PCR In Blood Samples Research Articles

Utility of an in-house real-time PCR in whole blood samples as a minimally invasive method for early and accurate diagnosis of invasive mould infections.

Invasive mould infections (IMIs) are a leading cause of death in patients with compromised immune systems. Proven invasive mould infection requires detection of a fungus by histopathological analysis of a biopsied specimen, sterile culture, or fungal DNA amplification by PCR in tissue. However, the clinical performance of a PCR assay on blood samples taken from patients suspected of invasive mould disease has not been fully evaluated, particularly for the differential diagnosis of invasive aspergillosis (IA) and invasive Mucormycosis (IM). To assess the diagnostic utility of our previously validated in-house real-time PCR in blood samples for diagnosis of invasive aspergillosis and mucormycosis in patients with suspected invasive mould infection. All patients with suspected invasive mould infection were prospectively enrolled from May 2021 to July 2021. Conventional fungal diagnosis was performed using tissue and respiratory samples. In-house PCR was performed on blood samples and its diagnostic performance evaluated. A total of 158 cases of suspected invasive mould infection were enrolled in the study. The sensitivity and specificity of in-house PCR performed on blood samples was found to be 92.5% and 81.4% respectively for diagnosis of probable IA, and 65% and 84.62% respectively for diagnosis of proven and probable IM. It was also able to detect 3 out of 5 cases of possible IM where no other microbiological evidence of IM was obtained. This assay could be helpful in minimally invasive diagnosis of IMIs for patients in whom invasive sampling is not feasible, especially as a preliminary or screening test. It can help in early diagnosis, anticipating conventional laboratory confirmation by days or weeks. Possible correlation between fungal load and mortality can help in initiating aggressive treatment for patients with high initial fungal load.

Lessons from a Multilaboratorial Task Force for Diagnosis of a Fatal Toxoplasmosis Outbreak in Captive Primates in Brazil.

Toxoplasmosis is an important zoonotic disease caused by the parasite Toxoplasma gondii and is especially fatal for neotropical primates. In Brazil, the Ministry of Health is responsible for national epizootic surveillance, but some diseases are still neglected. Here, we present an integrated investigation of an outbreak that occurred during the first year of the COVID-19 pandemic among eleven neotropical primates housed at a primatology center in Brazil. After presenting non-specific clinical signs, all animals died within four days. A wide range of pathogens were evaluated, and we successfully identified T. gondii as the causative agent within four days after necropsies. The liver was the most affected organ, presenting hemorrhage and hepatocellular necrosis. Tachyzoites and bradyzoite cysts were observed in histological examinations and immunohistochemistry in different organs; in addition, parasitic DNA was detected through PCR in blood samples from all specimens evaluated. A high prevalence of Escherichia coli was also observed, indicating sepsis. This case highlights some of the obstacles faced by the current Brazilian surveillance system. A diagnosis was obtained through the integrated action of researchers since investigation for toxoplasmosis is currently absent in national guidelines. An interdisciplinary investigation could be a possible model for future epizootic investigations in animals.

Blood transcriptome sequencing identifies biomarkers able to track disease stages in spinocerebellar ataxia type 3.

Transcriptional dysregulation has been described in spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD), an autosomal dominant ataxia caused by a polyglutamine expansion in the ataxin-3 protein. As ataxin-3 is ubiquitously expressed, transcriptional alterations in blood may reflect early changes that start before clinical onset and might serve as peripheral biomarkers in clinical and research settings. Our goal was to describe enriched pathways and report dysregulated genes, which can track disease onset, severity or progression in carriers of the ATXN3 mutation (pre-ataxic subjects and patients). Global dysregulation patterns were identified by RNA sequencing of blood samples from 40 carriers of ATXN3 mutation and 20 controls and further compared with transcriptomic data from post-mortem cerebellum samples of MJD patients and controls. Ten genes-ABCA1, CEP72, PTGDS, SAFB2, SFSWAP, CCDC88C, SH2B1, LTBP4, MEG3 and TSPOAP1-whose expression in blood was altered in the pre-ataxic stage and simultaneously, correlated with ataxia severity in the overt disease stage, were analysed by quantitative real-time PCR in blood samples from an independent set of 170 SCA3/MJD subjects and 57 controls. Pathway enrichment analysis indicated the Gαi signalling and the oestrogen receptor signalling to be similarly affected in blood and cerebellum. SAFB2, SFSWAP and LTBP4 were consistently dysregulated in pre-ataxic subjects compared to controls, displaying a combined discriminatory ability of 79%. In patients, ataxia severity was associated with higher levels of MEG3 and TSPOAP1. We propose expression levels of SAFB2, SFSWAP and LTBP4 as well as MEG3 and TSPOAP1 as stratification markers of SCA3/MJD progression, deserving further validation in longitudinal studies and in independent cohorts.

