Kinetics of Early Donor Chimerism after Nonmyeloablative Haematopoietic Stem Cell Transplantation Monitored with High-Resolution Real Time Quantitative PCR.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3669-3669
Author(s):  
Tania N. Masmas ◽  
Soren L. Petersen ◽  
Hans O. Madsen ◽  
Lars Ryder ◽  
Ebbe Dickmeiss ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Resolution ◽  
Real Time ◽  
Quantitative Pcr ◽  
Haematopoietic Stem Cell ◽  
Logrank Test ◽  
Post Transplant ◽  
Real Time Quantitative Pcr ◽  
Donor Chimerism ◽  
Kinetics Of

Abstract Purpose: From May 2003 to March 2005, 37 patients with haematological malignancies were transplanted with peripheral blood stem cells after nonmyeloablative conditioning with fludarabine 30 mg/m2 i.v. once daily on day −4 to −2 and 200 cGY of total body irradiation on day 0. Post-transplant immunosuppression consisted of oral cyclosporin 12.5 mg/kg/day and mycophenolate mofetil 30 mg/kg/day. Three patients were not eligible for follow-up due to lack of informed consent or informative markers for chimerism measurement. The purpose of this study was to evaluate kinetics of early chimerism of CD4+, CD8+, and, CD15+ cells immediately post transplant and furthermore to identify factors associated with rejection of graft. Methods: Blood samples were collected post transplant on day +1–3, +5–7, and then weekly. Samples were separated with immunomagnetic beads and DNA was salt extracted. Chimerism analysis was performed by automated high-resolution real-time quantitative PCR based on short insertion or deletion polymorphisms or single nucleotide polymorphisms. Results: The kinetics of CD15+ chimerism differed from that of CD4+ and CD8+ chimerism (Figure). CD15+ donor chimerism was low with a median of 0.9% donor cells on day +1–3, and remained low until around day +14. Subsequently the donor chimerism increased rapidly towards 100%. The CD4+ and CD8+ donor chimerism in contrast started higher with a median of 16.3% and 15.1% respectively on day +1–3, increased slowly, and took months to reach 100%. CD4+, CD8+ or CD15+ donor chimerism on day +1–3 did not predict later development of acute graft versus host disease. Two patients had primary rejection of the donor stem cells, while four patients had secondary rejection. The patients with primary rejection had no measurable donor chimerism in any cell lineages from day +1–3. No patient with CD15+ donor chimerism above the median (0.9%) on day +1–3 rejected, while 40% (6/15) of the patients with CD15+ donor chimerism below the median rejected (LogRank test, p=0.01). No differences were found in total number of nucleated cells, CD34+, CD3+, CD4+, or CD8+ cells in the donor harvest between patient who rejected and those who did not. Conclusions: The kinetics of early CD15+ donor chimerism differed from the kinetics of early CD4+ and CD8+ donor chimerism. Early CD15+ donor chimerism was markedly lower than CD4+ and CD8+ donor chimerism. CD15+ donor chimerism increased toward 100% donor chimerism more rapidly than the CD4+ and CD8+ chimerism. Primary rejection of donor stem cells can be identified early, as these patients have no measurable donor CD4+ or CD8+ chimerism on day +1–3. Furthermore, patients with CD15+ donor chimerism above the median on day +1–3 have a low risk for rejection. Figure Figure

scholarly journals Assessment of the Kinetics of Treponema pallidum Dissemination into Blood and Tissues in Experimental Syphilis by Real-Time Quantitative PCR

2007 ◽  
Vol 75 (6) ◽  
pp. 2954-2958 ◽  
Author(s):  
Juan C. Salazar ◽  
Asha Rathi ◽  
Nelson L. Michael ◽  
Justin D. Radolf ◽  
Linda L. Jagodzinski
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Whole Blood ◽  
Mononuclear Cells ◽  
Treponema Pallidum ◽  
Blood Components ◽  
Peripheral Blood Mononuclear ◽  
Real Time Quantitative Pcr ◽  
Kinetics Of

