How to choose the right real-time RT-PCR primer sets for the SARS-CoV-2 genome detection?

Objectives: The SARS-CoV-2 pandemic has created an unprecedented need for rapid large-scale diagnostic testing to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays recommended by the World Health Organization are being used by clinical and public health laboratories and typically target regions of the RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) coding region. However, it is currently unclear if results from different tests are comparable.

Detection of Human Microchimerism following Allogeneic Cell Transplantation Using Droplet Digital PCR.

Background: Cell transplantation is in clinical development for the treatment of various ailments including acquired and inborn hepatic diseases. Detection and quantification of the donor cells after infusion remain difficult. Traditional methods (sex-based FISH, HLA mismatch, and Short Tandem Repeat PCR) can only achieve low levels of sensitivity (1%) and therefore are seldom used.

Evaluation of minimal disseminated disease in cryopreserved ovarian tissue from bone and soft tissue sarcoma patients.

Study Question: What is the risk of finding malignant cells in cryopreserved ovarian tissue from sarcoma patients?

Summary Answer: Minimal disseminated disease (MDD) was not detected in frozen-thawed ovarian tissue from 26 patients by any of the sensitive methods applied.

What Is Known Already: In case of leukemia, the risk of malignant cell transmission through the graft is well known and widely documented. However, for bone cancer, like Ewing sarcoma or osteosarcoma, only a small number of case reports, have been published.

A novel approach for BCR-ABL1 standardization to improve International Scale estimation.

Introduction: Standardization of BCR-ABL1 messenger RNA quantification by real-time PCR on the International Scale (IS) is critical for monitoring therapy response in chronic myelogenous leukaemia. Since 2006, BCR-ABL1 IS standardization is propagated along reference laboratories by calculating a laboratory-specific conversion factor (CF), co-ordinated in Europe through the European Treatment and Outcome Study project. Although this process has proven successful to some extent, it has not been achievable for all laboratories due to the complexity of the process and the stringent requirements in terms of numbers of samples to be exchanged.

Expression of lineage markers using real-time quantitative polymerase chain reaction (RT-qPCR) in normal and in leukemia bone marrow.

Background: The study of lineage markers by real-time quantitative polymerase chain reaction (RT-qPCR) at diagnosis enables differentiation between acute myeloblastic leukemia, B- or T-lineage acute lymphoblastic leukemia, without cell sorting. Our objective was to assess the relationship between protein expression and the amount of lineage marker mRNA in acute leukemia samples and to determine whether four lineage markers could be used to differentiate between normal and acute leukemia bone marrow (BM) without cell sorting.

Methods: Quantification of the mRNA of CD19, CD79a, CD3e, and myeloperoxidase was performed by RT-qPCR on 130 acute leukemia BM samples at diagnosis and on 20 BM samples from healthy donors, without cell sorting.

Cholesterol-sensitive modulation of transcytosis.

Cholesterol-rich membrane domains (e.g., lipid rafts) are thought to act as molecular sorting machines, capable of coordinating the organization of signal transduction pathways within limited regions of the plasma membrane and organelles.

Early immunological monitoring after pediatric liver transplantation: cytokine immune deviation and graft acceptance in 40 recipients.

Cytokine deviation may be a factor contributing to graft acceptance. We analyze, in the context of liver transplantation, circulating cytokine levels and their mRNA precursors in liver biopsy samples to study a putative correlation with early immunologic outcome. Forty primary pediatric liver recipients were submitted to a prospective immune monitoring protocol, including 8 of 40 patients with an early, biopsy-proven acute rejection episode.

Increased galectin-3 expression and intra-epithelial neutrophils in small airways in severe COPD.

Galectins-1 and -3 regulate epithelial proliferation/apoptosis and neutrophil activation, and are implicated in lung cancer and asthma. The role of galectins in chronic obstructive pulmonary disease (COPD), characterised by epithelial changes and neutrophil infiltration, remains unknown. In the present study, galectin-1 and -3 expression was assessed by immunohistology in the bronchial epithelium of lung specimens from eight severe COPD patients and compared with nine nonsmokers and six smokers without COPD.