The presence of malignant ovine babesiosis in Bosnia and Herzegovina indicates a possible emerging risk for Balkan region

Malignant ovine babesiosis or ovine babesiosis caused by Babesia ovis is an important hemoprotozoan tick-borne disease of sheep. After 70 years, a study was conducted in Bosnia and Herzegovina, in the geographical areas of Podrinje and Eastern Herzegovina, aiming to molecularly identify and determine the presence of B. ovis infection in the sheep population. From 2019–2021, a clinical and molecular study was conducted in 53 flocks, and 192 sheep were examined, divided into two groups: clinically suspected and asymptomatic sheep. The presence of B. ovis was confirmed by PCR in blood samples of 75 % and 11.21 % of clinically affected and asymptomatic sheep, respectively. The majority of clinical cases of malignant ovine babesiosis were confirmed in the Rudo epidemiological unit (78.78 %) within the Podrinje region indicating typical seasonal pattern of disease occurrence and endemic focus. Rhipicephalus bursa was only tick species identified in Podrinje and Eastern Herzegovina. Acute form of disease was observed with dominant clinical signs of depression, fever, loss of appetite and respiratory distress. Fatal outcome was recorded in 12.28 % of cases. This study describes the molecular detection of B. ovis in sheep in Bosnia and Herzegovina for the first time. To the authors knowledge, this survey represents a report of the highest number of clinical cases of malignant ovine babesiosis in Europe. Since the disease has been recorded in the south-eastern border of Bosnia and Herzegovina, there is a high risk of disease transmission to a wider area of the Balkan region.

Böbrek nakli yapılan hastalarda nakil öncesi ve nakil sonrası enfeksiyon etkenlerinin sıklığı

Amaç: Böbrek nakli, böbrek yetmezliğinin en önemli ve başarılı tedavi yöntemidir. Bu çalışmada, böbrek nakli alıcılarında nakil öncesi ve nakil sonrası ilk altı ayda Cytomegalovirus (CMV), BK virüs (BKV) ve bakteriyel etkenlerin sıklığının araştırılması amaçlanmıştır. 
 Gereç ve Yöntem: Fakültemiz Organ Nakli Merkezinde böbrek nakli yapılan hastalardan, nakilden bir hafta önce ve nakilden sonra birinci, üçüncü ve altıncı aylarda alınan kan örneklerinde Real-time PCR ile CMV ve BKV araştırıldı. Kan, idrar, balgam/yara (gerekiyor ise) kültürleri yapıldı. İdrar sitolojisinde Decoy hücreleri değerlendirildi. 
 Bulgular: Böbrek nakli alıcılarının yaş ortalaması 32,60±11,71 28 (% 62,2)’i erkek ve 39 (% 86,7)’u akraba olan canlı vericili, altısı kadavra vericili idi. Nakil sonrası 11/38 (% 28,9) hastada BKV, 25/41 (% 60,9) hastada CMV, 11/31 (% 35,4) hastada Decoy hücre pozitiflikleri saptandı. Real-time PCR pozitifliği en yüksek oran BKV için üçüncü ve altıncı aylarda iken, CMV için birinci ay olup altıncı aya doğru giderek azalma gösterdi. İdrar kültüründe Escherichia coli, Klebsiella pneumoniae, albicans-dışı Candida, Enterococcus faecalis, kan kültüründe Staphylococcus hominis, Streptecoccus epidermidis, solunum yolu örneklerinin kültüründe Acinetobacter baumannii, Klebsiella pneumoniae, Aspergillus fumigatus ve Candida albicans üredi..
 Sonuç: Böbrek nakli alıcılarımızda bakteriyel enfeksiyonlar erken dönemde gelişti. Real-time PCR pozitiflik oranı en yüksek BKV için üçüncü ve altıncı aylarda iken CMV için birinci ay olup altıncı aya doğru giderek azalma gösterdi. Böbrek nakil hastalarında Decoy hücre pozitifliği de BKV enfeksiyonu tanısı için önemli olabilir.

