scholarly journals Necrotrophic Growth of Legionella pneumophila

2006 ◽  
Vol 72 (6) ◽  
pp. 4323-4328 ◽  
Author(s):  
R. Temmerman ◽  
H. Vervaeren ◽  
B. Noseda ◽  
N. Boon ◽  
W. Verstraete
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Legionella Pneumophila ◽  
Real Time Pcr ◽  
Water Systems ◽  
Saccharomyces Boulardii ◽  
Wall Structure ◽  
Lower Growth ◽  
Average Growth ◽  
Microbial Cells

ABSTRACT This study examined whether Legionella pneumophila is able to thrive on heat-killed microbial cells (necrotrophy) present in biofilms or heat-treated water systems. Quantification by means of plate counting, real-time PCR, and flow cytometry demonstrated necrotrophic growth of L. pneumophila in water after 96 h, when at least 100 dead cells are available to one L. pneumophila cell. Compared to the starting concentration of L. pneumophila, the maximum observed necrotrophic growth was 1.89 log units for real-time PCR and 1.49 log units for plate counting. The average growth was 1.57 � 0.32 log units (n = 5) for real-time PCR and 1.14 � 0.35 log units (n = 5) for plate counting. Viability staining and flow cytometry showed that the fraction of living cells in the L. pneumophila population rose from the initial 54% to 82% after 96 h. Growth was measured on heat-killed Pseudomonas putida, Escherichia coli, Acanthamoeba castellanii, Saccharomyces boulardii, and a biofilm sample. Gram-positive organisms did not result in significant growth of L. pneumophila, probably due to their robust cell wall structure. Although necrotrophy showed lower growth yields compared to replication within protozoan hosts, these findings indicate that it may be of major importance in the environmental persistence of L. pneumophila. Techniques aimed at the elimination of protozoa or biofilm from water systems will not necessarily result in a subsequent removal of L. pneumophila unless the formation of dead microbial cells is minimized.

scholarly journals 1622. Clinical and Environmental Surveillance of Legionella pneumophila in a Tertiary Healthcare Center in India

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S592-S592
Author(s):  
Sreenath k ◽  
Rama Chaudhry ◽  
Vinayaraj Ev ◽  
A B Dey ◽  
S K Kabra ◽  
...  
Keyword(s):  
Real Time ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Real Time Pcr ◽  
Water Systems ◽  
Environmental Surveillance ◽  
Healthcare Center ◽  
Tertiary Healthcare ◽  
Sporadic Cases ◽  
Pcr Targeting

Abstract Background Legionellosis is a form of pneumonia caused by Gram-negative bacilli belonging to the Legionella genus. In India, sporadic cases of legionellosis have been reported, but the incidence of this infection is still believed to be underestimated. We conducted a proactive clinical–environmental surveillance in a tertiary healthcare center to determine the frequency of legionellosis, and to identify the pathogen in the hospital water systems. Methods During February 2015–February 2019, we enrolled 533 cases (310 males, 223 females) with a diagnosis of pneumonia; a respiratory secretion was collected from each patient and tested for L.pneumophila by using a real-time PCR targeting mip gene. To identify Legionella spp. present in hospital water systems, we collected 201 hospital water samples and were analyzed by cultivation in BCYE agar. Legionella speciation and identification of Lp1 was done by real-time PCR assay. Results Among 533 cases, 11(2.1%) [6 male, 5 female] tested positive for L.pneumophila by real-time PCR. Of these, all were community-acquired sporadic cases not associated with a cluster or outbreak. Risk factors including smoking, alcohol use, malignancy, underlying respiratory disease, hypertension were identified in 8 (72.7%) cases. The duration of hospitalization for Legionella patients was 8–24 days; 5/11 (45.5%) patients were admitted to intensive care units. Of 11 patients 8 (72.7%) survived, and 3(27.3%) died. Among the 201 water samples tested, 38 (18.9%) tested positive for L.pneumophila by culture. The presence of Lp1 was detected in 25 (12.4%) water samples. Legionella spp. was recurrently isolated from patient areas, cooling towers, residential areas, and other areas inside the hospital campus. Conclusion The study indicates a low prevalence of legionellosis in this region. Even though Legionella colonization was detected in the hospital water system, no cases of hospital-acquired legionellosis were discovered during the study period. However, considering the risk of nosocomial legionellosis to patients we formulated Legionella control strategies in this hospital. Point-of-use filters were installed to the potable water units from where Legionella was isolated and repeat sampling from these sites were found to be negative for the contagion. Disclosures All authors: No reported disclosures.