Degalactosylated and/or denatured IgA, but not native IgA in any form, bind to mannose-binding lectin.

Mannose-binding lectin (MBL) is reported to bind to agalactosyl IgG, but not to normally galactosylated (native) IgG. It was recently reported that serum polymeric IgA in its native form reacts with MBL, whereas a more recent report has claimed that native IgD and IgE, and possibly IgM, do not. This led us to investigate whether IgA is truly reactive with MBL.

Mucosal immunity in asthma and chronic obstructive pulmonary disease: a role for immunoglobulin A?

Despite our knowledge on the role of IgA in mucosal homeostasis and host defense and clinical evidence suggesting deficient first-line defense mechanisms in chronic airway disorders, little is known regarding its role in asthma and chronic obstructive pulmonary disease (COPD). Studies suggest that the mucosal IgA response is impaired in COPD, and a deficient transport of IgA across the bronchial epithelium in COPD has been identified, possibly involving neutrophil proteinases, which may degrade the Ig receptor mediating this transepithelial routing. In contrast, the IgA response to allergens in patients with asthma may play a pathogenic role through eosinophil activation.

Evaluation of real-time PCR data.

If real-time PCR is to be of much worth to its user, some idea regarding the reliability of its data is essential. We discuss here some of the problems associated with interpreting numerical real-time PCR data that lend themselves to analytical evaluation. We translate into the language of molecular biology some of the criteria which are used to evaluate the performance of any new method (linearity, precision, specificity, limit of detection and quantification).

Endoplasmic reticulum resident, immunoglobulin joining chain, can be secreted by perturbation of the calcium concentration in the endoplasmic reticulum.

We established a transient human joining (J)-chain gene expression system in the baby hamster kidney (BHK) cell. The J-chain was detected as a 29-kDa single band on Western blotting. Immunofluorescent staining of the transfectant revealed an exclusive localization of the J-chain in the endoplasmic reticulum (ER).

Characterization of the interaction of the pneumococcal surface protein SpsA with the human polymeric immunoglobulin receptor (hpIgR).

Background & Objectives: The polymeric immunoglobulin receptor (pIgR) is produced by mucosal epithelial cells and plays a crucial role in mucosal immunity. At the basolateral surface of mucosal cells, the pIgR binds predominantly polymeric immunoglobulins, such as dimeric IgA and polymeric IgA (pIgA) and mediates their transport across the polarized cells. This results in apical release of secretory component (SC), either free or bound covalently to IgA, forming secretory IgA (SIgA).

Differentiation of acute myeloid leukemia from B- and T-lineage acute lymphoid leukemias by real-time quantitative reverse transcription-PCR of lineage marker mRNAs.

Background: Flow cytometry of lineage markers is useful in the classification of leukemias. Our aim was to assess whether the study of lineage genes at the RNA level would enable differentiation of acute myeloid leukemias (AMLs) from B-and T-lineage acute lymphoid leukemias (ALLs).

Methods: We measured mRNA of four lineage markers [CD19, CD79a, CD3e, and myeloperoxidase (MPO)] by reverse transcription followed by real-time quantitative (RTQ)-PCR.

Free fetal DNA concentration in maternal plasma during normal labour at term.

Objective: Our aim was to investigate the effect of uterine contractions on free fetal DNA concentration in maternal plasma at term.

Study Design: Nine pregnant women were admitted for elective induction of labour between 38 and 40 weeks of gestation. All patients carrying male fetuses and without history of pre-term labour and membrane rupture were selected.

Ectodomains 3 and 4 of human polymeric Immunoglobulin receptor (hpIgR) mediate invasion of Streptococcus pneumoniae into the epithelium.

Streptococcus pneumoniae binds to the ectodomain of the human polymeric Ig receptor (pIgR), also known as secretory component (SC), via a hexapeptide motif in the choline-binding protein SpsA. The SpsA-pIgR interaction mediates adherence and internalization of the human pathogen into epithelial cells. In this study the results of SpsA binding to human, mouse, and chimeric SC strongly supported the human specificity of this unique interaction and suggested that binding sites in the third and fourth Ig-like domain of human SC (D3 and D4, respectively) are involved in SpsA-pIgR complex formation.

