The role of real-time PCR testing in the investigation of paediatric patients with community-onset osteomyelitis and septic arthritis

2019 ◽  
Vol 188 (4) ◽  
pp. 1289-1295
Author(s):  
Sadhbh O’Rourke ◽  
Mary Meehan ◽  
Désirée Bennett ◽  
Nicola O’Sullivan ◽  
Robert Cunney ◽  
...  
Keyword(s):  
Septic Arthritis ◽  
Real Time ◽  
Real Time Pcr ◽  
Paediatric Patients ◽  
Community Onset

Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽  
2007 ◽  
Vol 2 (3) ◽  
pp. 271-279 ◽  
Author(s):  
Koray Ergunay ◽  
Gulcin Altinok ◽  
Bora Gurel ◽  
Ahmet Pinar ◽  
Arzu Sungur ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
San Francisco ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Parvovirus B19 ◽  
Etiologic Agent ◽  
Hydrops Fetalis ◽  
Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

scholarly journals A Proof of Concept of the Role of TDM-Based Clinical Pharmacological Advices in Optimizing Antimicrobial Therapy on Real-Time in Different Paediatric Settings

2021 ◽  
Vol 12 ◽  
Author(s):  
Milo Gatti ◽  
Pier Giorgio Cojutti ◽  
Caterina Campoli ◽  
Fabio Caramelli ◽  
Luigi Tommaso Corvaglia ◽  
...  
Keyword(s):  
Drug Interactions ◽  
Real Time ◽  
Antimicrobial Therapy ◽  
Turnaround Time ◽  
Antimicrobial Treatment ◽  
Therapeutic Drug ◽  
Proof Of Concept ◽  
Paediatric Patients ◽  
Dosing Regimens

Introduction: Antimicrobial treatment is quite common among hospitalized children. The dynamic age-associated physiological variations coupled with the pathophysiological alterations caused by underlying illness and potential drug-drug interactions makes the implementation of appropriate antimicrobial dosing extremely challenging among paediatrics. Therapeutic drug monitoring (TDM) may represent a valuable tool for assisting clinicians in optimizing antimicrobial exposure. Clinical pharmacological advice (CPA) is an approach based on the correct interpretation of the TDM result by the MD Clinical Pharmacologist in relation to specific underlying conditions, namely the antimicrobial susceptibility of the clinical isolate, the site of infection, the pathophysiological characteristics of the patient and/or the drug-drug interactions of cotreatments. The aim of this study was to assess the role of TDM-based CPAs in providing useful recommendations for the real-time personalization of antimicrobial dosing regimens in various paediatric settings.Materials and methods: Paediatric patients who were admitted to different settings of the IRCCS Azienda Ospedaliero-Universitaria of Bologna, Italy (paediatric intensive care unit [ICU], paediatric onco-haematology, neonatology, and emergency paediatric ward), between January 2021 and June 2021 and who received TDM-based CPAs on real-time for personalization of antimicrobial therapy were retrospectively assessed. Demographic and clinical features, CPAs delivered in relation to different settings and antimicrobials, and type of dosing adjustments were extracted. Two indicators of performance were identified. The number of dosing adjustments provided over the total number of delivered CPAs. The turnaround time (TAT) of CPAs according to a predefined scale (optimal, <12 h; quasi-optimal, between 12–24 h; acceptable, between 24–48 h; suboptimal, >48 h).Results: Overall, 247 CPAs were delivered to 53 paediatric patients (mean 4.7 ± 3.7 CPAs/patient). Most were delivered to onco-haematological patients (39.6%) and to ICU patients (35.8%), and concerned mainly isavuconazole (19.0%) and voriconazole (17.8%). Overall, CPAs suggested dosing adjustments in 37.7% of cases (24.3% increases and 13.4% decreases). Median TAT was 7.5 h (IQR 6.1–8.8 h). Overall, CPAs TAT was optimal in 91.5% of cases, and suboptimal in only 0.8% of cases.Discussion: Our study provides a proof of concept of the helpful role that TDM-based real-time CPAs may have in optimizing antimicrobial exposure in different challenging paediatric scenarios.