JAK2 mutation may predict response and guide first line treatment in rheumatoid arthritis

BackgroundJAK (Janus kinase) inhibitors work by inhibiting the activity of one or more of the enzyme Janus kinase with a therapeutic application for treatment of cancer and inflammatory disorders such as rheumatoid arthritis (RA). We aimed to study impact of JAK2 mutation in serum of rheumatoid arthritis patients on response to first line with conventional synthetic disease-modifying anti-rheumatic drug (csDMARDS) at 3rd month by evaluating DAS28 and ACR response criteria. The study included 85 newly diagnosed rheumatoid arthritis patients and 50 matched controls. Basal JAK2 mutation assessed by PCR in blood samples, TNF-α and IL 6 were measured by ELISA in serum of patient and control groups. All patients started therapy with csDMARDs. Response assessment at 3rd month was evaluated by DAS28 and ACR response criteria. JAK2 mutation was correlated with different clinical and laboratory parameters of patients.ResultsSeventeen females (83.5%) and 14 males (16.5%) with age mean ± SD (years); (48.7 ± 7.2). Pretreatment JAK2 mutation, TNF-α and IL 6 were significantly high in patients. JAK2 mutation was detected in 45 (52.9%) patients while 40 (47.1%) patients were JAK2 non-mutant. Mutant JAK2 was significantly linked to severity of disease evaluated by DAS28; 14 (70%) of patients with DAS28 (≤ 2.6) were non-mutant JAK2 vs sex (30%) patients mutant JAK2 while 19 (73.1%) of patients with DAS28 (> 5.1) were mutant JAK2 vs 7 (26.9%) patients non-mutant JAK2 (P 0.02). JAK2 mutation found to be significantly correlated with ACR 20, 50, and 70 response criteria; 68.2% of patients with non-mutant JAK2 showed ACR 70 vs 31.8% in mutant group, 52% of patients with non-mutant JAK2 showed ACR 50 vs 48% in mutant group while 31.6% of patients with non-mutant JAK2 showed ACR 20 vs 68.4% in mutant group (P 0.02). JAK2 mutation were more presented in young age patients (mean ± SD; 47.1 ± 7.2 vs 50.4 ± 6.9 in mutant vs non-mutant JAK2 patients, respectively with P 0.03). JAK2 mutation was associated with high pretreatment TNFα and IL6 level in serum. Mean ± SD of TNFα; 49.4 ± 41.9 in mutant vs 26 ± 24.4 pg/ml in non-mutant group, with P (0.003) while mean ± SD of IL6; 83.5 ± 56.8 in mutant vs 47 ± 46.9 pg/ml in non-mutant group, with P (0.002).ConclusionsAdult RA with pretreatment JAK2 mutation significantly showed high disease activity and high pretreatment TNFα and IL6 levels. Patients with JAK2 mutation found to be linked to poor response to 1st line csDMARDs including MTX so they could get more benefit with early introduction of JAK inhibitors as first line monotherapy or when combined with csDMARDS especially those with moderate to severe active RA.Trial registrationInstitutional Research Board (IRB)-Faculty of Medicine: IRB Proposal Code: R.20.11.1075-2020/11/16. Clinicaltrials.gov registration date: 8/12/2020, code: NCT04667988.

The expression and prognostic value of miR-146a and miR-155 in Turkish patients with multiple sclerosis

ABSTRACT Multiple sclerosis (MS) is an inflammatory, autoimmune demyelinating, and neurodegenerative disorder of the central nervous system. Interactions between environmental factors, predisposition genes, and determining genes appear to be involved in its etiology. Epigenetic mechanisms such as microRNA-mediated gene regulation can determine the susceptibility and severity of autoimmune diseases. Therefore, to determine the role of miR-146a and miR-155 in MS and its developmental stages, the expression levels in the serum of MS and clinically isolated syndrome (CIS) patients were compared with those of healthy controls. In the present study, the expression levels of miR-146a and miR-155 were assessed using quantitative Real-Time PCR in blood samples of 15 CIS patients and 61 relapsing-remitting multiple sclerosis (RRMS) patients alongside 32 healthy patients as controls. Furthermore, any associations with the clinicopathologic variables of the patients were also evaluated. Dysregulations were found only in the miR-146a and miR-155 expressions in the RRMS-Control group. When the RRMS patients were evaluated in terms of the characteristics of sex, annual attack rate, age of diagnosis, duration of follow-up, and immunomodulatory treatments used, no significant differences were observed. However, significant dysregulations were identified in miRNA expression in the vitamin D level, EDSS values, and the number of attacks. ROC curve analysis showed that miR-146a and miR-155 were significant in the RRMS-Control group for the area under the curve (AUC). It is possible that miR-146a may be associated with vitamin D deficiency and disease disability, while miR-155 may be associated with the number of attacks.

Low prevalence of SARS-CoV-2 in plasma of COVID-19 patients presenting to the emergency department.