ABSTRACT Little is known about the size and kinetics of treponemal burdens in blood and tissues during acquired or experimental syphilitic infection. We used real-time quantitative PCR to measure Treponema pallidum DNA levels in rabbits infected intratesticularly with the prototype Nichols strain. At the outset, we performed a series of in vitro blood spiking experiments to determine the effect of blood processing procedures on the distribution of treponemes in various blood components. T. pallidum DNA levels in plasma and whole blood were approximately 10-fold higher than those in serum and more than 200-fold greater than those in peripheral blood mononuclear cells (PBMCs). Ten rabbits were inoculated intratesticularly with doses of treponemes ranging from 4 × 107 to 2 × 108 organisms. In five rabbits, T. pallidum DNA levels were measured sequentially in serum, plasma, whole blood, and PBMCs until sacrifice at peak orchitis, at which time brain, kidney, liver, spleen, and testicles were harvested; blood and organs were also harvested at orchitis from the other five rabbits. T. pallidum DNA was detected in plasma within 24 h postinfection. Treponeme levels in whole blood and blood components increased significantly with the development of peak orchitis. Overall, levels in serum and PBMCs were lower than those in plasma and whole blood; this disparity was particularly marked at early time points. Significantly greater numbers of spirochetes were found in the spleen than in liver, kidney, or brain tissue at the time of sacrifice. Our findings highlight the remarkable capacity of T. pallidum to disseminate from the site of infection to blood and tissues, and they identify the spleen as a prime target for treponemal invasion.

scholarly journals A gold standard method for human cell quantification after xenotransplantation

2019 ◽  
Author(s):  
Mona Bensalah ◽  
Pierre Klein ◽  
Ingo Riederer ◽  
Soraya Chaouch ◽  
Laura Muraine ◽  
...  
Keyword(s):  
Stem Cells ◽  
Real Time ◽  
Quantitative Pcr ◽  
Gold Standard ◽  
Human Cells ◽  
Muscle Fibres ◽  
Human Stem Cells ◽  
Gold Standard Method ◽  
Immunodeficient Mice ◽  
Real Time Quantitative Pcr

AbstractXenotransplantation of human cells into immunodeficient mouse models is a very powerful tool and an essential step for the pre-clinical evaluation of therapeutic cell-and gene-based strategies. Here we describe an optimized protocol combining immunofluorescence and real-time quantitative PCR to both quantify and visualize the fate and localization of human myogenic cells after injection in regenerating muscles of immunodeficient mice. Whereas real-time quantitative PCR-based method provides an accurate quantification of human cells, it does not document their specific localization. The addition of an immunofluorescence approach using human-specific antibodies recognizing engrafted human cells gives information on the localization of the human cells within the host muscle fibres, in the stem cell niche or in the interstitial space.These two combined approaches offer an accurate evaluation of human engraftment including cell number and localization and should provide a gold standard to compare results obtained either using different types of human stem cells or comparing healthy and pathological muscle stem cells between different research laboratories worldwide.

Myocardial Patch Formation by Three-Dimensional Culture of Bone Marrow Mesenchymal Stem Cells with 3-Hydroxybutyrate-Co-4-Hydroxybutyrate Under Hypoxia

2020 ◽  
Vol 10 (7) ◽  
pp. 922-929
Author(s):  
Mu Junsheng ◽  
Tian Kun ◽  
Zhou Fan ◽  
Bo Ping
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Real Time ◽  
Quantitative Pcr ◽  
Troponin T ◽  
Mouse Bone Marrow ◽  
Real Time Quantitative Pcr ◽  
Marrow Mesenchymal Stem Cells ◽  
Hypoxic Group