scholarly journals In Vitro Screening and Transfection Concentration Optimization of Cynomolgus Monkey IκBα-siRNA

2020 ◽  
Vol 2020 ◽  
pp. 1-12 ◽  
Author(s):  
Zhaoxing Ou ◽  
Rui Zeng ◽  
Yifan Lin ◽  
Si Zhang ◽  
Mohammad Alzogool ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Real Time Pcr ◽  
Cynomolgus Monkey ◽  
Transfection Efficiency ◽  
In Vitro Screening ◽  
Sirna Transfection ◽  
Inhibitory Rate ◽  
Inhibitory Efficiency

Purpose. To seek for a small interfering RNA (siRNA) sequence targeting a cynomolgus monkey inhibitor of nuclear factor kappa B α (IκBα) that can specifically and effectively suppress IκBα gene expression of cynomolgus monkey ciliary muscle (CM) cells and trabecular meshwork (TM) cells in vitro and screen for optimal siRNA transfection concentration. Methods. Three IκBα-specific double-stranded siRNAs were designed and synthesized. They were transfected into primarily cultured cynomolgus monkey CM cells and TM cells. The mRNA and protein levels of IκBα were examined by using real-time quantitative polymerase chain reaction (real-time PCR) and western blot to screen a pair of candidate valid sequences with the highest inhibitory rate. Both cells were transfected with Cy5-labeled nonspecific control-siRNA (NC-siRNA) of four different concentrations (10, 20, 50, and 100 nmol/L(nM)), and flow cytometry was used to assess transfection efficiency. Then, cells were transfected with the candidate valid IκBα -siRNA of the same four concentrations, and the cytotoxicity was detected by using Cell Counting Kit-8 (CCK8), and the inhibitory efficiency of IκBα was identified via real-time PCR to find out optimal siRNA transfection concentration. Results. The suppression effect of the siRNA targeting the GCACTTAGCCTCTATCCAT of IκBα gene was most obvious by in vitro screening. The inhibitory rate of IκBα was 82% for CM cells and 82% for TM cells on the mRNA level and 98% for CM cells and 93% for TM cells on the protein level, respectively. The results of flow cytometry showed that the transfection efficiency was the highest at 100 nM, which was 89.0% for CM cells and 48.2% for TM cells, respectively. The results of CCK8 showed that there was no statistically significant difference in cell viability after transfection of different concentrations of IκBα-siRNA. The results of real-time PCR indicated that there was no statistical difference in the inhibitory efficiency of IκBα after transfection of different concentrations of IκBα-siRNA. Conclusion. It proves that the siRNA targeting the GCACTTAGCCTCTATCCAT of IκBα gene is the valid sequence to suppress cynomolgus monkey IκBα expression of CM cells and TM cells by RNAi. 10 nM is the optimal transfection concentration.

scholarly journals Quantitative Detection of Legionella pneumophila in Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of the dotA Gene

2005 ◽  
Vol 71 (7) ◽  
pp. 3433-3441 ◽  
Author(s):  
M. A. Yáñez ◽  
C. Carrasco-Serrano ◽  
V. M. Barberá ◽  
V. Catalán
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Immunomagnetic Separation ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Specific Sequence

ABSTRACT A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.

Low Expression of hOCT1 May Be a Key Point Mediating Low Intracellular Imatinib Accumulation in Imatinib Mesylate Resistance K562 Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4547-4547
Author(s):  
Huanling Zhu ◽  
Ting Liu ◽  
Yongqian Jia
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
K562 Cells ◽  
Pcr Analysis ◽  
Sensitive Cell ◽  
Imatinib Resistance ◽  
Low Expression

