Mass Spectrometry for Identification, Monitoring, and Minimal Residual Disease Detection of M-Proteins

BACKGROUND: Monoclonal gammopathies (MGs) are plasma cell disorders defined by the clonal expansion of plasma cells, resulting in the characteristic excretion of a monoclonal immunoglobulin (M-protein). M-protein detection and quantification are integral parts of the diagnosi

Development of a rapid and quantitative lateral flow assay for the simultaneous measurement of serum κ and λ immunoglobulin free light chains (FLC):inception of a new near-patient FLC screening tool

Item does not contain fulltextBACKGROUND: Serum free light chains (FLC) are sensitive biomarkers used for the diagnosis and management of plasma cell dyscrasias, such as multiple myeloma (MM), and are central to clinical screening algorithms and therapy response criteria. We have developed a portable, near-patient, lateral-flow test (Seralite(R)) that quantitates serum FLC in 10 min, and is designed to eliminate sample processing delays and accelerate decision-making in the clinic. METHODS: Assay interference, imprecision, lot-to-lot variability, linearity, and the utility of a competitive-inhibition design for the elimination of antigen-excess ('hook effect') were assessed. Reference ranges were calculated from 91 healthy donor sera. Preliminary clinical validation was conducted by retrospective analysis of sera from 329 patients. Quantitative and diagnostic results were compared to Freelite(R). RESULTS: Seralite(R) gave a broad competitive-inhibition calibration curve from below 2.5 mg/L to above 200 mg/L, provided good assay linearity (between 1.6 and 208.7 mg/L for kappa FLC and between 3.5 and 249.7 mg/L for lambda FLC) and sensitivity (1.4 mg/L for kappa FLC and 1.7 mg/L for lambda FLC), and eliminated anomalous results from antigen-excess. Seralite(R) gave good diagnostic concordance with Freelite(R) (Roche Hitachi Cobas C501) identifying an abnormal FLC ratio and FLC difference in 209 patients with newly diagnosed MM and differentiating these patients from normal healthy donors with polyclonal FLC. CONCLUSIONS: Seralite(R) sensitively quantitates FLC and rapidly identifies clinical conditions where FLC are abnormal, including MM

Selective cancer-germline gene expression in pediatric brain tumors

Cancer-germline genes (CGGs) code for immunogenic antigens that are present in various human tumors and can be targeted by immunotherapy. Their expression has been studied in a wide range of human tumors in adults. We measured the expression of 12 CGGs in pediatric brain tumors, to identify targets for therapeutic cancer vaccines. Real Time PCR was used to quantify the expression of genes MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-C2, NY-ESO-1 and GAGE-1,2,8 in 50 pediatric brain tumors of different histological subtypes. Protein expression was examined with immunohistochemistry. Fifty-five percent of the medulloblastomas (n = 11), 86% of the ependymomas (n = 7), 40% of the choroid plexus tumors (n = 5) and 67% of astrocytic tumors (n = 27) expressed one or more CGGs. Immunohistochemical analysis confirmed qPCR results. With exception of a minority of tumors, the overall level of CGG expression in pediatric brain tumors was low. We observed a high expression of at least one CGG in 32% of the samples. CGG-encoded antigens are therefore suitable targets in a very selected group of pediatric patients with a brain tumor. Interestingly, glioblastomas from adult patients expressed CGGs more often and at significantly higher levels compared to pediatric glioblastomas. This observation is in line with the notion that pediatric and adult glioblastomas develop along different genetic pathways

