Comparison of Quantitative Cytomegalovirus Real-time PCR in Whole Blood and pp65 Antigenemia Assay: Clinical Utility of CMV Real-time PCR in Hematopoietic Stem Cell Transplant Recipients

Successful preemptive therapy for cytomegalovirus (CMV) infection in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infection. Although the pp65 antigenemia assay has been widely used for this purpose, real-time quantification of CMV DNA has recently been recognized as an alternative diagnostic approach. However, the guidelines for antiviral therapy based on real-time quantitative polymerase chain reaction (RQ-PCR) have yet to be established. From November 2004 to March 2005, a total of 555 whole blood samples from 131 hematopoietic stem cell transplant (HSCT) recipients were prospectively collected. RQ-PCR was conducted using an Artus® CMV LC PCR kit (QIAGEN). Both qualitative and quantitative correlations were drawn between the two methods. Exposure to the antiviral agent influenced the results of the two assays. Additionally, the discrepancy was observed at low levels of antigenemia and CMV DNA load. Via ROC curve analysis, the tentative cutoff value for preemptive therapy was determined to be approximately 2×104 copies/mL (sensitivity, 80.0%; specificity, 50.0%) in the high risk patients, and approximately 3×104 copies/mL (sensitivity, 90.0%; specificity, 70.0%) in the patients at low risk for CMV disease. Further study to validate the optimal cutoff value for the initiation of preemptive therapy is currently underway

Development of a thermal sensor to probe cell viability and concentration in cell suspensions

This paper presents a novel biothermal sensor to probe cell viability and concentration of a cell suspension. The sensing technique exploits the thermophysical properties of the suspension, so no labeling of suspended cells is required. When the sensor is periodically heated, the amplitude and phase of the thermal signal are dependent on the thermal properties of the cell suspension, particularly the thermal conductivity k. We measured k of HeLa, hepatocyte, and NIH-3T3 J2 cell suspensions with various concentrations and viabilities. The results demonstrate that the k of a cell suspension has a strong correlation with its concentration and viability. Accordingly, k can be employed as an index of cell concentration and viability. Furthermore, without data processing to obtain k, the electric signal that reflects the thermal response of the sensor can be used as a tool to probe viability of a cell suspension in real time. The proposed thermal sensing technique offers label-free, non-invasive, long-term, and real-time means to probe the viability and concentration of cells in a suspension

Immune gene expression networks in sepsis: A network biology approach.

To study the dysregulated host immune response to infection in sepsis, gene expression profiles from the Gene Expression Omnibus (GEO) datasets GSE54514, GSE57065, GSE64456, GSE95233, GSE66099 and GSE72829 were selected. From the Kyoto Encyclopedia of Genes and Genomes (KEGG) immune system pathways, 998 unique genes were selected, and genes were classified as follows based on gene annotation from KEGG, Gene Ontology, and Reactome: adaptive immunity, antigen presentation, cytokines and chemokines, complement, hematopoiesis, innate immunity, leukocyte migration, NK cell activity, platelet activity, and signaling. After correlation matrix formation, correlation coefficient of 0.8 was selected for network generation and network analysis. Total transcriptome was analyzed for differentially expressed genes (DEG), followed by gene set enrichment analysis. The network topological structure revealed that adaptive immunity tended to form a prominent and isolated cluster in sepsis. Common genes within the cluster from the 6 datasets included CD247, CD8A, ITK, LAT, and LCK. The clustering coefficient and modularity parameters were increased in 5/6 and 4/6 datasets in the sepsis group that seemed to be associated with functional aspect of the network. GSE95233 revealed that the nonsurvivor group showed a prominent and isolated adaptive immunity cluster, whereas the survivor group had isolated complement-coagulation and platelet-related clusters. T cell receptor signaling (TCR) pathway and antigen processing and presentation pathway were down-regulated in 5/6 and 4/6 datasets, respectively. Complement and coagulation, Fc gamma, epsilon related signaling pathways were up-regulated in 5/6 datasets. Altogether, network and gene set enrichment analysis showed that adaptive-immunity-related genes along with TCR pathway were down-regulated and isolated from immune the network that seemed to be associated with unfavorable prognosis. Prominence of platelet and complement-coagulation-related genes in the immune network was associated with survival in sepsis. Complement-coagulation pathway was up-regulated in the sepsis group that was associated with favorable prognosis. Network and gene set enrichment analysis supported elucidation of sepsis pathogenesis

Regulatory Mechanism between Ferritin and Mitochondrial Reactive Oxygen Species in Spinal Ligament-Derived Cells from Ossification of Posterior Longitudinal Ligament Patient

