Przydatność kliniczna cyfrowej PCR kropli do monitorowania transkryptów BCR-ABL1 pacjentów z ostrą białaczką limfoblastyczną z dodatnim chromosomem Philadelphia Put up-chimeryczny receptor antygenowy 19/22 T-Cell Cocktail Remedy
Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) accounts for 20-30% of grownup sufferers with ALL, characterised by translocation of t(9, 22). Tyrosine kinase inhibitors (TKIs) have considerably improved the result although there are nonetheless some issues together with relapse as a result of drug-resistant mutations and suboptimal molecular remission depth. Beforehand, we reported the security and efficacy of sequential infusion of CD19/22 chimeric antigen receptor T-cell (CAR-T) immunotherapy within the therapy of relapsed/refractory (R/R) B-cell neoplasms together with circumstances with Ph+ ALL. Given doable deeper response, extra sufferers have been anticipated to achieve optimum minimal residual illness (MRD) response.
An alternate technique, duplex droplet digital PCR (ddPCR) with excessive sensitivity was established, which may present absolute quantification of MRD with out the necessity for calibration curves. Right here, we retrospectively collected 95 bone marrow samples from 10 sufferers with R/R Ph+, who acquired 19/22 CAR-T-cell cocktail remedy. Notably, sequential molecular remission for greater than three months (SMR3), a major indicator primarily based on ddPCR after CAR-T infusion was established, which was outlined as a sequential molecular remission for not <three months with damaging MRD. On this cohort, no recurrence was noticed in six sufferers reaching SMR3, the place 4 of whom accepted allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CAR-T cell routine.
Sadly, the opposite 4 sufferers who didn’t attain SMR3 relapsed, and didn’t obtain further particular therapy besides CAR-T routine. To sum up, ddPCR could also be another, particularly when nucleic acid was inadequate in medical apply. No achievement of SMR3 could be an early warning of potential relapse after CAR-T and indicating the initiation of different therapies together with allo-HSCT.
Description: The dendritic cell lysosomal-associated membrane protein (DC-LAMP)/CD208 is a type I integral transmembrane glycoprotein mostly homologous to CD68, of about 45 kDa in mouse and 90 kDa in human (glycosylation), with a bipartite C-terminal structure divided by a serine/proline rich region, a transmembrane domain and a conserved tyrosine-based lysosomal targeting motif in its cytoplasmic tail. Initially cloned as a specific marker of human mature dendritic cells (DCs), DC-LAMP has been subsequently shown to be expressed in alveolar type II pneumocytes. In both cell types, the molecule is found in the limiting membrane of intracellular multi-lamellar bodies, known as MIIC (MHC class II compartments) in human mature DCs and as lung surfactant-containing lamellar bodies in type II pneumocytes. In the latter cell type, DC-LAMP expression is also detected at the cell surface.