Abstract Objective To establish an imatinib resistance cell line and to study its resistant principia. Methods K562 cells were cultured in imatinib at gradually increased concentrations to generate their resistance cell line. Clone imatinib resistance cell lines by limited dilution culture. MTT assay, real time PCR and Semi-quantity PCR, flow cytometry and HPLC were used to clarify the possible mechanisms of the resistance. Results Imatinib resistance cell line K562R was successfully induced by continuous culture in the presence of gradually increasing doses of imatinib up to 5μmol/L. K562R cells were maintained in the media containing 5μmol/L imatinib. Proliferation data showed that cell growth of K562R was not inhibited in 5 μmol/L imatinib, whereas the parental sensitive cell was significantly inhibited by up to 2μM imatinib. The IC50 of K562R was about 7.5μmol/L which was ten times higher than that of the parental cell. HPLC revealed that the intracellular imatinib concentration of K562R was strikingly lower than that of the parental cells (up to 27.8-fold). MDR1 were not detected in mRNA (by RT-PCR)and protein(by flow cytometry) levels on K562R cell, whereas hOCT1 level measured by semi-quantity PCR showed lower expression in K562R cell lines than that of parental sensitive cell, indicating that low intracellular imatinib concentration may be due to lower affluence of imatinib by low level of hOCT1. (5) Real time PCR analysis showed no BCR-ABL/G6PD gene amplification and sequence analysis of the 374bp ABL kinase domain showed no mutation in K562R cell lines. Conclusion An imatinib resistance cell line K562R has been successfully established. Low expression of hOCT1 may be a key point mediating low intracellular imaitnib accumulation in K562R cell lines.

Long-Term Increase in Fetal Hemoglobin Expression in Nonhuman Primates Following Transplantation of Autologous Bcl11a Nuclease-Edited HSCs

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2035-2035 ◽  
Author(s):  
Olivier Humbert ◽  
Hans-Peter Kiem
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Real Time Pcr ◽  
Peripheral Blood ◽  
Nonhuman Primates ◽  
Pcr Analysis ◽  
Beta Globin ◽  
Gamma Globin ◽  
Cd34 Cells

Abstract Elevated levels of fetal hemoglobin (HbF) ameliorate the clinical symptoms of beta-thalassemia and sickle cell anemia. The transcription factor B-cell lymphoma/leukemia 11A (BCL11A) is required for silencing of gamma-globin expression in adult erythroid cells and functions as a switch from fetal to adult hemoglobin production in humans. BCL11A therefore constitutes a therapeutic target for the treatment of hemoglobinopathies. We inactivated BCL11A function by double-strand DNA break-induced mutagenesis using Transcription Activator-Like Effector Nucleases (TALENs). 20 to 30% gene editing could be achieved in vitro in human and nonhuman primate CD34+ cells by TALEN mRNAs electroporation targeting exon 2 of Bcl11a. Colony-forming efficiency was slightly lower in Bcl11a-edited CD34+ cells but lineage differentiation potential was unchanged. Erythroid differentiation of CD34+ cells in culture showed increased Fetal to Beta hemoglobin ratio in both human and primate Bcl11a-modified cells as compared to control cells, thus validating our editing approach to increase HbF production. To determine if Bcl11a-edited hematopoietic stem cells (HSCs) could be engrafted and give rise to HbF-producing erythrocytes, we transplanted a pigtail macaque with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. We detected about 1 % gene disruption in vivo early post-transplant and disruption frequency gradually declined to reach a set point of about 0.3% starting at day 28 post-transplantation. In this analysis, which we have so far taken out to 42 days, single clones could be tracked based on their mutation signature, and we found that several clones persisted over time, confirming engraftment of Bcl11a-modified cells. Since the transplantation procedure and chemo-radiotherapy conditioning can raise HbF production, three control animals that were transplanted using similar conditions as with the Bcl11a-edited HSCs and one untransplanted animal were also included in our analysis. Flow cytometry measurement of HbF in peripheral blood showed a rapid increase in F-cell production in all animals, reaching levels that ranged from 10% to 40% by 30 days, while the untransplanted control showed basal HbF expression of about 0.5% (Fig. 1A). The peak for HbF expression lasted for about 140 days and eventually returned to basal levels that averaged 0.5% for all control animals. In comparison, the animal transplanted with Bcl11a-edited cells showed significantly higher HbF levels starting at day 140 post-treatment (1-1.5%), and HbF production has remained constant for at least 150 days. This result was confirmed by hemoglobin mRNA analysis in peripheral blood using real-time PCR. We found a rapid increase in gamma globin expression following transplantation, before returning to near basal levels. As compared to controls, the animal transplanted with Bcl11a-edited cells showed a 5 to 10-fold increase in gamma to beta globin ratio at day 140 and this ratio has remained constant ever since (Fig. 1B). We are currently working on ways to enhance Bcl11a-editing and to select for Bcl11a-modified HSCs using targeted integration of the chemoselection cassette P140K MGMT to ultimately achieve curative HbF production. Potential TALEN off-target sites will also be examined as well as any side effect associated with the inactivation of BCL11A. Overall, our data demonstrate that transplantation of Bcl11a-edited HSCs results in elevated HbF production in nonhuman primates. Furthermore, we show that nonhuman primates can serve as a useful model for novel gene editing strategies toward the treatment of hemoglobinopathies. Figure 1. In vivo monitoring of HbF expression by flow cytometry and real-time PCR. (A) Intracellular HbF staining of peripheral blood measured by flow cytometry. (B) Real-time PCR analysis of hemoglobin transcripts in RNA isolated from peripheral blood. Expression was normalized to GAPDH and %HbG is calculated as HbG/(HbG+HbB). HbG=gamma globin; HbB=beta globin. Black line=Bcl11a transplant; grey line=control transplant; dashed line=untransplanted control. Figure 1. In vivo monitoring of HbF expression by flow cytometry and real-time PCR. (A) Intracellular HbF staining of peripheral blood measured by flow cytometry. (B) Real-time PCR analysis of hemoglobin transcripts in RNA isolated from peripheral blood. Expression was normalized to GAPDH and %HbG is calculated as HbG/(HbG+HbB). HbG=gamma globin; HbB=beta globin. Black line=Bcl11a transplant; grey line=control transplant; dashed line=untransplanted control. Disclosures No relevant conflicts of interest to declare.

