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Pleuropneumonia-Like Organisms

Pleuropneumonia-Like Organisms (PPLO) are a group of small, fastidious bacteria that lack a cell wall and are closely related to the Mycoplasma genus.
They are known to cause respiratory and genital tract infections in various animal species, including humans.
PPLO are challenging to cultivate and identify, requiring specialized laboratory techniques.
Understanding their biology and developing effective diagnostic and treatment protocols is crucial for managing infections caused by these elusive microorganisms.

Most cited protocols related to «Pleuropneumonia-Like Organisms»

1
Isolation and Culture of Bovine Mycoplasma
The HB0801 strain was isolated from the lesioned lung of an infected beef cattle from Yingcheng city in Hubei province, China by this laboratory [4] and stored at the China Center for Type Culture Collection (CCTCC # M2010040) at Wuhan University, Wuhan, China. HB1007 was isolated from milk of a dairy cow with mastitis in Hubei in 2010.
The strain was cultured on a pleuropneumonia-like organisms (PPLO) agar plate at 37°C, in a 5% CO2 atmosphere for 3 days or in PPLO broth (2.5 g glucose, 10.5 g PPLO, 2.5 g yeast, 50 mL donor equine serum (Thermo Fisher Scientific, Waltham, MA, USA), 5 mL10% arginine, 5 mL 10×MEM, 5 mL of 80,000 IU/mL penicillin-G, and 500 µL 1% phenol red) at 37°C for 3 days on an orbital shaker.
Qi J., Guo A., Cui P., Chen Y., Mustafa R., Ba X., Hu C., Bai Z., Chen X., Shi L, & Chen H. (2012). Comparative Geno-Plasticity Analysis of Mycoplasma bovis HB0801 (Chinese Isolate). PLoS ONE, 7(5), e38239.
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Publication 2012
Agar Arginine Atmosphere Beef Cattle Donors Equus caballus Glucose Lung Mastitis Milk, Cow's Penicillin G Pleuropneumonia-Like Organisms Serum Strains Yeast, Dried
2
Isolation and Culture of RA Synovial Fibroblasts
Synovial tissues were obtained from synovial biopsies of six patients with RA undergoing joint surgery (synovectomy or joint replacement by prosthesis implantation), who all met the criteria of the American College of Rheumatology [27 (link)]. The tissue samples were obtained during routine surgery at the Department of Orthopedics of the University of Regensburg, where approved by the local ethics committee and patients involved gave informed consent. Culture of SF was performed as described recently [5 (link)]. Following enzymatic digestion, fibroblasts were grown in DMEM (Biochrom, Berlin, Germany) containing 10% heat inactivated FCS (Gibco Life Technologies, Grand Island, NY, USA), 100 U/ml penicillin and streptomycin (PAA Laboratories GmbH, Linz, Austria) and cultured for four passages at 37°C in 10% carbon dioxide. The SF were stained for a fibroblast marker by immunohistochemistry. More than 95% could be stained positively for the fibroblast enzyme prolyl 4-hydroxylase and none were positive for the macrophage marker CD68 or the neutrophil marker cathepsin G after the second passage of cultivation with enzymatic digestion equalling passage 0 (data not shown). Routine tests for mycoplasms were negative. At 85 to 95% confluency, cells were passaged 1:2 and a part of the cells was harvested. Total RNA was extracted and stored at -70°C. Culture conditions were (and have to be) kept constant during the experiments. Passaging of the cells was performed at 85 to 95% confluency as fibroblasts exhibit contact inhibition (unpublished observations) and were passaged 1:2 to provide cell-cell contacts between the cells.
Neumann E., Riepl B., Knedla A., Lefèvre S., Tarner I.H., Grifka J., Steinmeyer J., Schölmerich J., Gay S, & Müller-Ladner U. (2010). Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts. Arthritis Research & Therapy, 12(3), R83.
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Publication 2010
Arthroplasty, Replacement Biopsy Carbon dioxide Cathepsin G Cells Contact Inhibition Digestion Enzymes Fibroblasts Immunohistochemistry Joints Macrophage Neutrophil Operative Surgical Procedures Orthopedic Surgical Procedures Patients Penicillins Pleuropneumonia-Like Organisms Procollagen-Proline Dioxygenase Regional Ethics Committees Streptomycin Synovectomy Synovial Membrane Tissues
3
Comparing MODY1 iPSCs and Human Islets
Human islets (n = 3) were isolated as described previously29 (link) from pancreata obtained from two female and one male brain-dead deceased donors after informed consent from relatives for organ donation and for use in research at the islet isolation facility of Oslo University Hospital, Oslo, Norway. In this study, we present data from two human islets samples, one of the samples were excluded due to technical issues. All experiments with human islets was approved by the regional committee for medical and health research ethics central in Norway (2011/782).