P5374Genetic deletion of IL-22 increased cardiac rupture after myocardial infarction in mice

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
M Yamamoto ◽  
H Yasukawa ◽  
J Takahashi ◽  
S Nohara ◽  
T Sasak ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Real Time ◽  
Real Time Pcr ◽  
Tissue Repair ◽  
Repair Process ◽  
Cardiac Rupture ◽  
Blue Dye ◽  
Border Area ◽  
Infarct Area

Abstract Background Interleukin-22 (IL-22) is a member of the IL-10 cytokine family, which mainly targets epithelial cells and does not target immune cells. Recently, it has been reported that IL-22 play roles in tissue repair in the skin and the liver; however, role of IL-22 in the process of tissue repair after myocardial infarction (MI) is unknown. Here, we investigated the role of IL-22 in tissue repair process after MI. Methods and results First, we examined the expression of IL-22 and its receptor IL-22RA1 in the wild type (WT) mice by real-time PCR. The expression of IL-22 and IL-22RA1 in the hearts were significantly increased 3 days after MI (p<0.05). To clarify the role of IL-22 in the heart after MI, we produced MI model in the WT mice and IL-22 knockout (KO) mice. We found that the IL-22 KO mice had significantly higher mortality than the WT mice after MI (p<0.05). Approximately 80% of the IL-22 KO mice died with cardiac rupture after MI. The infarct size which was estimated by evans blue dye and triphenyltetrazolium chloride staining at 3 days after MI was comparable between the IL-22 KO mice and the WT mice. Next, we performed real time PCR and PCR array analysis for tissue fibrosis and repair genes. We found that alpha-smooth muscle actin (aSMA), NF-kB, TNF-a and MMP13 (also known as collagenase-3) were significantly increased in the infarct area of IL-22 KO mice compared to WT mice. Immunostaining showed that the myofibroblast marker aSMA positive cells in the border area after MI were markedly higher in the IL-22 KO mice compared with the WT mice (p<0.05). Approximately 70% of cardiac rupture after MI in the IL-22 KO mice were occurred in the infarct area adjacent to the border area. Furthermore, we found aSMA positive cells and MMP13 positive cells around the ruptured site of the heart. Conclusion Thus, IL-22 KO mice exhibit high mortality and increased cardiac rupture after MI. And expression of aSMA and MMP13 were highly expressed in the ruptured site after MI in the IL-22 KO mice. These results suggest that IL-22 may play an important role in the tissue repair process after MI.

128 THE ROLE OF ENDOTHELINS IN REGULATING BOVINE GRANULOSA CELLS STEROIDOGENESIS

2016 ◽  
Vol 28 (2) ◽  
pp. 194 ◽  
Author(s):  
L. F. Schütz ◽  
J. E. Ervin ◽  
L. Zhang ◽  
C. Robinson ◽  
M. Totty ◽  
...  
Keyword(s):  
Growth Factor ◽  
Real Time ◽  
Granulosa Cells ◽  
Real Time Pcr ◽  
Linear Models ◽  
Rna Extraction ◽  
Insulin Like Growth Factor ◽  
Steroid Production ◽  
Oestradiol Production

Endothelins are a group of vasoactive 21 amino acid peptides reported to play roles in steroidogenesis, folliculogenesis, and ovulation (Bridges et al. 2012 Life Sci. 91, 501–506). Nevertheless, the role of endothelins in regulating steroidogenesis in the bovine species requires further investigation. Thus, the objective of this study was to investigate the effects of endothelin 1 (ET-1) and endothelin 2 (ET-2) on bovine granulosa cell (GC) steroidogenesis. Bovine ovaries were obtained from a local abattoir. Follicular fluid was aspirated from small (1–5 mm) follicles and GC were isolated and exposed to various treatments (ET-1, ET-2, or ET-1 plus ET-2 with FSH and with or without insulin-like growth factor-1). In replicated experiments, culture medium was removed and analysed for steroid production via radioimmunoassay. Granulosa cells were either harvested with trypsin and counted using a Coulter Counter or collected with Trizol for RNA extraction and quantification via real-time PCR (18S rRNA was used as a housekeeping gene). Steroid production was expressed as nanograms (in the case of progesterone) and picograms (in the case of oestradiol) per 105 cells per 24 h. Relative quantity of target gene mRNA was expressed as 2–ΔΔCt using the relative comparative threshold cycle (Ct) method. Data were analysed via ANOVA and the general linear models (GLM) procedure of SAS for Windows (SAS Institute Inc., Cary, NC). If a significant main effect was identified, differences among means were determined by Fisher’s protected least significant differences test. The values were reported as least squares means ± standard error of the mean. In the presence of insulin-like growth factor-1, ET-1 significantly inhibited oestradiol production at 300 ng mL–1 (100.30 ± 11.05; P < 0.05), but not at 30 ng mL–1 (114.47 ± 11.05; P > 0.05) in comparison to the control (141.21 ± 11.05), whereas no differences were observed for progesterone production at 300 ng mL–1 (60.11 ± 7.11; P > 0.05) or at 30 ng mL–1 (64.02 ± 7.11; P > 0.05) in comparison to control (76.75 ± 7.11). ET-2 also significantly inhibited oestradiol production at 300 ng mL–1 (91.08 ± 11.87; P < 0.01), but not at 30 ng mL–1 (112.77 ± 11.87; P > 0.05) in comparison to the control in the presence of insulin-like growth factor-1. No significant effect of ET-1 and ET-2 was observed on steroidogenesis of granulosa cells cultured without insulin-like growth factor-1. Consistent with steroids production data, real-time PCR results indicated that, in the presence of IGF-1, ET-1 (5.66 ± 1.05) and ET-2 (5.65 ± 1.05) inhibited (P < 0.05) aromatase gene expression compared to controls (11.33 + 1.05), and ET-1 plus ET-2 (2.42 ± 1.05) reduced (P < 0.05) expression below that observed with either alone. No effect of ET-1 (4.38 ± 0.95; P > 0.05), ET-2 (5.94 ± 0.95; P > 0.05), or ET-1 plus ET-2 (4.57 ± 0.95; P > 0.05) was observed for side-chain cleavage enzyme (CYP11A1) in comparison to controls (4.4 ± 1.07). Altogether, these results indicate that endothelins are involved in the regulation of steroidogenesis of bovine GC.

