Tumor-Specific miRNA Signatures in Combination with CA19-9 for Liquid Biopsy-Based Detection of PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most aggressive malignancies and has high mortality and poor survival rates. Therefore, there is an urgent need to discover non-invasive biomarkers for early detection before PDAC reaches the incurable stage. We hypothesized that liquid biopsy of PDAC-derived extracellular vesicles (PDEs) containing abundant microRNAs (miRNAs) could be used for early diagnosis of PDAC because they can be selectively enriched and because they are biologically stable. We isolated PDEs by immunocapture using magnetic beads, and we identified 13 miRNA candidates in 20 pancreatic cancer patients and 20 normal controls. We found that expression of five miRNAs, including miR-10b, miR-16, miR-155, miR-429, and miR-1290, was markedly higher in PDEs. Furthermore, the miRNA signatures along with serum carbohydrate antigen 19-9 (CA19-9) were optimized by logistic regression, and the miRNA signature and CA19-9 combination markers (CMs) were effective at differentiating PDAC patients from normal controls. As a result, the CMs represented a high sensitivity (AUC, 0.964; sensitivity, 100%; specificity, 80%) and a high specificity (AUC, 0.962; sensitivity, 85.71%; specificity, 100%). These findings suggest that five miRNAs expressed in PDEs and CA19-9 are valuable biomarkers for screening and diagnosis of pancreatic cancer by liquid biopsy.

Cryopreserved cord blood mononuclear cells in DMSO are healthy for at least 6 hours after thawing.

PURPOSES: We investigated the impact of time, storage temperature, and dimethyl sulfoxide (DMSO) on the viability of HSCs, as well as on apoptotic changes in thawed CB. MATERIALS & METHODS: Thirteen units of cryopreserved CB were thawed and half of each sample was stored at room temperature (RT) and the other half at 4℃, without removing or diluting DMSO. Flow cytometry was employed to enumerate total nucleated cells (TNCs), total/viable CD34+ cells, and early/late apoptotic cells using anti-CD45, anti-CD34, and annexin V(AnV), 7-amino actinomycin D(AAD) staining, respectively. RESULTS: In CBs stored at 4℃ there were no significant changes in numbers of TNCs, total/viable CD34+ cells, or early/late apoptotic cell up to 48 h. However, the numbers of these cells declined significantly at RT. Total and viable CD34+ cell counts did not change for up to 6 h at RT but viable CD34+ cells decreased significantly after 24 h, and total CD34+ cell after 48 h. Early and late apoptosis tended to increase with time at RT, and numbers of viable CD34+ cells and early apoptotic cells differed significantly between RT and 4℃ after 48 h. CONCLUSIONS: There are no significant changes of viability and apoptosis in CBs stored in DMSO at 4℃ until 48 h after thawing, while at RT, there are no significant changes of total/viable CD34+ cell counts or in the proportion of apoptotic cells for at least 6 h after thawing.

Repurposing the public cord blood bank inventory in Korea to enhance cord blood use.

To enhance public cord blood (CB) use, we examined the current status of CB banking and tried to suggest revision of the banking standard. We retrospectively analyzed the use of stored public CB units between 2011 and 2016 using data from the CB information center in Korea. A total of 19,871 CB units were registered, and 363 units were selected for transplantation. The transplanted CB units contained significantly higher numbers of CD34+ cells than the average numbers in the stored CB units (5.5 × 10^6 vs. 3.2 × 10^6, p < 0.01). They also contained more total nucleated cells (TNCs) than the average of the stored CB units (13.7 × 10^8 vs. 10.7 × 10^8, p < 0.01). Only 49% of the stored CB units contained>10 × 10^8 TNCs, while 81% of the units transplanted contained >10 × 10^8 TNCs. The average length of cryopreservation of the transplanted CB units was 4.58 years and 95% of them had been stored for less than 10 years. During the study period, 18,763 CB units were requested for research, but only 5,888 were released. This discrepancy was mostly due to errors in regulatory and/or networking elements of the CB supply system. The data suggest that preserving CB units for less than 10 years and increasing the required minimum TNC count to 10 × 10^8 would produce an inventory containing units that were more useful for CBT. CB units that did not meet the requisite quality standards could be used for research, and systems for their fair distribution to researchers are needed.