Secretory component is cleaved by neutrophil serine proteinases but its epithelial production is increased by neutrophils through NF-kappa B- and p38 mitogen-activated protein kinase-dependent mechanisms.

We previously showed that expression of polymeric immunoglobulin receptor (pIgR)/secretory component (SC), the epithelial receptor assuming transport of polymeric IgA in mucosal secretions, is strongly decreased in severe chronic obstructive pulmonary disease. Here, we evaluated in vitro the effects of polymorphonuclear neutrophil (PMN) mediators on pIgR/SC. On polyacrylamide gel electrophoresis analysis, soluble SC was rapidly cleaved by supernatants from phorbol-myristate-acetate-activated PMN, through a serine proteinase activity.

Oxidative burst in lipopolysaccharide-activated human alveolar macrophages is inhibited by interleukin-9.

Interleukin (IL)-9 is known to regulate many cell types involved in T-helper type 2 responses classically associated with asthma, including B- and T-lymphocytes, mast cells, eosinophils and epithelial cells. In contrast, target cells mediating the effects of IL-9 in the lower respiratory tract remain to be identified. Therefore, the authors evaluated the activity of IL-9 on human alveolar macrophages (AM) from healthy volunteers.

Rab11 family interacting protein 2 associates with Myosin Vb and regulates plasma membrane recycling.

Plasma membrane recycling is an important process necessary for maintaining membrane composition. The motor protein myosin Vb regulates plasma membrane recycling through its association with Rab11a. Overexpression of the tail of myosin Vb disrupts trafficking out of plasma membrane recycling systems and leads to the accumulation of Rab11a in both polarized and non-polarized cells.

MAL2, a novel raft protein of the MAL family, is an essential component of the machinery for transcytosis in hepatoma HepG2 cells.

Transcytosis is used alone (e.g., hepatoma HepG2 cells) or in combination with a direct pathway from the Golgi (e.

Different temperature sensitivity of endosomes involved in transport to lysosomes and transcytosis in rat hepatocytes: analysis by free-flow electrophoresis.

Endocytosis at reduced temperature has been used to define and characterize endosome subpopulations. Thus, the temperature sensitivity of endosome subpopulations involved in transport to lysosomes and transcytosis in rat hepatocytes was analyzed applying endosome labeling in the isolated perfused rat liver with route-specific ligands in combination with temperature shift protocols. Free-flow electrophoresis (FFE) that separates membranes and organelles based on their surface charge was then applied to isolate functional endosomes.

IL-9 inhibits oxidative burst and TNF-alpha release in lipopolysaccharide-stimulated human monocytes through TGF-beta.

IL-9 is a Th2 cytokine that exerts pleiotropic activities on T cells, B cells, mast cells, hematopoietic progenitors, and lung epithelial cells, but no effect of this cytokine has been reported so far on mononuclear phagocytes. Human blood monocytes preincubated with IL-9 for 24 h before LPS or PMA stimulation exhibited a decreased oxidative burst, even in the presence of IFN-gamma. The inhibitory effect of IL-9 was specifically abolished by anti-hIL-9R mAb, and the presence of IL-9 receptors was demonstrated on human blood monocytes by FACS.

Effect of IgA on respiratory burst and cytokine release by human alveolar macrophages: role of ERK1/2 mitogen-activated protein kinases and NF-kappaB.

Human alveolar macrophages (HAM) express FcalphaR receptors for immunoglobulin (Ig)A which could link humoral and cellular branches of lung immunity. Here, we investigate the effects of polymeric (p-IgA) and secretory (S-IgA) IgA interaction with Fc(alpha)R on lipopolysaccharide (LPS)- and phorbol myristate acetate (PMA)-activated respiratory burst and TNF-alpha release by HAM. Activation of HAM with LPS and PMA increases the respiratory burst and TNF-alpha release through activation of the extracellular signal-related protein kinases 1 and 2 (ERK1/2) pathway, because these effects are inhibited by treatment of HAM with PD98059, a selective inhibitor of mitogen-activated protein (MAP)/ERK kinases (MEK) pathway.