formula [weight (kg)/height (m)2]. Obesity was defined as the presence of a BMI above the 95th percentile for age and sex based on established percentile curves for Turkish children , phosphorus (P), magnesium (Mg), alkaline phosphatase (ALP) and glucose levels were measured using the enzymatic colorimetric method (Roche Integra 800), while serum 25 hydroxy vitamin D [25(OH)D] levels were measured by high-performance liquid chromatography (Shimadzu UFLC). Serum levels of ferritin, insulin and intact parathyroid hormone (iPTH) were evaluated using the electrochemiluminescence method (Advia Centaur XP). Insulin resistance for each patient was estimated by the homeostatic model assessment method insulin ratio (HOMA-IR) using the formula [HOMA-IR=(glucose X insulin)/405]. Vitamin D levels were estimated immediately after taking the blood sample. The same assays were used throughout the study. For evaluation of the results, the subjects were divided into three groups according to their vitamin D status   and alsoStatistical analysis was performed using SPSS 13.0 for Windows . One-way ANOVA was used in comparing variables with normal distribution. Pearson’s chi-square and Fisher’s exact tests were used for comparisons of categorical variables. Correlations between two variables were evaluated by Pearson’s correlation coefficient. Values for all parameters, except gender, were expressed as mean±standard deviation, and a p-value of less than 0.05 was considered indicative of statistical significance.We retrospectively reviewed the records of a total of 440 children and adolescents aged between 0 and 16 years of age. Overall, 40% of patients had 25(OH)D levels lower than 20 ng/mL with 110 patients (25%) having deficiency and 66 patients (15%) having insufficiency. Comparisons between age groups in terms of vitamin D levels revealed that infants had a higher mean level of 25(OH)D compared to older children, with fewer of them having insufficiency D levels . On the other hand, children with vitamin D deficiency had significantly higher BMI compared to patients with higher serum levels of 25(OH)D . CorrelaWe found a remarkably high prevalence of poor vitamin D status among the 0-16 years age group included in this sample. This finding clearly indicates that vitamin D deficiency is an important health problem in children living in Ankara. However, one weak point of our study was that the prevalence was not determined on a population basis. We believe that a nationwide study would give more accurate results on the true prevalence. Vitamin D deficiency was found to be higher in girls than in boys, especially in the adolescent age group. It was also noted that the frequency of vitamin D deficiency increased with age. Studies on adults and children have shown that vitamin D insufficiency is more prevalent among overweight and obese people ,13,14,15We observed lower deficiency rates in the younger age group in our sample. This finding is probably related to preventive measures in primary health care conducted by the Ministry of Health in Turkey. Since 2005, vitamin D supplements are distributed to every newborn throughout their infancy at no financial cost . SimilarIt has been reported that vitamin D insufficiency is more prevalent among girls ,21. In aThe limited number of studies conducted on this issue have shown a positive correlation between the levels of 25(OH)D and ferritin. Constantini et al  reportedIn conclusion, vitamin D deficiency and insufficiency were found to be highly prevalent in our study group of children aged 0-16 years. This finding is in line with the high rate of vitamin D insufficiency recorded in Turkish infants and children, especially in girls and adolescents reported recently. The insufficiency state may be caused by low sunshine exposure, skin pigmentation, air pollution, skin covering and low vitamin D intake. According to our study, vitamin D supplementation should be provided not only for infants, but also for children of all ages, including adolescents.We concluded that vitamin D deficiency and insufficiency are highly prevalent in children aged 0 to 16. In our country, a high rate of vitamin D insufficiency is recorded in all age groups of children, especially in girls and adolescents. It may be caused by low sunshine exposure, skin pigmentation, air pollution, skin covering and low vitamin D intake. According to our study, it seems that vitamin D supplement should be provided not only for infants, but also for all the children, especially for those of in adolescent period.