Description: IL3 exerts its biologic activity through its interaction with a cell surface receptor that consists of two subunits. The a subunit (CD123) specifically binds IL3, whereas the ß subunit is required for signaling and is common to the GMCSFR and IL5-R. 107D2.08 and 106C2.02 mAbs were obtained after mouse immunization with sorted human tonsillar PDC. Both clones strongly stain PDCs and basophils, weakly stain monocytes, CD34+ derived DCs and CD11c+ DC, while no staining is observed on T, B, NK cells as well as on mono-derived DCs. Staining with 107D2.08 and 106C2.02 mAbs are maintained on sorted PDC cultured in the presence of IL3 and CD40L, but lost when IL3 alone is added to the culture. The recognition of the IL3Ra chain by 107D2.08 and 106C2.02 was confirmed by transfection studies. 107D2.08 appeared to be the most appropriate clone for in situ studies. 107D2.08 allowed the first observation of IL3Ra+ cells in breast tumor microenvironment
Description: Langerin/CD207 is a transmembrane C-type lectin receptor (CLR) of epidermal and mucosal Langerhans cells (LCs) that induces Birbeck's granule formation. Langerin features a single carbohydrate recognition domain (CRD) with mannose-type specificity in its extracellular portion. Langerin is unique among the CLRs in that it contains an intracellular domain with a proline-rich motif. Langerin expression has not been reported outside the DC system. (Valladeau J et al, 1999, Eur.J.Immunol., 29:2695-2704; Valladeau J et al, 2000 Immunity, 12 : 71-81; Kashihara M et al, 1986, J.Invest.Derm., 87 :602-607 Ito T et al, 1999, J.Immunol., 163 :1409-1419 ;Saeland S & Valladeau J, CD207 (Langerin) Workshop reports 2002, Leukocyte-Typing VII, White Cell Diff Antigens, D. Mason et al, Eds, Oxford University Press:306-307)
Description: The IL7-R consists of 2 chains, IL-7R known as CD127 and common cytokine receptor chain known as CD132. A 75 to 80kDa human IL-7 receptor has been cloned that belongs to hematopoietic cytokinereceptor super family. R34-34, raised against human leukemic pre-B cells, recognized a molecule expressed on normal B cell precursors but not on mature B cells. This antibody specifically reverted IL-7 mediated growth inhibition of leukemic BCP (normal B cells precursors) and mature T cells. IL-7R expression is dramatically influenced by cytokines and antigens. This IL-7R displays both high and low affinity for its ligand (IL-7). Inhibitory and proliferative effects of IL-7 can be mediated through the same receptor on various lineages. CD4+ memory T cells express high level of IL-7R Subsets that express it generally require it, including progenitors of T and B cells, naïve and memory T cells. (Pandrau-Garcia D et al, 1994, Blood, 83, 3613-9 Mazzucchelli R et al, Nat. Review Immunol., 2007,7, 144-54)
Description: CD40 (48 to 50 kDa) is a transmembrane glycoprotein mainly expressed on the surface of B cells and also expressed on monocytes, dendritic cells, and thymic epithelium. CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily, which includes the low affinity nerve growth factor (NGF) receptor and CD95/Fas. CD40 is the receptor for CD40 ligand. CD40L (CD40L, CD154, gp39, and TRAM) belongs to the TNF gene family and is expressed more widely than CD40, predominantly on activated CD4+ T cells. Following interaction with CD40 ligand, CD40 mediates a number of major immunoregulatory functions, central to the control of thymus dependent humoral immunity and may be critical in the development of cell mediated immune responses. Other biological actions include B cell homotypic adhesion, proliferation, immunoglobulin isotype switch, and secretion. Activation of CD40 has also been shown to inhibit the growth of certain B cell lymphomas and to induce the death of transformed cells of mesenchymal or epithelial origin
Description: The dendritic cell lysosomal-associated membrane protein (DC-LAMP)/CD208 is a type I integral transmembrane glycoprotein mostly homologous to CD68, of about 45 kDa in mouse and 90 kDa in human (glycosylation), with a bipartite C-terminal structure divided by a serine/proline rich region, a transmembrane domain and a conserved tyrosine-based lysosomal targeting motif in its cytoplasmic tail. Initially cloned as a specific marker of human mature dendritic cells (DCs), DC-LAMP has been subsequently shown to be expressed in alveolar type II pneumocytes. In both cell types, the molecule is found in the limiting membrane of intracellular multi-lamellar bodies, known as MIIC (MHC class II compartments) in human mature DCs and as lung surfactant-containing lamellar bodies in type II pneumocytes. In the latter cell type, DC-LAMP expression is also detected at the cell surface.