The human induced pluripotent stem cells iPSCs used in this study were episomal reprogrammed from four family members with MODY1 diabetes, including a healthy family control. The iPSCs cell lines were characterized and confirmed to have normal karyotype, and to be mycoplasma free using a MycoAlert Mycoplasm Detection Kit (Lonza, LT07-418). All hiPS cell lines were subjected to Stage-specific embryonic antigen 4 (SSEA4) enrichment (SSEA4 microbeads, MACS Miltenyi Biotec) before proceeding to differentiation.
All samples used for differentiation and further analyses throughout the manuscript were generated from a MODY1 quadro, i.e. three HNF4A mutation carriers (two with overt diabetes and one non-diabetic mutation carrier) and their family control (healthy male sibling without mutation).
All experiments with hiPSCs were approved by the Regional Committee of Medical and Health Research Ethics (REK 2010/2295), and all methods were performed in accordance with the Helsinki Declaration.
Vethe H., Bjørlykke Y., Ghila L.M., Paulo J.A., Scholz H., Gygi S.P., Chera S, & Ræder H. (2017). Probing the missing mature β-cell proteomic landscape in differentiating patient iPSC-derived cells. Scientific Reports, 7, 4780.
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Publication 2017
Brain Death Cell Lines Coxa Diabetes Mellitus Donors Episomes Family Member Females Homo sapiens Human Induced Pluripotent Stem Cells Induced Pluripotent Stem Cells isolation Karyotyping Males Maturity-Onset Diabetes of the Young, Type 1 Microspheres Mutation Mycoplasma Organ Transplantation Pancreas Pleuropneumonia-Like Organisms stage-specific embryonic antigen-4
4
Mycoplasma Detection and Elimination in THP-1 Cells
Human monocytic THP-1 cell lines were cultured in RPMI 1640 (MediaTech, Inc) containing 10% fetal bovine serum (endotoxin-free; Atlanta Biologicals) and 1% penicillin-streptomycin solution (Invitrogen Life Technologies). Mycoplasma-positive and negative THP-1 cells were stimulated either with E. coli LPS (100 ng/ml) or Francisella novicida (strain JSG2401; U112) for 15 hours in antibiotics-free media. The release of IL-1β and IL-8 in the culture media was evaluated by ELISA developed in our laboratory [9] (link). RNA was extracted using Trizol (Invitrogen Life Technologies) and evaluated for gene expression using RT-qPCR as described previously [10] (link). Briefly, the expression of the genes of interest was calculated in relative copy numbers (RCN), which is relative to the average expression value of two housekeeping genes. Data were analyzed within one group and between two groups using paired t-test. For all analyses, p<0.05 was considered to be significant. To check for mycoplasmal contamination, genomic DNA was isolated using a Puregen kit (Qiagen; Puregen Accessories), and verified by PCR where a high quality genomic DNA is an important factor. The combination of the following three sets of primers allowed detection of most mycoplasma species [11] (link), [12] (link), [13] (link): Myco-280 forward 5′- gggagaaacaggattagataccct-3′ and reverse 5′-tgcaccatctgtcactctgttaacctc-3′; Myco-500 forward 5′-ggcgaatgggtgagtaacacg-3′ and reverse; and Myco-717 forward F: 5′-actcctacgggaggcagcagta-3′ and reverse 5′-tgcaccatctgtcactctgtt-3′. PCR conditions were 5 min 95°C and then 35 cycles at 94°C for 15 s, at 60°C for 15 sec, and at 72 for °C for 15 sec.
To eliminate mycoplasma contamination, mycoplasma positive THP-1 cells were treated with 25 µg/ml of Plasmocin (InvivoGen) for three weeks. Aliquots were taken every three to four days for detection of mycoplasma by PCR. When cells were confirmed to be a mycoplasma negative, they were further maintained in RPMI 1640 media in the absence of Plasmocin.