scholarly journals Role of real time PCR and specific IgG in identifying patients with Aspergillus colonisation

2010 ◽  
Vol 9 ◽  
pp. S35
Author(s):  
C.G. Baxter ◽  
A.M. Jones ◽  
K. Webb ◽  
A. Moody ◽  
S. Follett ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Specific Igg

HOXB7 Overexpression in Mesenchymal Cells Stimulates the Production of Pro-Angiogenic Molecules: Potential Role in Multiple Myeloma Associated Angiogenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2743-2743
Author(s):  
Simona Colla ◽  
Nicola Giuliani ◽  
Paola Storti ◽  
Mirca Lazzaretti ◽  
Katia Todoerti ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Cell Line ◽  
Real Time ◽  
Real Time Pcr ◽  
Potential Role ◽  
Angiogenic Switch ◽  
Bone Biopsies

Abstract Bone marrow (BM) neo-angiogenesis has a critical role in multiple myeloma (MM) progression. It is well established that the angiogenic process in MM is mainly due to an overproduction of pro-angiogenic molecules by MM cells and the BM microenvironment cells. However the molecular mechanisms at the basis of the angiogenic process in MM are currently under investigation. The deregulation of the homeobox genes has been previously associated to tumor progression and neoangiogenesis. Particularly, overexpression of the homeobox HOXB7 is critical in tumor-associated angiogenic switch in solid tumors as breast cancer. Actually the potential role of HOXB7 in MM-induced angiogenesis is not known. In this study we have investigated the expression of HOXB7 by MM and BM microenvironment cells and its potential role in the regulation of the angiogenic process. First, by microarray analysis in a large database of MM patients (n°= 132) we found that HOXB7 was overexpressed by MM cells in about 10% of patients as compared to healthy donors and MGUS subjects. On the other hand HOXB7 mRNA was expressed in 18 out of 23 human myeloma cell lines tested. Moreover, we found that isolated BM mesenchymal (MSC) and osteoblastic (OB) cells, obtained from bone biopsies in a subgroup of MM patients (n°=24) expressed HOXB7 gene by microarray analysis and real time PCR. HOXB7 expression was also investigated at protein level by immunohistochemistry on bone biopsies of MM patients finding that MSC and OB as well as endothelial cells expressed HOXB7 protein mainly at nuclear level. In order to investigate the potential role of HOXB7 in the angiogenic process we enforced HOXB7 expression by lentivirus vectors in MSC using both primary BM MSC and the human MSC cell line HS-5 to obtain a stable transduced cell line. The overexpression of HOXB7 in HOXB7 transduced MSC as compared to the empty vector-transduced MSC cells was confirmed by real time PCR, western blot and immunohistochemistry. By Gene chips U133 plus 2.0 (Affymetrix) we evaluated the gene expression profiling of HOXB7 over-expressing MSC finding that proangiogenic cytokines, metalloproteinases and chemokines were significantly modulated in HOXB7-transduced MSC cells as compared to control cells. Data were validated either by real time PCR or by western blot and by an angiogenesis antibody array showing that bFGF and VEGF production was induced in MSC by HOXB7 overexpression. Consistently, we found that conditioned media of HOXB7-transduced MSC cells significantly stimulated vessel formation as compared to controls using an in vitro angiogenic model. Finally we observed that the angiogenic in vitro differentiation of HOXB7-transduced MSC was significantly increased as compared to controls. In conclusion our data suggest the HOXB7 overexpression in MSC regulates the angiogenic switch and could be a potential therapeutic target in MM-induced angiogenesis.