Description: The dendritic cell lysosomal-associated membrane protein (DC-LAMP)/CD208 is a type I integral transmembrane glycoprotein mostly homologous to CD68, of about 45 kDa in mouse and 90 kDa in human (glycosylation), with a bipartite C-terminal structure divided by a serine/proline rich region, a transmembrane domain and a conserved tyrosine-based lysosomal targeting motif in its cytoplasmic tail. Initially cloned as a specific marker of human mature dendritic cells (DCs), DC-LAMP has been subsequently shown to be expressed in alveolar type II pneumocytes. In both cell types, the molecule is found in the limiting membrane of intracellular multi-lamellar bodies, known as MIIC (MHC class II compartments) in human mature DCs and as lung surfactant-containing lamellar bodies in type II pneumocytes. In the latter cell type, DC-LAMP expression is also detected at the cell surface.
Description: IL3 exerts its biologic activity through its interaction with a cell surface receptor that consists of two subunits. The a subunit (CD123) specifically binds IL3, whereas the ß subunit is required for signaling and is common to the GMCSFR and IL5-R. 107D2.08 and 106C2.02 mAbs were obtained after mouse immunization with sorted human tonsillar PDC. Both clones strongly stain PDCs and basophils, weakly stain monocytes, CD34+ derived DCs and CD11c+ DC, while no staining is observed on T, B, NK cells as well as on mono-derived DCs. Staining with 107D2.08 and 106C2.02 mAbs are maintained on sorted PDC cultured in the presence of IL3 and CD40L, but lost when IL3 alone is added to the culture. The recognition of the IL3Ra chain by 107D2.08 and 106C2.02 was confirmed by transfection studies. 107D2.08 appeared to be the most appropriate clone for in situ studies. 107D2.08 allowed the first observation of IL3Ra+ cells in breast tumor microenvironment
Description: IL3 exerts its biologic activity through its interaction with a cell surface receptor that consists of two subunits. The a subunit (CD123) specifically binds IL3, whereas the ß subunit is required for signaling and is common to the GMCSFR and IL5-R. 107D2.08 and 106C2.02 mAbs were obtained after mouse immunization with sorted human tonsillar PDC. Both clones strongly stain PDCs and basophils, weakly stain monocytes, CD34+ derived DCs and CD11c+ DC, while no staining is observed on T, B, NK cells as well as on mono-derived DCs. Staining with 107D2.08 and 106C2.02 mAbs are maintained on sorted PDC cultured in the presence of IL3 and CD40L, but lost when IL3 alone is added to the culture. The recognition of the IL3Ra chain by 107D2.08 and 106C2.02 was confirmed by transfection studies. 107D2.08 appeared to be the most appropriate clone for in situ studies. 107D2.08 allowed the first observation of IL3Ra+ cells in breast tumor microenvironment
Description: Langerin/CD207 is a transmembrane C-type lectin receptor (CLR) of epidermal and mucosal Langerhans cells (LCs) that induces Birbeck's granule formation. Langerin features a single carbohydrate recognition domain (CRD) with mannose-type specificity in its extracellular portion. Langerin is unique among the CLRs in that it contains an intracellular domain with a proline-rich motif. Langerin expression has not been reported outside the DC system. (Valladeau J et al, 1999, Eur.J.Immunol., 29:2695-2704; Valladeau J et al, 2000 Immunity, 12 : 71-81; Kashihara M et al, 1986, J.Invest.Derm., 87 :602-607 Ito T et al, 1999, J.Immunol., 163 :1409-1419 ;Saeland S & Valladeau J, CD207 (Langerin) Workshop reports 2002, Leukocyte-Typing VII, White Cell Diff Antigens, D. Mason et al, Eds, Oxford University Press:306-307)