Correct and reliable identification of SARS-CoV-2 in COVID-19 suspected patients is essential for diagnosis. Respiratory samples should always be tested with real-time PCR for SARS-CoV-2. In addition, blood samples have been tested, but without consistent results and therefore the added value of this sample type is unknown. The aim of this study was to determine the prevalence of SARS-CoV-2 by real-time PCR in blood samples obtained from PCR-proven COVID-19 patients and in addition to elaborate on the potential use of blood for diagnostics. In this single center study, blood samples drawn from patients at the emergency department with proven COVID-19 infection based on a positive SARS-CoV-2 PCR in respiratory samples were tested for the presence of SARS-CoV-2. Samples from 118 patients were selected, of which 102 could be included in the study (median age was 65 (IQR 10), 65.7 % men). In six (5.9 %) of the tested samples, SARS-CoV-2 was identified by real-time PCR. In conclusion, SARS-CoV-2 can be detected by real-time PCR in plasma samples from patients with proven COVID-19, but only in a minority of the patients. Plasma should therefore not be used as primary sample in an acute phase setting to identify SARS-CoV-2 infection. These findings are important to complete the knowledge on possible sample types to test to diagnose COVID-19.

Mitochondrial DNA Copy Number in Peripheral Blood as a Potential Non-invasive Biomarker for Multiple Sclerosis.

The impaired mitochondrial function has been implicated in the pathogenicity of multiple sclerosis(MS), a chronicinflammatory, demyelinating,andneurodegenerative disease of the CNS. Circulating mtDNA copy number in body fluids has been proposed as an indicator for several neurodegenerative diseases, and the altered cerebrospinal fluid mtDNA has been shown as a promising marker for MS. The aim of this study was to determine changes and biomarker potential of circulating mtDNA in peripheral blood in MS. The mtDNA copy number was quantified by real-time PCR in blood samples from 60 patients with relapsing-remitting MS (RRMS) and 64 healthy controls. The RRMS patients had significantly lower circulating mtDNA copy number compared to controls. Subgroup analysis with stratification of RRMS patients based on disease duration under or over 10years revealed that the mtDNA copy number was significantly lower in the group with longer disease duration. A negative correlation was observed between mtDNA copy number and disease duration. The ROC curve analysis indicated a significant ability of mtDNA copy number to separate RRMS patients from controls with an AUC of 0.859. This is the first study to measure peripheral blood mtDNA copy number in MS patients. Current data suggest that the reduction in peripheral blood mtDNA copy number may be an early event in MS and correlate with the disease progression. The findings of this study indicate that circulating blood-based mtDNA copy number may be a potential non-invasive candidate biomarker for mitochondria-mediated neurodegeneration and MS. This can put forward the clinical applicability over other invasive markers.

Increase in Streptococcus pneumoniae serotype 3 associated parapneumonic pleural effusion/empyema after the introduction of PCV13 in Germany

IntroductionPediatric pneumococcal pneumonia complicated by parapneumonic pleural effusion/empyema (PPE/PE) remains a major concern despite general immunization with pneumococcal conjugate vaccines (PCVs). MethodsIn a nationwide pediatric hospital surveillance study in Germany we identified 584 children <18 years of age with bacteriologically confirmed PPE/PE from October 2010 to June 2018. Streptococcus pneumoniae was identified by culture and/or PCR of blood samples and/or pleural fluid and serotyped. ResultsS. pneumoniae was identified in 256 of 584 (43.8%) children by culture (n = 122) and/or PCR (n = 207). The following pneumococcal serotypes were detected in 114 children: serotype 3 (42.1%), 1 (25.4%), 7F (12.3%), 19A (7.9%), other PCV13 serotypes (4.4%) and non-PCV13 serotypes (7.9%). Between October 2010 and June 2014 serotype 1 (38.1%) and serotype 3 (25.4%) were most prevalent, whereas between July 2014 and June 2018 serotype 3 (62.7%) and non-PCV13 serotypes (15.7%) were dominant. Compared to children with other pneumococcal serotypes, children with serotype 3 associated PPE/PE were younger (median 3.2 years [IQR 2.1–4.3 years] vs. median 5.6 years [IQR 3.8–8.2 years]; p < 0.001) and more frequently admitted to intensive care (43 [89.6%] vs. 48 [73.8%]; p = 0.04). Seventy-six of 114 (66.7%) children with pneumococcal PPE/PE had been vaccinated with pneumococcal vaccines. Thirty-nine of 76 (51.3%) had received a vaccine covering the serotype detected. Thirty of these 39 breakthrough cases were age-appropriately vaccinated with PCV13 and considered vaccine failures, including 26 children with serotype 3, three children with serotype 19A and one child with serotype 1. ConclusionFollowing the introduction of PCV13 in general childhood vaccination we observed a strong emergence of serotype 3 associated PPE/PE in the German pediatric population, including a considerable number of younger children with serotype 3 vaccine breakthrough cases and failures. Future PCVs should not only cover newly emerging serotypes, but also include a more effective component against serotype 3.