Zakharova E., Grandhi J., Wewers M.D, & Gavrilin M.A. (2010). Mycoplasma Suppression of THP-1 Cell TLR Responses Is Corrected with Antibiotics. PLoS ONE, 5(3), e9900.
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Publication 2010
Antibiotics Biological Factors Cells Culture Media Endotoxins Enzyme-Linked Immunosorbent Assay Escherichia coli Fetal Bovine Serum Francisella tularensis subsp. novicida Gene Expression Genes, Housekeeping Genome Homo sapiens Interleukin-1 beta Monocytes Mycoplasma Oligonucleotide Primers Penicillins plasmocin Pleuropneumonia-Like Organisms Strains Streptomycin THP-1 Cells trizol
5
Generation of High-Titer CAR-Expressing PG13 Cells
A clinical grade high-titer PG13 clone expressing the 1928z chimeric antigen receptor (CAR) was generated by transiently transfecting Phoenix-eco cells with the plasmid encoding the gammaretroviral vector SFG-1928z12 (link) and subsequently infecting PG13 cells with cell-free vector stocks from the transfected Phoenix-eco cells. The PG13-1928z cell population was subsequently subcloned by limiting dilution. Clones were isolated and titers were determined by infecting HeLa cells under standardized conditions. High titer clones were identified by fluorescence activated cell sorting (FACS) using the anti-1928z CAR hamster monoclonal antibody 19E3 that was generated in-house by the MSKCC monoclonal antibody core facility. The high titer PG13-1928z clone 34 was subjected to a second round of subcloning by limiting dilution. The subclone PG13-1928z cl.3 was demonstrated to express the 19-28zCAR and was selected for its ability to efficiently transduce peripheral blood mononuclear cells (PBMCs). Integrity of the retroviral vector construct was demonstrated and a single copy of the integrated proviral vector was detected by Southern blot analysis in the genomic DNA extracted from PG13-1928z clone 3 (data not shown). The PG13-1928z clone 3 was expanded to generate a seed bank (SB) that was tested for absence of mycoplasm, replication competent retrovirus (RCR), and for sterility. The SB passed all required tests.
Hollyman D., Stefanski J., Przybylowski M., Bartido S., Borquez-Ojeda O., Taylor C., Yeh R., Capacio V., Olszewska M., Hosey J., Sadelain M., Brentjens R.J, & Rivière I. (2009). Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy. Journal of immunotherapy (Hagerstown, Md. : 1997), 32(2), 169-180.
Publication 2009
Antibodies, Anti-Idiotypic Cells Clone Cells Cloning Vectors DNA Replication Genome Hamsters HeLa Cells Monoclonal Antibodies PBMC Peripheral Blood Mononuclear Cells Plasmids Pleuropneumonia-Like Organisms Receptor, Chimeric Antigen Retroviridae Seed Bank Southern Blotting Sterility, Reproductive Technique, Dilution

Most recents protocols related to «Pleuropneumonia-Like Organisms»

1
Induction and Quantification of Cancer Stem Cell Spheres
HEK 293T (RRID:CVCL_0063), A549 (RRID:CVCL_0023) and H1299 (RRID:CVCL_0060) cell lines were acquired from the American Type Culture Collection (ATCC), either directly or from colleagues, kept frozen at liquid Nitrogen after received and used in culture for a maximum of 4 months. ATCC cell lines were characterized by Short Tandem Repeat (STR) profiling and Mycoplasm contamination was evaluated monthly by PCR. All cell lines were cultured in complete Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin 100U.ml-1 /streptomycin 100 µg.ml-1 and L-glutamine 2 mM in 5% CO2 humidified atmosphere at 37 °C.
For sphere induction, LC cells were grown to 90% confluence, trypsinized, and plated in stem cell (SC) medium in ultra-low adhesion multi-well plates (Corning) [9 (link)]. After 7 days, the number of spheres was quantified using 10× magnifications under a phase contrast microscope (Carl-Zeiss, AxioObserverZ1).
Muñoz-Bernart M., Budnick N., Castro A., Manzi M., Monge M.E., Pioli J., Defranchi S., Parrilla G., Santilli J.P., Davies K., Espinosa J.M., Kobayashi K., Vigliano C, & Perez-Castro C. (2023). S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) inhibits lung cancer tumorigenesis by regulating cell plasticity. Biology Direct, 18, 8.