Description: Langerin/CD207 is a transmembrane C-type lectin receptor (CLR) of epidermal and mucosal Langerhans cells (LCs) that induces Birbeck's granule formation. Langerin features a single carbohydrate recognition domain (CRD) with mannose-type specificity in its extracellular portion. Langerin is unique among the CLRs in that it contains an intracellular domain with a proline-rich motif. Langerin expression has not been reported outside the DC system. (Valladeau J et al, 1999, Eur.J.Immunol., 29:2695-2704; Valladeau J et al, 2000 Immunity, 12 : 71-81; Kashihara M et al, 1986, J.Invest.Derm., 87 :602-607 Ito T et al, 1999, J.Immunol., 163 :1409-1419 ;Saeland S & Valladeau J, CD207 (Langerin) Workshop reports 2002, Leukocyte-Typing VII, White Cell Diff Antigens, D. Mason et al, Eds, Oxford University Press:306-307)
Description: The IL7-R consists of 2 chains, IL-7R known as CD127 and common cytokine receptor chain known as CD132. A 75 to 80kDa human IL-7 receptor has been cloned that belongs to hematopoietic cytokinereceptor super family. R34-34, raised against human leukemic pre-B cells, recognized a molecule expressed on normal B cell precursors but not on mature B cells. This antibody specifically reverted IL-7 mediated growth inhibition of leukemic BCP (normal B cells precursors) and mature T cells. IL-7R expression is dramatically influenced by cytokines and antigens. This IL-7R displays both high and low affinity for its ligand (IL-7). Inhibitory and proliferative effects of IL-7 can be mediated through the same receptor on various lineages. CD4+ memory T cells express high level of IL-7R Subsets that express it generally require it, including progenitors of T and B cells, naïve and memory T cells. (Pandrau-Garcia D et al, 1994, Blood, 83, 3613-9 Mazzucchelli R et al, Nat. Review Immunol., 2007,7, 144-54)
Description: The IL7-R consists of 2 chains, IL-7R known as CD127 and common cytokine receptor chain known as CD132. A 75 to 80kDa human IL-7 receptor has been cloned that belongs to hematopoietic cytokinereceptor super family. R34-34, raised against human leukemic pre-B cells, recognized a molecule expressed on normal B cell precursors but not on mature B cells. This antibody specifically reverted IL-7 mediated growth inhibition of leukemic BCP (normal B cells precursors) and mature T cells. IL-7R expression is dramatically influenced by cytokines and antigens. This IL-7R displays both high and low affinity for its ligand (IL-7). Inhibitory and proliferative effects of IL-7 can be mediated through the same receptor on various lineages. CD4+ memory T cells express high level of IL-7R Subsets that express it generally require it, including progenitors of T and B cells, naïve and memory T cells. (Pandrau-Garcia D et al, 1994, Blood, 83, 3613-9 Mazzucchelli R et al, Nat. Review Immunol., 2007,7, 144-54)
Buffer: Liquid in PBS containing 50% glycerol, 0.5% BSA and 0.02% sodium azide. The antibody was affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogen.
Description: A polyclonal antibody against GPR12. Recognizes GPR12 from Human. This antibody is Unconjugated. Tested in the following application: WB, IF, ELISA;WB:1/500-1/2000.IF:1/200-1/1000.ELISA:1/5000
Buffer: Preservative: 0.03% Proclin 300 Constituents: 50% Glycerol, 0.01M PBS, pH 7.4 >95%, Protein G purified
Description: A polyclonal antibody against GPR12. Recognizes GPR12 from Human. This antibody is Unconjugated. Tested in the following application: ELISA, WB, IHC, IF; Recommended dilution: WB:1:500-1:5000, IHC:1:20-1:200, IF:1:50-1:200
Immunogen information: Synthesized peptide derived from the N-terminal region of human GPR12 at AA rangle: 10-90
Applications tips:
Description: A polyclonal antibody for detection of GPR12 from Human. This GPR12 antibody is for WB, IF, ELISA. It is affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogenand is unconjugated. The antibody is produced in rabbit by using as an immunogen synthesized peptide derived from the N-terminal region of human GPR12 at AA rangle: 10-90
Immunogen information: Synthesized peptide derived from the N-terminal region of human GPR12 at AA rangle: 10-90
Applications tips:
Description: A polyclonal antibody for detection of GPR12 from Human. This GPR12 antibody is for WB, IF, ELISA. It is affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogenand is unconjugated. The antibody is produced in rabbit by using as an immunogen synthesized peptide derived from the N-terminal region of human GPR12 at AA rangle: 10-90