Herein we researched the effects of a hypoxic microenvironment on bone marrow mesenchymal stem cells (BM-MSCs) on poly 3-hydroxybutyrate-co-4-hydroxybutyrate [P(3HB-co-4HB)] and present a theoretical basis for development of cell transplantation. Mouse bone marrow mesenchymal stem cells were isolated by whole bone marrow culture and surface antigens were analyzed by flow cytometry of passage 5 cells. P(3HB-co-4HB) and bone marrow mesenchymal stem cells were prepared as stem cell patches randomly divided into normoxia (control, 20% oxygen) and hypoxia (3% oxygen) groups. After 24 h, the patch was used for experiments. Cell proliferation was determined by CCK-8 assays. Adhesion, survival, and growth of cells on patches were observed by scanning electron microscopy. Expression of hypoxia-inducible factor-1α (HIF-1α) was tested by real-time quantitative PCR and western blotting. At 2 weeks after addition of cardiomyocyte differentiation inducer 5-azacytidine, cardiac troponin T (cTnT) expression was detected by immunofluorescence. After 24 h, the proliferation of the hypoxic group was considerably greater compared with the normoxic group (n = 12,P < 0 05). SEM demonstrated that the number of viable cells in the hypoxic group was higher than that in the normoxic group. Adhesion between cells and the patch was firm and cell morphology was normal in the hypoxic group. Significant upregulation of HIF-1α mRNA was observed by real-time quantitative PCR after 12 h (P < 0 05). HIF-1αprotein expression in the hypoxia group was considerably higher than that in the normoxia group. cTnT expression in the hypoxic group was more pronounced than that in the normoxic group. Our results show that a hypoxic microenvironment promotes the adhesion, survival, proliferation, and myocardial differentiation of bone marrow mesenchymal stem cells on a P(3HB-co-4HB) patch, which may be mediated by the HIF-1α; pathway.

Population dynamics and in situ kinetics of nitrifying bacteria in autotrophic nitrifying biofilms as determined by real-time quantitative PCR

2006 ◽  
Vol 94 (6) ◽  
pp. 1111-1121 ◽  
Author(s):  
Tomonori Kindaichi ◽  
Yoshiko Kawano ◽  
Tsukasa Ito ◽  
Hisashi Satoh ◽  
Satoshi Okabe
Keyword(s):  
Population Dynamics ◽  
Real Time ◽  
Quantitative Pcr ◽  
Nitrifying Bacteria ◽  
Real Time Quantitative Pcr ◽  
Kinetics Of

Replication kinetics of duck virus enteritis vaccine virus in ducklings immunized by the mucosal or systemic route using real-time quantitative PCR

2009 ◽  
Vol 86 (1) ◽  
pp. 63-67 ◽  
Author(s):  
Xuefeng Qi ◽  
Xiaoyan Yang ◽  
Anchun Cheng ◽  
Mingshu Wang ◽  
Yufei Guo ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Vaccine Virus ◽  
Real Time Quantitative Pcr ◽  
Kinetics Of ◽  
Duck Virus Enteritis

scholarly journals Quantification and Stability of Human Adenoviruses and Polyomavirus JCPyV in Wastewater Matrices

2006 ◽  
Vol 72 (12) ◽  
pp. 7894-7896 ◽  
Author(s):  
Silvia Bofill-Mas ◽  
Nestor Albinana-Gimenez ◽  
Pilar Clemente-Casares ◽  
Ayalkibet Hundesa ◽  
Jesus Rodriguez-Manzano ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
High Stability ◽  
Wastewater Sludge ◽  
Human Polyomavirus ◽  
Sewage Effluent ◽  
Viral Contamination ◽  
Human Adenoviruses ◽  
Urban Sewage ◽  
Real Time Quantitative Pcr

ABSTRACT Human adenoviruses (HAdV) and human polyomavirus JCPyV have been previously proposed as indicators of fecal viral contamination in the environment. Different wastewater matrices have been analyzed by applying real-time quantitative PCR procedures for the presence, quantity, and stability of a wide diversity of excreted HAdV and JCPyV. High quantities of HAdV and JCPyV were detected in sewage, effluent wastewater, sludge, and biosolid samples. Both viruses showed high stability in urban sewage. These results confirm the suitability of both viruses as indicators of human fecal viral pollution.

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