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Publication 2023
Atmosphere Cell Culture Techniques Cell Lines Cells Eagle Fetal Bovine Serum Freezing Glutamine Microscopy Microscopy, Phase-Contrast Nitrogen Penicillins Pleuropneumonia-Like Organisms Short Tandem Repeat Stem Cells Streptomycin
2
Generating Formative Cells from Mouse ESCs
Mouse ESCs were cultured in Knockout DMEM (Thermo Fisher, CAT#10829018) supplemented with 15% FBS, L-Glutamine, Penicillin/Streptamycin, NEAA, LIF (1000U/mL), and 2i (1μM MEK inhibitor PD0325901 and 3μM GSK3 inhibitor CHIR99021). Wildtype and dCD cells were R1 ESCs from a 129 strain background. MLL3−/−; MLL4fl/fl ESCs were a mixed background of C57BL/6J and 129 strains. Formative cells were generated by removal of LIF and 2i. Briefly, 5e4 ESCs were plated per well of a 6-well plate on day −1 in LIF+2i media. To initiate differentiation, LIF and 2i were removed 24 h after seeding (day 0). Formative cells were collected on day 3 of differentiation, 63 h after removal of LIF and 2i. To overcome proliferation defects, 7.5e4 MLL3/4 DKO cells were plated per well of a 6-well plate. Naive cells were passaged and staged appropriately for simultaneous harvest. The expected amino acid substitutions in MLL3/4 dCD cells were validated using Sanger sequencing on amplicons generated from genomic PCR. To generate DKOs from MLL3−/−; MLL4fl/fl ESCs, we transfected with Rosa26-CRE-ERT2 plasmid linearized by EcoRI, selected with puromycin, and genotyped using genomic PCR. We then added tamoxifen for 3 days and picked clones to validate knockout of MLL4 floxed allele. Primers for genotyping listed in Additional file 3. Lines consistently tested negative for mycoplasm.
Boileau R.M., Chen K.X, & Blelloch R. (2023). Loss of MLL3/4 decouples enhancer H3K4 monomethylation, H3K27 acetylation, and gene activation during embryonic stem cell differentiation. Genome Biology, 24, 41.
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Publication 2023
Alleles Amino Acid Substitution Chir 99021 Clone Cells Deoxyribonuclease EcoRI Enhanced S-Cone Syndrome Genome Glutamine Glycogen Synthase Kinase 3 KMT2B protein, human mitogen-activated protein kinase 3, human MLL3 protein, human Mus Oligonucleotide Primers PD-0325901 Penicillins Plasmids Pleuropneumonia-Like Organisms Puromycin Strains Tamoxifen
3
Bovine Respiratory Pathogen Profiling
For each case, 3 pooled samples each of fresh cranioventral and caudodorsal lung
were submitted for routine bacterial culture and for specialized mycoplasmal
culture to the Animal Health Laboratory, University of Guelph (Supplemental Methods). For routine cultures, all bacterial
growth was identified using matrix-assisted laser-desorption ionization
time-of-flight mass spectrometry. For mycoplasmal culture, isolates were
identified by indirect fluorescent antibody tests. For each case, 3 pooled
samples of fresh caudodorsal lung were routinely tested using real-time PCR for
bovine alphaherpesvirus 1 (BHV-1), bovine respiratory syncytial virus (BRSV),
bovine parainfluenzavirus 3 (BPIV3), bovine coronavirus (BCV), and bovine viral
diarrhea virus (BVDV) types 1 and 2 (Supplemental Methods). At a later time, samples from 12 BIP, 20
BP, and 10 AIP cases were tested by PCR for bovine hokovirus (Ungulate
tetraparvovirus 1), based on results of high-throughput sequencing
on 3 cases per group (data not shown; Supplemental Methods), and bovine adenovirus serotypes 1 to 8
(Supplemental Methods).
Haydock L.A., Fenton R.K., Sergejewich L., Veldhuizen R.A., Smerek D., Ojkic D, & Caswell J.L. (2023). Bronchopneumonia with interstitial pneumonia in beef feedlot cattle: Characterization and laboratory investigation. Veterinary Pathology, 60(2), 214-225.