Immunogen information: Synthesized peptide derived from the N-terminal region of human GPR12 at AA rangle: 10-90
Applications tips:
Description: A polyclonal antibody for detection of GPR12 from Human. This GPR12 antibody is for WB, IF, ELISA. It is affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogenand is unconjugated. The antibody is produced in rabbit by using as an immunogen synthesized peptide derived from the N-terminal region of human GPR12 at AA rangle: 10-90
Buffer: Preservative: 0.03% Proclin 300 Constituents: 50% Glycerol, 0.01M PBS, pH 7.4 >95%, Protein G purified
Description: A polyclonal antibody against GPR12. Recognizes GPR12 from Human. This antibody is HRP conjugated. Tested in the following application: ELISA
Buffer: Preservative: 0.03% Proclin 300 Constituents: 50% Glycerol, 0.01M PBS, pH 7.4 >95%, Protein G purified
Description: A polyclonal antibody against GPR12. Recognizes GPR12 from Human. This antibody is FITC conjugated. Tested in the following application: ELISA
Buffer: Preservative: 0.03% Proclin 300 Constituents: 50% Glycerol, 0.01M PBS, pH 7.4 >95%, Protein G purified
Description: A polyclonal antibody against GPR12. Recognizes GPR12 from Human. This antibody is Biotin conjugated. Tested in the following application: ELISA
Description: A polyclonal antibody raised in Rabbit that recognizes and binds to Human GPR12 (Extracellular Domain). This antibody is tested and proven to work in the following applications:
Description: A polyclonal antibody raised in Rabbit that recognizes and binds to Human GPR12 (N-Terminus). This antibody is tested and proven to work in the following applications:
Ilościowe wykrywanie i monitorowanie Colletotrichum siamense na drzewach kauczukowych przy użyciu PCR w czasie rzeczywistym
Colletotrichum siamense is without doubt one of the most necessary pathogens of rubber timber in Asia. The right detection and quantification of C. siamense populations in rubber timber are of significance for monitoring the epidemics of the illness. On this research, we developed an ITS-based real-time PCR technique to effectively detect C. siamense infecting rubber tree, which reliably detected as little as 100 fg genomic DNA, 100 copies of goal DNA and 20 conidia.
The actual-time PCR protocol acknowledged all C. siamense isolates collected from three provinces in China, whereas no amplification was noticed with rubber tree and its different pathogens. Detection and quantification of C. siamense have been carried out in artificially and naturally contaminated rubber leaves. We may nonetheless detect C. siamense in plant mixes of which solely 0.0001% of the tissue contaminated. An accumulation of C. siamense DNA was noticed throughout the entire an infection course of in any respect three leaf phenological levels, suggesting the real-time PCR technique can be utilized to watch C. siamense improvement in rubber timber. Lastly, the tactic allowed the detection of C. siamense in naturally contaminated and symptomless leaves of rubber timber within the fields. In contrast with earlier detection strategies, the real-time PCR technique is extra particular and extra delicate, and might be of nice use for research aiming to achieve a greater understanding of the epidemiology of Colletotrichum leaf illness, in addition to the prediction of illness danger and the management proposal.
Ilościowy pomiar liczby kopii transpozonu przy użyciu cyfrowej PCR kropli
Spontaneous proliferation of transposable parts contributes to genetic range at various ranges reminiscent of somatic mosaicism, genetic divergence in inhabitants, and genome evolution. Such genetic range is important for vegetation’ adaptation to altering atmosphere and serves as a useful useful resource for crop enchancment. Subsequently, measuring the copy quantity variation of transposable parts with precision and effectivity is necessary to grasp the extent of their proliferation.
