Presepsin production in monocyte/macrophage-mediated phagocytosis of neutrophil extracellular traps.

Presepsin, a biomarker discovered in Japan, has been clinically applied as a diagnostic aid for sepsis. Recently, however, it has been reported that presepsin levels are elevated in patients with severe systemic lupus erythematosus without infection, suggesting the existence of a production mechanism that does not involve bacterial phagocytosis. In this study, we aimed to elucidate the mechanism of presepsin production without bacterial phagocytosis and explore the clinical significance of presepsin. Neutrophil extracellular traps (NETs) were induced by Escherichia coli and phorbol myristate acetate (PMA) in neutrophils isolated from the peripheral blood of healthy subjects. NET induction alone did not increase presepsin levels, but co-culturing with monocytes significantly increased them. The addition of a NET formation inhibitor also suppressed presepsin levels, suggesting that presepsin production is greatly influenced by monocyte phagocytosis of NETs. Phagocytosis of NETs by THP-1 and U937 cells, which was induced by CD14 expression, also increased presepsin levels. This study suggests that presepsin can be used to assess the severity of inflammatory diseases, such as autoimmune diseases, and monitor treatment effects.

Usefulness of presepsin / creatinine ratio as a new index that corrects for renal function.

Background : Presepsin (P-SEP) is a highly specific sepsis marker, and its fluctuation with respect to advanced renal impairment or sample agitation has not been fully investigated. We evaluated several renal function-corrected P-SEP indices to establish a simple index and its reference range. Methods : Blood samples for P-SEP measurement were collected with minimal agitation. P-SEP levels were measured using the rapid automated immunoanalyzer "PATHFAST." This study included 85 chronic kidney disease (CKD) patients, 65 healthy volunteers, and 4 sepsis patients. Results : Patients stratified by estimated glomerular filtration rate (GFR) had significantly higher P-SEP levels for CKD stage G3, especially the advanced GFR stage. We evaluated presepsin / creatinine (P-SEP / CRE) and P-SEP / eGFR ratios as possible indices for renal function. The P-SEP / CRE ratio exhibited no increase correlating with the GFR stage and was identical in the normal and CKD groups ;  P-SEP / eGFR decreased if GFR stage worsened. The P-SEP / CRE ratio became significantly higher in sepsis patients and was a more useful index with a reference range of 67-263. Conclusions : P-SEP levels were inversely correlated with renal function, indicating the necessity to consider the influence of renal impairment in CKD patients. The P-SEP / CRE ratio is helpful for sepsis diagnosis, even in patients with renal impairment. J. Med. Invest. 68 : 105-111, February, 2021.

The ADVIA2120i parameter Revised %MICRO is a surrogate marker of schistocyte formation after hematopoietic stem cell transplantation.

Objectives : Hematopoietic stem cell transplantation (HSCT)-associated thrombotic microangiopathy (TA-TMA) is an important early post-treatment condition. This study evaluated the Revised %MICRO, a parameter obtained from the ADVIA 2120i automated blood cell counter, as a surrogate marker of the schistocyte ratio. We hypothesized that individual differences between the %MICRO value and schistocyte ratio would remain constant. Design and Methods: EDTA-2K-treated peripheral blood samples were collected from 19 patients who underwent allogeneic HSCT from April 2014 to September 2018. First, the baseline difference, X, was calculated using a sample from the first day after HSCT as X = %MICRO (first day) - schistocyte ratio (first day). Next, the Revised %MICRO for each subsequent day was calculated as Revised %MICRO = %MICRO - X. We evaluated correlations of the schistocyte ratio with the calculated %MICRO and Revised %MICRO and the RBC fragment, RBC distribution width, %MICRO and Revised %MICRO data obtained from the ADVIA 2120i. Results : The mean schistocyte percentage and Revised %MICRO were both 0.4% ±â€…0.6. RBC fragments correlated weakly with the %MICRO and schistocyte ratio, respectively (r = 0.162 and r = 0.771, respectively), whereas the Revised %MICRO correlated strongly with the schistocyte ratio (r = 0.893). Conclusion : The Revised %MICRO appears to be a good surrogate of the schistocyte ratio in a clinical setting. J. Med. Invest. 67 : 250-254, August, 2020.

Clinical evaluation of presepsin considering renal function.