Regulation of MYCN expression in human neuroblastoma cells

Contains fulltext : 81722.pdf (publisher's version ) (Open Access)BACKGROUND: Amplification of the MYCN gene in neuroblastoma (NB) is associated with a poor prognosis. However, MYCN-amplification does not automatically result in higher expression of MYCN in children with NB. We hypothesized that the discrepancy between MYCN gene expression and prognosis in these children might be explained by the expression of either MYCN-opposite strand (MYCNOS) or the shortened MYCN-isoform (DeltaMYCN) that was recently identified in fetal tissues. Both MYCNOS and DeltaMYCN are potential inhibitors of MYCN either at the mRNA or at the protein level. METHODS: Expression of MYCN, MYCNOS and DeltaMYCN was measured in human NB tissues of different stages. Transcript levels were quantified using a real-time reverse transcriptase polymerase chain reaction assay (QPCR). In addition, relative expression of these three transcripts was compared to the number of MYCN copies, which was determined by genomic real-time PCR (gQPCR). RESULTS: Both DeltaMYCN and MYCNOS are expressed in all NBs examined. In NBs with MYCN-amplification, these transcripts are significantly higher expressed. The ratio of MYCN:DeltaMYCN expression was identical in all tested NBs. This indicates that DeltaMYCN and MYCN are co-regulated, which suggests that DeltaMYCN is not a regulator of MYCN in NB. However, the ratio of MYCNOS:MYCN expression is directly correlated with NB disease stage (p = 0.007). In the more advanced NB stages and NBs with MYCN-amplification, relatively more MYCNOS is present as compared to MYCN. Expression of the antisense gene MYCNOS might be relevant to the progression of NB, potentially by directly inhibiting MYCN transcription by transcriptional interference at the DNA level. CONCLUSION: The MYCNOS:MYCN-ratio in NBs is significantly correlated with both MYCN-amplification and NB-stage. Our data indicate that in NB, MYCN expression levels might be influenced by MYCNOS but not by DeltaMYCN

Maturation of monocyte-derived dendritic cells with Toll-like receptor 3 and 7/8 ligands combined with prostaglandin E2 results in high interleukin-12 production and cell migration

Dendritic cells (DC) are professional antigen-presenting cells of the immune system that play a key role in regulating T cell-based immunity. In vivo, the capacity of DC to activate T cells depends on their ability to migrate to the T cell areas of lymph nodes as well as on their maturation state. Depending on their cytokine-secreting profile, DC are able to skew the immune response in a specific direction. In particular, IL-12p70 producing DC drive T cells towards a T helper 1 type response. A serious disadvantage of current clinical grade ex vivo generated monocyte-derived DC is the poor IL-12p70 production. We have investigated the effects of Toll-like receptor (TLR)-mediated maturation on ex vivo generated human monocyte-derived DC. We demonstrate that in contrast to cytokine-matured DC, DC matured with poly(I:C) (TLR3 ligand) and/or R848 (TLR7/8 ligand) are able to produce vast amounts of IL-12p70, but exhibit a reduced migratory capacity. The addition of prostaglandin E2 (PGE2) improved the migratory capacity of TLR-ligand matured DC while maintaining their IL-12p70 production upon T cell encounter. We propose a novel clinical grade maturation protocol in which TLR ligands poly(I:C) and R848 are combined with PGE2 to generate DC with both high migratory capacity and IL-12p70 production upon T cell encounter

Septic AKI in ICU patients. diagnosis, pathophysiology, and treatment type, dosing, and timing: a comprehensive review of recent and future developments

Evidence is accumulating showing that septic acute kidney injury (AKI) is different from non-septic AKI. Specifically, a large body of research points to apoptotic processes underlying septic AKI. Unravelling the complex and intertwined apoptotic and immuno-inflammatory pathways at the cellular level will undoubtedly create new and exciting perspectives for the future development (e.g., caspase inhibition) or refinement (specific vasopressor use) of therapeutic strategies. Shock complicating sepsis may cause more AKI but also will render treatment of this condition in an hemodynamically unstable patient more difficult. Expert opinion, along with the aggregated results of two recent large randomized trials, favors continuous renal replacement therapy (CRRT) as preferential treatment for septic AKI (hemodynamically unstable). It is suggested that this approach might decrease the need for subsequent chronic dialysis. Large-scale introduction of citrate as an anticoagulant most likely will change CRRT management in intensive care units (ICU), because it not only significantly increases filter lifespan but also better preserves filter porosity. A possible role of citrate in reducing mortality and morbidity, mainly in surgical ICU patients, remains to be proven. Also, citrate administration in the predilution mode appears to be safe and exempt of relevant side effects, yet still requires rigorous monitoring. Current consensus exists about using a CRRT dose of 25 ml/kg/h in non-septic AKI. However, because patients should not be undertreated, this implies that doses as high as 30 to 35 ml/kg/h must be prescribed to account for eventual treatment interruptions. Awaiting results from large, ongoing trials, 35 ml/kg/h should remain the standard dose in septic AKI, particularly when shock is present. To date, exact timing of CRRT is not well defined. A widely accepted composite definition of timing is needed before an appropriate study challenging this major issue can be launched

Reference ranges of the Sebia free light chain ratio in patients with chronic kidney disease.

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