Human infection with Plasmodium knowlesi on the Laos-Vietnam border

BackgroundBorder malaria in the Greater Mekong region of Southeast Asia poses a serious threat to the health of the ethnic minority populations of the region. Traditionally thought to be caused primarily by the malaria parasites Plasmodium falciparum and Plasmodium vivax, recently a zoonotic parasite, Plasmodium knowlesi, has been identified in some countries of the region. The presence of this parasite poses a challenge to malaria control programmes, as it is maintained in a zoonotic reservoir of forest-dwelling macaque monkeys.MethodsA cross-sectional malaria parasite species prevalence survey was conducted along the Laos-Vietnam border in the central part of the two countries. Human blood samples were collected from Savannakhet in Laos and Quang Tri in Vietnam between August and October 2010 and assayed for the presence of human malaria parasite species and P. knowlesi. A PCR targeting the 18S small subunit ribosomal RNA gene and circumsporozoite protein gene was used for Plasmodium species identification.ResultsNine cases of P. knowlesi were detected by PCR in blood samples from the Laos side and three from the Vietnam side. All P. knowlesi infections were found in co-infection with P. vivax, with some triple infections of P. knowlesi, P. vivax and P. falciparum detected in Laos. Phylogenetic analysis of these parasites suggests that P. knowlesi is circulating in the Laos-Vietnam border region.ConclusionThis report shows that P. knowlesi is transmited on both sides of the Vietnam-Laos border. Continued monitoring of the range and prevalence of P. knowlesi on both the sides of Laos-Vietnam border is of importance to the National Malaria Control Programmes of both countries.

Soluble HLA-G Expression Inversely Correlates With Fetal Microchimerism Levels in Peripheral Blood From Women With Scleroderma.

Women with scleroderma (SSc) maintain significantly higher quantities of persisting fetal microchimerism (FMc) from complete or incomplete pregnancies in their peripheral blood compared to healthy women. The non-classical class-I human leukocyte antigen (HLA) molecule HLA-G plays a pivotal role for the implantation and maintenance of pregnancy and has often been investigated in offspring from women with pregnancy complications. However data show that maternal HLA-G polymorphisms as well as maternal soluble HLA-G (sHLA-G) expression could influence pregnancy outcome. Here, we aimed to investigate the underlying role of maternal sHLA-G expression and HLA-G polymorphisms on the persistence of FMc. We measured sHLA-G levels by enzyme linked immunosorbent assay in plasma samples from 88 healthy women and 74 women with SSc. Male Mc was quantified by DYS14 real-time PCR in blood samples from 58 women who had previously given birth to at least one male child. Furthermore, eight HLA-G 5′URR/3′UTR polymorphisms, previously described as influencing HLA-G expression, were performed on DNA samples from 96 healthy women and 106 women with SSc. Peripheral sHLA-G was at lower concentration in plasma from SSc (76.2 ± 48.3 IU/mL) compared to healthy women (117.5 ± 60.1 IU/mL, p < 0.0001), independently of clinical subtypes, autoantibody profiles, disease duration, or treatments. Moreover, sHLA-G levels were inversely correlated to FMc quantities (Spearman correlation, p < 0.01). Finally, women with SSc had lower sHLA-G independently of the eight HLA-G 5′URR/3′UTR polymorphisms, although they were statistically more often homozygous than heterozygous for HLA-G polymorphism genotypes −716 (G/T), −201 (G/A), 14 bp (ins/del), and +3,142 (G/A) than healthy women. In conclusion, women with SSc display less sHLA-G expression independently of the eight HLA-G polymorphisms tested. This decreased production correlates with higher quantities of persisting FMc commonly observed in blood from SSc women. These results shed some lights on the contribution of the maternal HLA-G protein to long-term persistent fetal Mc and initiate new perspectives in this field.