Serial Changes of Cytokines in Children with Cerebral Palsy Who Received Intravenous Granulocyte-colony Stimulating Factor Followed by Autologous Mobilized Peripheral Blood Mononuclear Cells.

BACKGROUND: This study was performed to assess serial cytokine changes and their clinical impact in children with cerebral palsy (CP) who received granulocyte-colony stimulating factor (G-CSF) followed by infusion of autologous mobilized peripheral blood mononuclear cells (mPBMCs). METHODS: Peripheral blood (PB) samples were collected from 16 CP children at enrollment, and 1 month and 7 months after G-CSF infusion as well as at the end of the study. Cytokine levels were measured by enzyme-linked immunosorbent assays with plasma samples. RESULTS: There were no significant differences in cytokine levels between the mPBMC and placebo groups over 6 months. However, when clinical responders and non-responders were compared, interleukin (IL)-6 (P = 0.050) as well as G-CSF (P = 0.010) were higher in the responders than the non-responders at 1 month, while brain-derived neurotrophic factor (BDNF) (P = 0.030) and insulin-like growth factor (IGF)-1 (P = 0.001) were lower. In addition, BDNF was higher at baseline in the responders than the non-responders (P = 0.030). CONCLUSION: The changes of G-CSF itself, as well as G-CSF-induced cytokines such as IL-6, may be associated with the clinical improvement of neurologic functions. The G-CSF-induced changes of IL-6, BDNF and IGF-1, and BDNF levels before treatment, could be used as prognostic factors in G-CSF trials in CP children.

Neuroregenerative potential of intravenous G-CSF and autologous peripheral blood stem cells in children with cerebral palsy: a randomized, double-blind, cross-over study.

OBJECTIVE: We performed a randomized, double-blind, cross-over study to assess the neuroregenerative potential of intravenous granulocyte colony-stimulating factor (G-CSF) followed by infusion of mobilized peripheral blood mononuclear cells (mPBMCs) in children with cerebral palsy (CP). METHODS: Children with non-severe CP were enrolled in this study. G-CSF was administered for 5 days, then mPBMCs were collected by apheresis and cryopreserved. One month later (M1), recipients were randomized to receive either mPBMCs or a placebo infusion, and these treatment groups were switched at 7 months (M7) and observed for another 6 months (M13). We assessed the efficacy of treatment by evaluating neurodevelopmental tests, as well as by brain magnetic resonance imaging-diffusion tensor imaging (MRI-DTI) and 18F-fluorodeoxyglucose (FDG) brain positron emission tomography-computed tomography (PET-CT) scanning to evaluate the anatomical and functional changes in the brain. RESULTS: Fifty-seven patients aged 4.3 ± 1.9 (range 2-10) years and weighing 16.6 ± 4.9 (range 11.6-56.0) kg were enrolled in this study. The administration of G-CSF as well as the collection and reinfusion of mPBMCs were safe and tolerable. The yield of mPBMCs was comparable to that reported in studies of pediatric donors without CP and patients with nonhematologic diseases. 42.6% of the patients responded to the treatment with higher neurodevelopmental scores than would normally be expected. In addition, larger changes in neurodevelopment test scores were observed in the 1 month after G-CSF administration (M0-M1) than during the 6 months after reinfusion with mPBMCs or placebo (M1-M7 or M7-M13). Patients who received G-CSF followed by mPBMC infusion at 7 months (T7 group) demonstrated significantly more neurodevelopmental improvement than patients who received G-CSF followed by mPBMC infusion at 1 month (T1 group). In contrast to the results of neurodevelopment tests, the results of MRI-DTI at the end of this study showed greater improvement in the T1 group. Although we observed metabolic changes to the cerebellum, thalamus and cerebral cortex in the 18F-FDG brain PET-CT scans, there were no significant differences in such changes between the mPBMC and placebo group or between the T1 and T7 group. CONCLUSIONS: Neurodevelopmental improvement was seen in response to intravenous G-CSF followed by mPBMC reinfusion, particularly to the G-CSF alone even without mPBMC reinfusion. Further studies using a larger number of mPBMCs for the infusion which could be collected by repeated cycles of apheresis or using repeated cycles of G-CSF alone, are needed to clarify the effect of mPBMC reinfusion or G-CSF alone (Trial registration: ClinicalTrials.gov, NCT02983708. Registered 5 December, 2016, retrospectively registered).