Droplet Digital PCR (ddPCR) is an correct and delicate method that permits measurement of copy quantity variation of a transposon. Briefly, genomic DNA is extracted, digested, and partitioned into 1000’s of nanoliter-scale droplets. The TaqMan real-time PCR adopted by the end-point fluorescence detection allows the quantitative measurement of copy variety of template DNAs. Right here on this chapter, we describe the step-by-step process of ddPCR utilizing EVADE retrotransposon of Arabidopsis for example.
Porównanie wyników tomografii komputerowej (CT) z RT-PCR w diagnostyce COVID-19 u dzieci
polymerase chain response (RT-PCR) check ends in kids with possible or definitive prognosis of coronavirus illness 2019 (COVID-19).
Strategies: On this retrospective archive research, pediatric sufferers who have been adopted up within the hospital with a doable or definitive prognosis of COVID-19 and who had chest CT at presentation have been included. CT scan photos of the sufferers have been reinterpreted by a pediatric radiologist and in contrast with their RT-PCR check outcomes.
Outcomes: Of the whole of 89 sufferers, 33 had damaging and 56 had constructive RT-PCR exams. The presence of pulmonary lesions and consolidation was statistically considerably greater within the RT-PCR damaging group than within the RT-PCR constructive group (p = 0.037 and 0.001, respectively). Lobe involvement of 0%-25% was greater within the RT-PCR constructive group (p = 0.001), and lobe involvements of 25%-50% and 50%-75% have been considerably greater within the RT-PCR damaging group (p = 0.001 and 0.005, respectively). Central and perihilar involvement was discovered to be statistically important in the RT-PCR damaging group (p = 0.008 and 0.005, respectively).
Conclusion: Chest CT findings could present some clues in predicting RT-PCR positivity in kids with a possible prognosis of COVID-19. Lobe involvement proportion of as much as 25% is a discovering in favor of sufferers with constructive RT-PCR check, whereas 25%-75% lobe involvement, central and perihilar involvement, and consolidation might be interpreted in favor of sufferers with damaging RT-PCR check.
Integracja przygotowania próbek mikroprzepływowych z detekcją PCR w celu zbadania skutków jednoczesnej separacji inhibitora DNA i wymiany roztworu DNA
On this paper, we utilized a curved-channel microfluidic system to separate DNA from PCR-inhibitor-containing water and concurrently wash them into clear water for detection utilizing a conveyable PCR thermocycler. Environmental DNA (eDNA) sampling has turn into an efficient surveying method for detecting uncommon organisms. Nonetheless, low focus eDNA molecules could also be masked by PCR inhibitors throughout amplification and detection, growing the chance of false negatives. Subsequently, applied sciences for on-site DNA separation and washing are urgently wanted. Our system consisted of a half-circle microchannel with a DNA-inhibitor pattern inlet, a clear buffer inlet, and a number of retailers. Through the use of the flow-induced inertial forces, 10 μm DNA-conjugated microparticles have been targeted on the inner-wall of the curved microchannel whereas separation from 1 μm inhibitor-conjugated microparticles and DNA washing have been achieved concurrently with the Dean circulation.
We achieved singleplex focusing, isolation and washing of 10 μm particles at an effectivity of 94.5 ± 2.0%. In duplex experiments with 1 μm and 10 μm particles, bigger particles have been washed with an effectivity of 92.1 ± 1.6% and a purity of 79 ± 2%. By surface-functionalizing the microparticles with affinity teams towards Atlantic salmon DNA and humic acid (HA), and processing samples of varied concentrations in our system, we achieved an efficient purification and detection of DNA molecules utilizing the transportable PCR thermocycler. Our technique considerably decreased PCR quantitation cycles from Cq > 38 to Cq = 30.35 ± 0.5, which confirmed enhancement of PCR amplification. The proposed system takes a promising step ahead in pattern preparation in the direction of an built-in system that can be utilized for simultaneous purification and resolution alternate of DNA in point-of-need environmental monitoring functions.
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