Preoperative assessment of HER-2/neu status in breast carcinoma: The role of quantitative real-time PCR on core-biopsy specimens

2010 ◽  
Vol 116 (2) ◽  
pp. 234-239 ◽  
Author(s):  
Tommaso Susini ◽  
Cecilia Bussani ◽  
Giulia Marini ◽  
Jacopo Nori ◽  
Simone Olivieri ◽  
...  
Keyword(s):  
Breast Carcinoma ◽  
Real Time ◽  
Real Time Pcr ◽  
Core Biopsy ◽  
Preoperative Assessment ◽  
Quantitative Real Time Pcr ◽  
Biopsy Specimens ◽  
Her 2

scholarly journals An in Vitro Study on the Role of Angiogenesis in Iron Deficiency Induced Reactive Thrombocytosis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2450-2450
Author(s):  
Pedro De Alarcon ◽  
Manu Gnanamony ◽  
Jessica Garcia
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Iron Deficiency ◽  
Real Time ◽  
Real Time Pcr ◽  
Tube Formation ◽  
Reactive Thrombocytosis ◽  
Serum Free

Abstract Introduction: Iron deficiency (ID) is one of the recognized causes of reactive thrombocytosis in children. Factors that are commonly associated with megakaryopoiesis such as thrombopoietin (TPO), interleukin 6 (IL-6) and IL-11 are not altered in patients with iron deficiency and thrombocytosis suggesting the role of alternate mechanisms in controlling this process. We have previously shown using an ID rat model that ID increased the number of megakaryocytes in the bone marrow. We have also shown an increase in VEGFR (FLT1) and CXCR4 staining in bone marrow slides of ID rats. This data suggests that angiogenesis plays a vital role in the development of reactive thrombocytosis in response to ID. In this report, we have expanded our study to identify specific angiogenic signaling molecules associated with ID and used functional assays to validate it. Methods: For this study, we used the megakaryoblast cell line MEG-01 as an in vitro model of megakaryopoiesis. MEG-01 cells were adapted to grow in chemically defined serum free medium containing iron (iron replete media). For iron deficiency, serum free iron free media was mixed with iron replete media at a 1% v/v concentration (iron deplete media). For our experiments, MEG-01 cells were grown in both iron replete and depleted media for 7 days. Cell viability was measured using the trypan blue exclusion assay. Messenger-RNA expression of iron-related markers (TFR1, TFR2, FLT1, FLT3, FTL, FTH1, TF, HMOX1 and HMOX2) and angiogenic markers (VEGFA, VEGFB, VEGFC, PDGF, ANGPTL1, ANGPTL2, FGF2) was studied using real time PCR. We performed functional validation of angiogenesis with an in vitro tube formation assay using human umbilical vein endothelial (HUVEC) cells. For statistical analysis of the data we performed the t test using graph pad prism software and we considered p<0.05 as statistically significant. Results: In low iron conditions, MEG-01 cells showed a significant increase in FLT1 (4 fold) and FLT3 (3 fold) expression using real time PCR (p<0.001). Iron deficiency also induced a 2 fold increase in the mRNA expression of angiogenic molecules VEGFB, VEGFC, FGF2 and PDGFA (p<0.001). Using the tube formation assay, we also show that conditioned media collected from iron deficient MEG-01 cells induced increased vessel formation in endothelial cells. Conclusion: In this study, we were able to validate our earlier in vivo findings on iron deficiency induced reactive thrombocytosis. We show that cells adapt to low iron conditions by upregulating FLT1, FLT3 and FTL. We also show that several markers in the angiogenesis pathway like VEGFB, VEGFC, FGF2 and PDGFA are upregulated in response to iron deficiency. We were also able to show that an increase in these angiogenic molecules induced increased vessel formation in endothelial cells. This report, along with our previous findings, points to the importance of the angiogenic pathway in reactive thrombocytosis induced by iron deficiency. Disclosures No relevant conflicts of interest to declare.

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