Effect of Intravenous Infusion of G-CSF-Mobilized Peripheral Blood Mononuclear Cells on Upper Extremity Function in Cerebral Palsy Children.

OBJECTIVE: To investigate the effect of intravenous infusion of peripheral blood mononuclear cells (mPBMC) mobilized by granulocyte-colony stimulating factor (G-CSF) on upper extremity function in children with cerebral palsy (CP). METHODS: Fifty-seven children with CP were enrolled. Ten patients were excluded due to follow-up loss. In total, 47 patients (30 males and 17 females) were analyzed. All patients' parents provided signed consent before the start of the study. After administration of G-CSF for 5 days, mPBMC was collected and cryopreserved. Patients were randomized into two groups 1 month later. Twenty-two patients were administered mPBMC and 25 patients received normal saline as placebo. Six months later, the two groups were switched, and administered mPBMC and placebo, respectively. Quality of Upper Extremity Skills Test (QUEST) and the Manual Ability Classification System (MACS) were used to evaluate upper motor function. RESULTS: All subdomain and total scores of QUEST were significantly improved after mPBMC and placebo infusion, without significant differences between mPBMC and placebo groups. A month after G-CSF, all subdomain and total scores of QUEST were improved. The level of MACS remained unchanged in both mPBMC and placebo groups. CONCLUSION: In this study, intravenously infused mPBMC showed no significant effect on upper extremity function in children with CP, as compared to placebo. The effect of mPBMC was likely masked by the effect of G-CSF, which was used in both groups and/or G-CSF itself might have other neurotrophic potentials in children with CP.

Mononuclear cells from the cord blood and granulocytecolony stimulating factor-mobilized peripheral blood: is there a potential for treatment of cerebral palsy?

To investigate a possible therapeutic mechanism of cell therapy in the field of cerebral palsy using granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (mPBMCs), we compared the expression of inflammatory cytokines and neurotrophic factors in PBMCs and mPBMCs from children with cerebral palsy to those from healthy adult donors and to cord blood mononuclear cells donated from healthy newborns. No significant differences in expression of neurotrophic factors were found between PBMCs and mPBMCs. However, in cerebral palsy children, the expression of interleukin-6 was significantly increased in mPBMCs as compared to PBMCs, and the expression of interleukin-3 was significantly decreased in mPBMCs as compared to PBMCs. In healthy adults, the expression levels of both interleukin-1ß and interleukin-6 were significantly increased in mPBMCs as compared to PBMCs. The expression of brain-derived neurotrophic factors in mPBMC from cerebral palsy children was significantly higher than that in the cord blood or mPBMCs from healthy adults. The expression of G-CSF in mPBMCs from cerebral palsy children was comparable to that in the cord blood but significantly higher than that in mPBMCs from healthy adults. Lower expression of pro-inflammatory cytokines (interleukin-1ß, interleukin-3, and -6) and higher expression of anti-inflammatory cytokines (interleukin-8 and interleukin-9) were observed from the cord blood and mPBMCs from cerebral palsy children rather than from healthy adults. These findings indicate that mPBMCs from cerebral palsy and cord blood mononuclear cells from healthy newborns have the potential to become seed cells for treatment of cerebral palsy.