APX3330 inhibition of the redox function of ape-1/ref-1 (Ref-1) in promyelocytic leukemia cells enhances retinoic acid (ATRA) induced myeloid differentiation and limits cell proliferation as an approach to the prevention of the retinoic acid syndrome (RAS)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14613-e14613
Author(s):  
K. A. Robertson ◽  
E. S. Colvin ◽  
M. R. Kelley ◽  
M. L. Fishel
Keyword(s):  
Flow Cytometry ◽  
Retinoic Acid ◽  
Cell Growth ◽  
Real Time ◽  
Real Time Pcr ◽  
Mapk Pathway ◽  
Promyelocytic Leukemia ◽  
Leukemia Cells ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation

e14613 Background: ATRA + chemotherapy has improved the treatment of promyelocytic leukemia(APL). However, 25% of ATRA treated APL patients experience toxicities that comprise the RAS (life-threatening respiratory distress, edema, renal failure, hypotension, coagulopathy and rising blast count). One approach to prevent RAS is to limit blast proliferation and enhance myeloid differentiation. Ref-1 is a DNA repair protein that functions in redox regulation of cellular proteins, such as Fos, Jun, p53, and NFkB. HL60 myeloid leukemia cells are promyeloblasts that respond to ATRA with granulocytic differentiation/growth arrest. Prior studies suggest Ref-1 redox control is integral to ATRA-induced differentiation. To define the role of the redox function of Ref-1, we used the Ref-1 specific drug, APX3330, to block Ref-1 redox function and examined the response of HL60 cells to ATRA. Methods: Cell growth assessed using trypan blue. Differentiation was evaluated by morphology and expression of CD11b by flow cytometry. Apoptosis was assayed by annexin-PI staining on flow cytometry and cell cycle analysis assayed with propidium iodide flow cytometry. To assess activation of the MAPK pathway, BLR-1 expression was determined by real time PCR. Results: 1) APX3330 blockade of Ref-1 redox function resulted in limited cell growth yet a profound increase in differentiation and a moderate increase in apoptosis. 2) dose dependent studies with ATRA showed a similar degree of differentiation in cells treated with 10 μM ATRA to cells treated with APX3330 + 0.01 μM ATRA; allowing HL60 cells + APX3330 to give a similar response to a 1000 fold lower dose of ATRA. APX3330 alone did not induce differentiation and induced only minimal apoptosis but in combination with ATRA, increased the number of cells in G1/G0 phase significantly. 3) APX3330 + ATRA increased BLR-1 expression significantly by real time PCR suggesting enhanced activation of the MAPK pathway. Conclusions: APX3330 + ATRA limits HL60 growth and dramatically enhances terminal granulocytic differentiation. These finding may provide a therapeutic approach for prevention of the RAS. No significant financial relationships to disclose.

Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry

2009 ◽  
Vol 84 (6) ◽  
pp. 1137-1147 ◽  
Author(s):  
Mateja Kramer ◽  
Nataša Obermajer ◽  
Bojana Bogovič Matijašić ◽  
Irena Rogelj ◽  
Vojko Kmetec
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Real Time Pcr ◽  
Probiotic Bacteria
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