Publication 2023
Adenoviruses Animals, Laboratory Bacteria Bovine parainfluenza virus 3 Cattle Coronavirus, Bovine Indirect Immunofluorescence Lung Mass Spectrometry Pleuropneumonia-Like Organisms Real-Time Polymerase Chain Reaction Respiratory Syncytial Virus, Bovine Virus
4
Characterizing Bovine Interstitial Pneumonia
Cases were a convenience sample of naturally occurring mortalities on 6
large-scale commercial beef feedlots in Alberta and Saskatchewan, Canada, from
January 2018 to January 2022. Based on gross and histopathological diagnoses,
the study included 18 cases of BIP, 24 cases of BP, and 13 cases of AIP. From
each case, lung samples were tested as follows: fresh tissue for routine
bacterial culture and mycoplasmal culture (cranioventral and caudodorsal lung,
separately), fresh tissue for polymerase chain reaction (PCR) testing for
viruses (caudodorsal lung only), and formalin fixation for histopathology
(cranioventral and caudodorsal lung, separately). Clotted blood was collected
from the heart, and the serum was used for cytokine analysis. Bronchoalveolar
lavage fluid was harvested from 13 cases for surfactant analysis (Supplemental Table S1).
Haydock L.A., Fenton R.K., Sergejewich L., Veldhuizen R.A., Smerek D., Ojkic D, & Caswell J.L. (2023). Bronchopneumonia with interstitial pneumonia in beef feedlot cattle: Characterization and laboratory investigation. Veterinary Pathology, 60(2), 214-225.
Publication 2023
Beef Blood Cytokine Diagnosis Formalin Heart Lung Pleuropneumonia-Like Organisms Polymerase Chain Reaction Pulmonary Surfactants Serum Tissues
5
Human Colon Cancer Cell Culture
Human colon cancer HCT116 (kindly provided by Prof. Ber Vogelstein, Johns Hopkins University, Baltimore, MD, USA) and RKO cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies-Invitrogen, Eggenstein, Germany), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Corning Life Sciences, New York, NY, USA), plus glutamine and antibiotics (Corning Life Sciences, New York, NY, USA) in a humidified atmosphere with 5% CO2 at 37 °C. Cells underwent routine testing to ensure that they were mycoplasm negative. The inhibitor of the antioxidant response Brusatol (Sigma-Aldrich, St Louis, MO, USA) [34 (link),35 (link)] was used at 100 µM for 4 h pre-treatment, as previously reported [36 (link)]; Bip/GRP78 inhibitor HA15 (Sigma-Aldrich, St Louis, MO, USA, SML2118) [37 (link),38 (link)] was used at 10 µM for 1 h pre-treatment, as previously reported [20 (link)].
Garufi A., Pettinari R., Marchetti F., Cirone M, & D’Orazi G. (2023). NRF2 and Bip Interconnection Mediates Resistance to the Organometallic Ruthenium-Cymene Bisdemethoxycurcumin Complex Cytotoxicity in Colon Cancer Cells. Biomedicines, 11(2), 593.
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Publication 2023
Antibiotics, Antitubercular Antioxidants Atmosphere brusatol Cancer of Colon Cells Eagle Fetal Bovine Serum Glucose Regulated Protein 78 kDa Glutamine Homo sapiens Pleuropneumonia-Like Organisms

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Penicillin/streptomycin is a commonly used antibiotic mixture for cell culture applications. It provides broad-spectrum antimicrobial activity to prevent bacterial contamination in cell culture experiments.

More about "Pleuropneumonia-Like Organisms"

Pleuropneumonia-Like Organisms (PPLO), also known as Mycoplasma, are a unique group of tiny, fastidious bacteria that lack a cell wall.
These elusive microorganisms are closely related to the Mycoplasma genus and are known to cause respiratory and genital tract infections in various animal species, including humans.
Understanding the biology of PPLO is crucial for managing infections, as they are challenging to cultivate and identify, requiring specialized laboratory techniques.
Effective diagnostic and treatment protocols are essential for addressing PPLO-related infections.
When studying PPLO, researchers often utilize specialized growth media like Fetal Bovine Serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), and supplements such as Penicillin/Streptomycin and Glutamine (GlutaMAX) to support their growth and maintenance.
Fetal calf serum, a common component of cell culture media, also plays a vital role in PPLO research.
The lack of a cell wall in PPLO makes them intrinsically resistant to antibiotics like Penicillin, which target the bacterial cell wall.
However, antibiotics like Streptomycin can be effective in treating PPLO infections.
By leveraging the power of AI-driven protocol optimization tools, such as PubCompare.ai, researchers can effectively identify the most accurate and efficient protocols for studying these elusive microorganisms, enhancing the accuracy and precision of their PPLO-related research.