Early changes in serum interleukin-6 levels in extremely premature newborns for detecting fetal inflammation.

BACKGROUND: Fetal inflammatory response syndrome (FIRS) is defined by elevated levels of inflammatory cytokines circulating in fetal blood, which may result in preterm morbidities. Serum interleukin-6 (IL-6) level has been reported to be a good indicator of FIRS; however, changes in IL-6 levels after birth remain to be elucidated. Herein, we characterized early changes in serum IL-6 levels in extremely premature newborns (EPNs, < 28 wks gestation), and then determined the cut-off values for detecting fetal inflammation at each postnatal epoch. METHODS: In this single-center study, 49 EPNs were retrospectively studied. Serum IL-6 measurements are routinely performed at delivery, 1-3, 6-12, and 24-36 h of life. Receiver operating characteristic (ROC) curve analyses were performed for detecting the presence of funisitis, the histologic counterpart of FIRS. RESULTS: Overall, serum IL-6 levels were significantly elevated at 1-3 (298 [31-4719] pg/mL) and 6-12 (29 [2-12,635] pg/mL) hours of life, then returned to at-delivery levels at 24-36 h of life. When comparing serum IL-6 levels at each postnatal epoch, the levels at delivery, 1-3, and 6-12 h of life were significantly higher in the EPNs with funisitis. Serum IL-6 cut-off values at delivery, 1-3, 6-12, and 24-36 h of life for the presence of funisitis were 20, 572, 290, and 13 pg/mL with area under ROCs of 0.75, 0.71, 0.68, and 0.53, respectively. CONCLUSIONS: Serum IL-6 levels in EPNs significantly increase early after birth, then decrease to at-delivery levels by 24-36 h of life. Therefore, postnatal age-dependent cut-off values of serum IL-6 might be considered for detecting fetal inflammation with confirmed funisitis.

Targeting of SIRPα as a potential therapy for Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare neoplastic disorder characterized by inflammatory lesions arising from the anomalous accumulation of pathogenic CD1a+ CD207+ dendritic cells (DCs). SIRPα is a transmembrane protein highly expressed in myeloid cells such as DCs and macrophages. Here we show that SIRPα is a potential therapeutic target for LCH. We found that SIRPα is expressed in CD1a+ cells of human LCH lesions as well as in CD11c+ DCs in the spleen, liver, and lung of a mouse model of LCH (BRAFV600ECD11c mouse), in which an LCH-associated active form of human BRAF is expressed in a manner dependent on the mouse Cd11c promoter. BRAFV600ECD11c mice manifested markedly increased numbers of CD4+ T cells, regulatory T cells, and macrophages as well as of CD11c+ MHCII+ DCs in the spleen. Monotherapy with a mAb to SIRPα greatly reduced the percentage of CD11c+ MHCII+ DCs in peripheral blood, LCH-like lesion size in the liver, and the number of CD11c+ MHCII+ DCs in the spleen of the mutant mice. Moreover, this mAb promoted macrophage-mediated phagocytosis of CD11c+ DCs from BRAFV600ECD11c mice, whereas it had no effects on the viability or CCL19-dependent migration of such CD11c+ DCs or on their expression of the chemokine genes Ccl5, Ccl20, Cxcl11, and Cxcl12. Our results thus suggest that anti-SIRPα monotherapy is a promising approach to the treatment of LCH that is dependent in part on the promotion of the macrophage-mediated killing of LCH cells.

Evaluation of the performance of GeneSoC®, a novel rapid real-time PCR system, to detect Staphylococcus aureus and methicillin resistance in blood cultures.

Staphylococcus aureus bacteremia results in substantial mortality. Rapid identification and the determination of methicillin susceptibility are crucial for immediate treatment with appropriate antibiotics. In the present study, we aimed to evaluate the basic assay performance of GeneSoC®, a novel rapid quantitative polymerase chain reaction (qPCR) method, for the detection of methicillin-susceptible (MS) or -resistant (MR) S. aureus in blood culture (BC) bottles. qPCR pimers and probes were desinged for femA and mecA genes to diagnose S. aureus and its methicilline-resistance status. GeneSoC® system can detect target genes within 12 min per sample using microfludic thermal cycling. A total of 100 BC-positive samples, showing clusters of gram-positive cocci using microscopy, were tested. The analytical sensitivity was demonstrated for the target sequence of femA and mecA genes at 10 copies/µL, respectively. The detection limit of the MRSA bacterial burden using this system was 104 and 103 CFU/mL for femA and mecA, respectively. Compared with culture-based identification and susceptibility testing, the sensitivity and specificity for the detection of femA (+)/mecA (+) MRSA using GeneSoC® were 90.9 and 98.9%, respectively, whereas the sensitivity and specificity for detection of femA (+)/mecA (-) MSSA were 96.2% and 97.3%, respectively. In conclusion, although this was a small sample and pilot study, the GeneSoC® system is beneficial for rapid, reliable, and highly sensitive real-time testing of MRSA and MSSA in BC bottles.