Presepsin, a glycoprotein produced during bacterial phagocytosis, is used as a sepsis marker for bacterial infections. However, presepsin levels are affected by renal function, and the evaluation criteria according to kidney function or in chronic kidney diseases remain controversial. Furthermore, presepsin may be increased by sample stirring, but no studies have evaluated this effect.In this study, we excluded the effect of stirring by standardizing the blood collection conditions, analyzed the influence of kidney function on presepsin concentrations, and recalculated the reference range based on the findings. EDTA-whole blood from 47 healthy subjects and 85 patients with chronic kidney disease was collected to measure presepsin by PATHFAST. Presepsin was found to be significantly correlated with the levels of creatinine (r = 0.834), eGFRcreat (r = 0.837), cystatin-C (r = 0.845), and eGFRcys (r = 0.879). Furthermore, in patients with CKD, presepsin levels stratified by eGFRcys showed a significant increase in the CKD G2 patient group and with advancing glomerular filtration rate stage. The following values were obtained: Normal: 97.6 ± 27.4 pg/mL, CKD G1: 100.2 ± 27.6 pg/mL, CKD G2: 129.7 ± 40.7 pg/mL, CKD G3: 208.1 ± 70.2 pg/mL, CKD G4: 320.2 ± 170.1 pg/mL, CKD G5: 712.8 ± 336.3 pg/mL. The reference range, calculated by a nonparametric method using 67 cases of healthy volunteers and patients with chronic kidney disease G1, was found to be 59-153 pg/mL, which was notably lower than the standard reference range currently used. Presepsin concentrations were positively correlated with a few biomarkers of renal function, indicating the necessity to consider the effect of renal function in patients with renal impairment. Using the recalculated reference range considering kidney function may improve the accuracy of evaluating presepsin for diagnosis of sepsis compared to the standard reference currently in use.

Combination with a defucosylated anti-HM1.24 monoclonal antibody plus lenalidomide induces marked ADCC against myeloma cells and their progenitors.

The immunomodulatory drug lenalidomide (Len) has drawn attention to potentiate antibody-dependent cellular cytotoxicity (ADCC)-mediated immunotherapies. We developed the defucosylated version (YB-AHM) of humanized monoclonal antibody against HM1.24 (CD317) overexpressed in multiple myeloma (MM) cells. In this study, we evaluated ADCC by YB-AHM and Len in combination against MM cells and their progenitors. YB-AHM was able to selectively kill via ADCC MM cells in bone marrow samples from patients with MM with low effector/target ratios, which was further enhanced by treatment with Len. Interestingly, Len also up-regulated HM1.24 expression on MM cells in an effector-dependent manner. HM1.24 was found to be highly expressed in a drug-resistant clonogenic "side population" in MM cells; and this combinatory treatment successfully reduced SP fractions in RPMI 8226 and KMS-11 cells in the presence of effector cells, and suppressed a clonogenic potential of MM cells in colony-forming assays. Collectively, the present study suggests that YB-AHM and Len in combination may become an effective therapeutic strategy in MM, warranting further study to target drug-resistant MM clonogenic cells.

Glycolysis inhibition inactivates ABC transporters to restore drug sensitivity in malignant cells.

Cancer cells eventually acquire drug resistance largely via the aberrant expression of ATP-binding cassette (ABC) transporters, ATP-dependent efflux pumps. Because cancer cells produce ATP mostly through glycolysis, in the present study we explored the effects of inhibiting glycolysis on the ABC transporter function and drug sensitivity of malignant cells. Inhibition of glycolysis by 3-bromopyruvate (3BrPA) suppressed ATP production in malignant cells, and restored the retention of daunorubicin or mitoxantrone in ABC transporter-expressing, RPMI8226 (ABCG2), KG-1 (ABCB1) and HepG2 cells (ABCB1 and ABCG2). Interestingly, although side population (SP) cells isolated from RPMI8226 cells exhibited higher levels of glycolysis with an increased expression of genes involved in the glycolytic pathway, 3BrPA abolished Hoechst 33342 exclusion in SP cells. 3BrPA also disrupted clonogenic capacity in malignant cell lines including RPMI8226, KG-1, and HepG2. Furthermore, 3BrPA restored cytotoxic effects of daunorubicin and doxorubicin on KG-1 and RPMI8226 cells, and markedly suppressed subcutaneous tumor growth in combination with doxorubicin in RPMI8226-implanted mice. These results collectively suggest that the inhibition of glycolysis is able to overcome drug resistance in ABC transporter-expressing malignant cells through the inactivation of ABC transporters and impairment of SP cells with enhanced glycolysis as well as clonogenic cells.

KRN5500, a spicamycin derivative, exerts anti-myeloma effects through impairing both myeloma cells and osteoclasts.

The spicamycin analogue KRN5500 alters glycoprotein processing and induces damage in the endoplasmic reticulum (ER)-Golgi apparatus in cancer cells. In the present study, we explored the cytotoxic effects of KRN5500 on multiple myeloma (MM) cells and the bone marrow microenvironment with special reference to ER stress. Cell proliferation assay showed that KRN5500 induced G1 arrest and apoptosis in MM cells in a time- and dose-dependent manner. KRN5500 enhanced ER stress independently of caspase activation in MM cells. This cell death was observed even in the presence of bone marrow stroma cells or osteoclasts. Notably, KRN5500 induced cell death also in osteoclasts. In vivo effects of KRN5500 were evaluated using two xenograft models established in severe combined immunodeficient (SCID) mice by either subcutaneous injection of RPMI 8226 cells or intra-bone injection of INA-6 cells to subcutaneously implanted rabbit bones (SCID-rab model). KRN5500 significantly inhibited tumour growth in both animal models, and decreased the number of osteoclasts, which resulted in prevention of bone destruction in the SCID-rab model. These results suggest that KRN5500 exerts anti-MM effects through impairing both MM cells and osteoclasts. Therefore, this unique mechanism of KRN5500 might be a useful therapeutic option in patients with MM.