Primary spinal ligament-derived cells (SLDCs) from cervical herniated nucleus pulposus tissue (control, Ctrl) and ossification of the posterior longitudinal ligament (OPLL) tissue of surgical patients were analyzed for pathogenesis elucidation. Here, we found that decreased levels of ferritin and increased levels of alkaline phosphatase (ALP), a bone formation marker, provoked osteogenesis in SLDCs in OPLL. SLDCs from the Ctrl and OPLL groups satisfied the definition of mesenchymal stem/stromal cells. RNA sequencing revealed that oxidative phosphorylation and the citric acid cycle pathway were upregulated in the OPLL group. SLDCs in the OPLL group showed increased mitochondrial mass, increased mitochondrial reactive oxygen species (ROS) production, decreased levels of ROS scavengers including ferritin. ROS and ferritin levels were upregulated and downregulated in a time-dependent manner, and both types of molecules repressed ALP. Osteogenesis was mitigated by apoferritin addition. We propose that enhancing ferritin levels might alleviate osteogenesis in OPLL

Impact of Integrated Genetic Information on Diagnosis and Prognostication for Myeloproliferative Neoplasms in the Next-Generation Sequencing Era

Since next-generation sequencing has been widely used in clinical laboratories, the diagnosis and risk stratification of hematologic malignancies are greatly dependent on genetic aberrations. In this study, we analyzed the genomic landscapes of 200 patients with myeloproliferative neoplasms (MPNs) and evaluated the impact of the genomic landscape on diagnosis and risk stratification. Mutations in JAK2, CALR and MPL were detected in 76.4% of MPNs. The proportion of patients with clonal genetic markers increased up to 86.4% when all detectable genetic aberrations were included. Significant co-occurring genetic aberrations potentially associated with phenotype and/or disease progression, including those in JAK2/SF3B1 and TP53/del(13q), del(5q), −7/del(7q) and complex karyotypes, were detected. We also identified genetic aberrations associated with patient outcomes: TP53 and −7/del(7q) were associated with an inferior chance of survival, RUNX1, TP53 and IDH1/2 were associated with leukemic transformation and SF3B1, IDH1/2, ASXL1 and del(20q) were associated with fibrotic progression. We compared risk stratification systems and found that mutation-enhanced prognostic scoring systems could identify lower risk polycythemia vera, essential thrombocythemia and higher risk primary myelofibrosis. Furthermore, the new risk stratification systems showed a better predictive capacity for patient outcome. These results collectively indicate that integrated genetic information can enhance diagnosis and prognostication in patients with myeloproliferative neoplasms

Genetic Variants Associated with Drug Resistance of Cytomegalovirus in Hematopoietic Cell Transplantation Recipients

Cytomegalovirus (CMV) infection is a serious complication in hematopoietic cell transplantation (HCT) recipients. Drug-resistant strains make it more challenging to treat CMV infection. This study aimed to identify variants associated with CMV drug resistance in HCT recipients and assess their clinical significance. A total of 123 patients with refractory CMV DNAemia out of 2271 HCT patients at the Catholic Hematology Hospital between April 2016 and November 2021 were analyzed, which accounted for 8.6% of the 1428 patients who received pre-emptive therapy. Real-time PCR was used to monitor CMV infection. Direct sequencing was performed to identify drug-resistant variants in UL97 and UL54. Resistance variants were found in 10 (8.1%) patients, and variants of uncertain significance (VUS) were found in 48 (39.0%) patients. Patients with resistance variants had a significantly higher peak CMV viral load than those without (p = 0.015). Patients with any variants had a higher risk of severe graft-versus-host disease and lower one-year survival rates than those without (p = 0.003 and p = 0.044, respectively). Interestingly, the presence of variants reduced the rate of CMV clearance, particularly in patients who did not modify their initial antiviral regimen. However, it had no apparent impact on individuals whose antiviral regimens were changed due to refractoriness. This study highlights the importance of identifying genetic variants associated with CMV drug resistance in HCT recipients for providing appropriate antiviral treatment and predicting patient outcomes

Genetic and epigenetic alterations of bone marrow stromal cells in myelodysplastic syndrome and acute myeloid leukemia patients

We evaluated the characteristics of bone marrow stromal cells (BMSCs) and hematopoietic cells (HCs) from patients of myelodysplastic syndrome (MDS, n = 21) and acute myeloid leukemia (AML, n = 58), and compared the results with control BMSCs derived from healthy donors (n = 8). The patient BMSCs had lower proliferative activity than that of the controls due to increased senescence. This retarded proliferation induced failure to obtain enough metaphase cells for karyotyping in patient BMSCs (10%). Patient BMSCs were genetically altered which was demonstrated by chromosome abnormalities in 5% of the patients (one MDS and three AML), whereas no clonal abnormalities were detected in the controls. The most common abnormality of the BMSCs was an extra chromosome 5, followed by an extra chromosome 7 and balanced translocations. The proportion of the abnormal metaphase cells was low (17.8%). We also analyzed the epigenetic changes of long interspersed nucleotide element 1 (LINE-1) repetitive element and CDKN2B using pyrosequencing. The quantitative measurement of global LINE-1 methylation demonstrated that patient BMSCs revealed global hypomethylation (68.2 ± 3.8) compared with controls (72.9 ± 3.4, P < 0.001) and that the global hypomethylation of BMSCs were more significant in AML than in MDS patients (67.9 ± 3.8, 69.4 ± 4.2, respectively). These findings seem worthy of further evaluation of their association with ineffective hematopoiesis and leukemogenesis