Body mass index and leukocyte telomere length dynamics among older adults: Results from the ESTHER cohort

ObjectiveTelomere length (TL) has been proposed as a biomarker of ageing, which might be used to identify individuals at higher risk of age-related diseases. Obesity is a well-known risk factor for several diseases. This study aims to analyse the associations of BMI with TL and the rate of TL change in older adults. MethodsLeukocyte TL (LTL) was measured by quantitative PCR in blood samples of 3600 older adults aged 50–75years obtained at the baseline examination of a population-based cohort study in Germany. For longitudinal analyses, measurements were repeated in blood samples obtained at 8-year follow-up from 1000 participants. Multivariate linear regression models were used to estimate associations of BMI with LTL and changes in LTL over time. ResultsLTL was inversely associated with age (r=−0.090, p<0.0001). BMI and LTL associations varied according to age (p for interaction=0.021). BMI was significantly inversely associated with LTL in those younger than 60years (−6basepairs per 1kg/m2 difference in BMI). In particular, weight gain during adulthood was inversely associated with LTL in a dose–response manner in this age group, with those having gained ≥30kg having significantly shorter telomeres (−209basepairs) than those who maintained their weight. No clear patterns were observed between any of BMI-related variables and the rate of LTL change. ConclusionsOur cross-sectional analysis supports suggestions that weight gain during adulthood and obesity may contribute to shorter telomere length below 60years of age, but this relationship could not be shown longitudinally.

Effects of cigarette smoking and nicotine metabolite ratio on leukocyte telomere length.

Studies of the effects of smoking on leukocyte telomere length (LTL) using cigarettes smoked per day or pack years smoked (PYS) present limitations. Reported high levels of smoking may not increase toxin exposure levels proportionally. Nicotine metabolism ratio (NMR) predicts total cigarette puff volume and overall exposure based on total N-nitrosamines, is highly reproducible and independent of time since the last cigarette. We hypothesized that smokers with higher NMRs will exhibit increased total puff volume, reflecting efforts to extract more nicotine from their cigarettes and increasing toxin exposure. In addition, higher levels of smoking could cause a gross damage in LTL. The urinary cotinine, 3-OH cotinine and nicotine levels of 147 smokers were analyzed using a LC/MS system Triple-Q6410. LTL and CYP2A6 genotype was determined by PCR in blood samples. We found a significant association between NMR and CYP2A6 genotype. Reduction in LTL was seen in relation to accumulated tobacco consumption and years smoking when we adjusted for age and gender. However, there were no significant differences between NMR values and LTL. In our study the higher exposure was associated with lower number of PYS. Smokers with reduced cigarette consumption may exhibit compensatory smoking behavior that results in no reduced tobacco toxin exposure. Our results suggest that lifetime accumulated smoking exposure could cause a gross damage in LTL rather than NMR or PYS. Nevertheless, a combination of smoking topography (NMR) and consumption (PYS) measures may provide useful information about smoking effects on health outcomes.

Evaluation of Hs-CRP Levels and Interleukin 18 (-137G/C) Promoter Polymorphism in Risk Prediction of Coronary Artery Disease in First Degree Relatives

BackgroundCoronary Artery Disease (CAD) is clearly a multifactorial disease that develops from childhood and ultimately leads to death. Several reports revealed having a First Degree Relatives (FDRS) with premature CAD is a significant autonomous risk factor for CAD development. C - reactive protein (CRP) is a member of the pentraxin family and is the most widely studied proinflammatory biomarker. IL-18 is a pleiotrophic and proinflammatory cytokine which is produced mainly by macrophages and plays an important role in the inflammatory cascade.Methods and ResultsHs-CRP levels were estimated by ELISA and Genotyping of IL-18 gene variant located on promoter -137 (G/C) by Allele specific PCR in blood samples of 300 CAD patients and 300 controls and 100 FDRS. Promoter Binding sites and Protein interacting partners were identified by Alibaba 2.1 and Genemania online tools respectively. Hs-CRP levels were significantly high in CAD patients followed by FDRS when compared to controls. In IL-18 -137 (G/C) polymorphism homozygous GG is significantly associated with occurrence of CAD and Hs-CRP levels were significantly higher in GG genotype subjects when compared to GC and CC. IL-18 was found to be interacting with 100 protein interactants.ConclusionOur results indicate that Hs-CRP levels and IL-18-137(G/C) polymorphism may help to identify risk of future events of CAD in asymptomatic healthy FDRS.

Anti-Apoptotic Pathways in Bone Marrow and Megakaryocytes in Myeloproliferative Neoplasia

Objective: Proliferative activity contributes to bone marrow cellularity in myeloproliferative neoplasia (MPN). Megakaryocytes are the most important cells in MPN bone marrow pathology. JAK2^V617F mutation constitutively activates JAK2, pErk (phosphorylating extracellular signal-regulated kinase) and PI3K (phosphatidylinositol 3-kinase)-Akt signaling. Erk is involved in megakaryocyte differentiation, PI3K-Akt inhibits megakaryocyte apoptosis via Bcl-x_L and two downstream effectors (p70S6k and Bnip3). Immunohistochemic expression of phosphorylated Erk, Akt, p70S6k and Bnip3 was studied along with microvessel density (MVD) in MPN bone marrow and megakaryocytes. Methods: 36 essential thrombocythemia (ET), 25 polycythemia vera and 45 primary myelofibrosis patients were analyzed for pErk, pAkt, Bnip3, p70S6k and MVD expression by immunostaining bone marrow biopsy sections followed by automated image analysis. JAK2^V617F was analyzed through real-time PCR in blood samples. Results: pErk and pAkt were significantly higher expressed in MPN megakaryocytes, mainly in ET patients, compared to controls. Bnip3 was higher expressed in bone marrow of control patients and in MPN megakaryocytes. Mainly in ET patients, MPN megakaryocytes showed higher p70S6k expression compared to controls. Conclusion: Increased bone marrow cellularity in MPN patients might be influenced by increased pErk, pAkt and decreased Bnip3 expression. A dominant role for megakaryocytes in ET patients was shown. Increased amounts of megakaryocytes in MPN patients can be due to increased pAkt and p70S6k.

Telomere Shortening At Diagnosis Of Chronic Phase Chronic Myelogenous Leukemia (CML-CP) Is Significantly More Pronounced In Patients With Less-Than-Optimal Versus Optimal Molecular Response (ELN criteria) After 12 Months Of Treatment With Nilotinib

IntroductionChronic myeloid leukemia (CML) is a clonal stem cell disorder characterized by the BCR-ABL translocation. Telomere length (TL) reflects the replicative history of eukaryotic cells and progressive telomere shortening is associated with genetic instability. Clinically, accelerated telomere shortening has been demonstrated in patients with CML and was found to correlate with disease progression and clinical risk score in the pre-tyrosine kinase inhibitor (TKI) era. The aim of the current study was to investigate whether telomere length (TL) at diagnosis might predict response to treatment in patients receiving nilotinib as first line treatment of chronic phase CML on the ENEST1st study (NCT01061177) Methods and PatientsTL analysis of peripheral blood leukocytes was analyzed using monochrome multiplex quantitative PCR in blood samples from 93 newly diagnosed CML patients enrolled on study in Germany. One extreme outlier was excluded from the analysis. 89 healthy controls were used for age-adaption of TL. Median age of the analyzed CML patients was 49.6 years (range: 19-83), Sokal (32 low, 31 intermediate, 13 high risk) and Euro (34 low, 36 intermediate, 6 high risk) scores were available in 76 patients. Response to treatment according to standard criteria was available at 3 month (mo, n=79 patients), 6 mo (n=75), 12 mo (n=71) and 18 mo (n=55) after treatment start. ResultsMean age-adjusted TL in CML patients was significantly shortened compared to normal individuals (ΔT/S ratio: -0.30 +/- 0.67, p=<0.001). Interestingly, whereas TL followed an expected linear decline over time in the control population of healthy individuals, this age-correlation was no longer detectable in CML patients pointing to a significantly more pronounced TL deficit in younger as opposed to older patients with CML. In univariate analysis, no significant correlation between age-adjusted TL at diagnosis and Euro or Sokal risk score nor with any standard individual prognostic parameters was detected with the exception of the peripheral blast count which was shown to be inversely correlated with age adjusted telomere (ΔT/S ratio) length by linear regression (p=0.01).When ΔT/S ratio measured at diagnosis was correlated with response to treatment at defined time points according to the ELN criteria 2013 (i.e. <10% BCR-ABL/ABL ratio at 3 mo, <1% at 6 mo, and <0.1% at 12 mo), we could demonstrate that while all cohorts had shortened telomere length compared to age-adjusted controls, less-than-optimal responders had more accelerated telomere shortening compared to optimal responders, i.e. ΔT/S -0.66 (n=4) vs. -0.25 (n=75) at 3 mo; ΔT/S -0.37 (n=10) vs. -0.28 (n=65) at 6 mo and ΔT/S -0.67 (n=15) vs. -0.21 (n=56) at 12 mo. While this pattern seems rather consistent, only the difference at the 12 mo time point reached statistical significance (p=0.028), potentially due to the imbalance of the groups induced by the low number of less-than-optimal responders to treatment according to ELN criteria observed under nilotinib first line treatment. ConclusionsThese data were generated on the first prospective study of the role of telomere length as a potential biomarker for TKI treatment in CML. We could confirm that TL is significantly shortened in CML patients at diagnosis. Even more so, telomere shortening seems to be significantly more accelerated in younger as opposed to older patients pointing to a threshold value of telomere length in CML similar to observations in patients with bone marrow failure syndromes. Furthermore, telomere shortening tends to be more pronounced in patients not meeting the criteria for optimal response according to ELN 2013. Further follow up will reveal whether TL has the potential to serve as a long-term predictive biomarker for frequency and durability of response as well as for the sustainability of treatment cessation in CML patients responding well to TKI treatment. Disclosures:Frank:Novartis: Employment. Walasek:Novartis: Employment. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Giles:Novartis: Consultancy, Research Funding. Koschmieder:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Brümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Bristol Myer Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Ariad: Consultancy.

Analysis of association between dopamine receptor genes’ methylation and their expression profile with the risk of schizophrenia

Schizophrenia (SCZ) is a type of psychotic disorder that affects ~1% of the population. Dopamine is one of the major neurotransmitters in the brain and its receptors are associated with a number of psychotic disorders, including SCZ. The aims of the present study were to analyze methylation and the expression profile of dopamine receptor DRD1, DRD2, DRD4, and DRD5 genes in patients with SCZ. Promoter methylation of DRD1, DRD2, DRD4, and DRD5 genes was assayed by a methylation-specific PCR in blood samples obtained from 80 SCZ cases and 71 healthy controls. Also, we investigated DRD1, DRD2, DRD4, and DRD5 mRNA levels using real-time reverse transcription PCR in 34 blood samples of healthy controls and cases. Promoter methylation of DRD4, DRD5, and DRD2 genes was statistically different in cases compared with healthy controls. The mRNA expression level results also showed statistically significant differences (P<0.0001) between cases and healthy controls for the DRD2, DRD4, and DRD5 genes, but not for DRD1. Analyses of DRD genes' methylation have highlighted the fact that the DRD gene network, overall, is actively involved in the increased risk of SCZ.

Epidemiological Studies ,Molecular Diagnosis of Anaplasma Marginale in Cattle and Biochemical Changes Associated with it in Kaliobia Governorate

This investigation is performed on 100 cattle in Kaliobia governorate Egypt aged from 1- 6years severity of illness increase with age , these animals suffered from fever (41°C) enlargement lymph node and drop in milk yield emaciation in progressive stages , cattle producers first notice the anemic anaplasmosis – infected animal when it becomes weak and lag behind the herd when these animals were subjected to microscopic examination the degree of parasitaema was recorded as the percentage of infected red blood cells in each blood smear 100 microscopic field wear examined .We report the detection of anaplasma marginale by PCR in blood samples obtained from cattle supposed to be infected. The assay employs primers specific for the gene encoding anaplasma marginale specific PCR using primers derived from msp5 gene .The PCR products for 26 positive samples were subjected to sequence (Labtechnology, Egypt) and BLAST analysis was used for identification of the genomic DNA of these parasites.<b> </b>changes associated with anaplasma marginale in these cattle particular emphasis to the oxidative stress the reduce TAC level may reflect adecrease in antioxidant capacity and CBC change . Blood collected from all animals on EDTA to microscopic examination and PCR to<b> </b>determine type of<b> </b>anaplasma<b>.</b>

Utilidad de la determinación sérica de microRNA-21 en la enfermedad inflamatoria del colon

MicroRNAs are 20-22 nucleotide molecular structures with post-transcriptional activity that are involved in the immune response, as well as in the inflammatory pathways of different cells and tissues. We present herein a prospective study in which serum microRNA-21 expression was determined in patients diagnosed with acute appendicitis as a model of bowel inflammation. A prospective cohort study of patients diagnosed with acute appendicitis was conducted. Serum microRNA-21 was analyzed through the PCR of blood samples taken from the patients prior to surgery. MicroRNA-21 values were compared with the analytic variables (leukocytes, hemoglobin, hematocrit, platelets, prothrombin activity, glucose, urea, and creatinine) and the anatomopathologic variables (normal appendix, phlegmonous, gangrenous, and perforated acute appendicitis). A total of 60 patients with acute appendicitis diagnosis were consecutively included in the study from June to October 2009. Sixty-six percent of the patients were men (40 men and 20 women), with a mean age of 26.2±14.8 years. The mean absolute level of microRNA-21 was 24.8±0.93, whereas the mean microRNA-21 gene expression was 1.04±0.28. No correlation between the analytic and anatomopathologic parameters evaluated was observed (P=.47). It is necessary to continue to search for the most appropriate microRNAs, so that their determination in serum can lead to greater precision in establishing the diagnosis and outcome of inflammatory disorders of the bowel.