Gestational Age Dependency of Umbilical Cord Serum IL-6 Levels for Detecting Fetal Inflammation.

OBJECTIVE: The fetal inflammatory response syndrome (FIRS) is characterized by elevated concentrations of inflammatory cytokines in fetal blood, with preterm delivery and morbidity. Umbilical cord serum interleukin-6 (UC-s-IL-6) is an ideal marker for detecting FIRS. However, the effect of gestational age (GA) on UC-s-IL-6 levels has not been reported. This study aimed to determine the relationship between GA and UC-s-IL-6 levels, and GA-dependent cutoff values of UC-s-IL-6 levels for detecting fetal inflammation. STUDY DESIGN: UC-s-IL-6 concentrations were measured in 194 newborns (44 extremely preterm newborns (EPNs) at 22-27 weeks' GA, 68 very preterm newborns (VPNs) at 28-31 weeks' GA, and 82 preterm newborns (PNs) at 32-34 weeks' GA). Linear regression analyses were used to correlate GA and UC-s-IL-6 levels. Receiver operating characteristic (ROC) curves analyses were performed for detecting the presence of funisitis, as the histopathological counterpart of FIRS. RESULTS: A significant negative correlation between GA and UC-s-IL-6 levels was found in newborns with severe funisitis (r s = - 0.427, p = 0.004) and those with mild funisitis (r s = - 0.396, p = 0.025). ROC curve analyses revealed the area under the curve for detecting funisitis were 0.856, 0.837, and 0.622 in EPNs, VPNs, and PNs, respectively. The UC-s-IL-6 cutoff value in EPNs (28.1 pg/mL) exceeded those in VPNs and PNs (3.7 and 3.0 pg/mL, respectively). CONCLUSION: UC-s-IL-6 levels were inversely correlated with GA especially in newborns with funisitis. Such GA dependency of UC-s-IL-6 should be considered for detecting fetal inflammation. KEY POINTS: · IL-6 levels inversely correlate with GA.. · Higher IL-6 levels strongly indicate funisitis.. · Detecting cutoff values differ depending on GA..

Nationwide multicenter questionnaire surveys on countermeasures against antimicrobial resistance and infections in hospitals.

BACKGROUND: The goals of the National Action Plan on Antimicrobial Resistance (AMR) of Japan include "implementing appropriate infection prevention and control" and "appropriate use of antimicrobials," which are relevant to healthcare facilities. Specifically, linking efforts between existing infection control teams and antimicrobial stewardship programs was suggested to be important. Previous studies reported that human resources, such as full-time equivalents of infection control practitioners, were related to improvements in antimicrobial stewardship. METHODS: We posted questionnaires to all teaching hospitals (n = 1017) regarding hospital countermeasures against AMR and infections. To evaluate changes over time, surveys were conducted twice (1st survey: Nov 2016, 2nd survey: Feb 2018). A latent transition analysis (LTA) was performed to identify latent statuses, which refer to underlying subgroups of hospitals, and effects of the number of members in infection control teams per bed on being in the better statuses. RESULTS: The number of valid responses was 678 (response rate, 66.7%) for the 1st survey and 559 (55.0%) for the 2nd survey. More than 99% of participating hospitals had infection control teams, with differences in activity among hospitals. Roughly 70% had their own intervention criteria for antibiotics therapies, whereas only about 60 and 50% had criteria established for the use of anti-methicillin-resistant Staphylococcus aureus antibiotics and broad-spectrum antibiotics, respectively. Only 50 and 40% of hospitals conducted surveillance of catheter-associated urinary tract infections and ventilator-associated pneumonia, respectively. Less than 50% of hospitals used maximal barrier precautions for central line catheter insertion. The LTA identified five latent statuses. The membership probability of the most favorable status in the 2nd study period was slightly increased from the 1st study period (23.6 to 25.3%). However, the increase in the least favorable status was higher (26.3 to 31.8%). Results of the LTA did not support a relationship between increasing the number of infection control practitioners per bed, which is reportedly related to improvements in antimicrobial stewardship, and being in more favorable latent statuses. CONCLUSIONS: Our results suggest the need for more comprehensive antimicrobial stewardship programs and increased surveillance activities for healthcare-associated infections to improve antimicrobial stewardship and infection control in hospitals.

Epithelial and ganglionic distribution at the distal rectal end in anorectal malformations: could it play a role in anastomotic adaptation?

PURPOSE: In anorectal malformations (ARMs), the epithelium of the distal rectal end is not well described. We histomorphologically evaluated epithelial and ganglionic distribution in the distal rectal end of ARMs resected during anorectoplasty to assess similarities and differences with normal anal canal structure. METHODS: In this single-center retrospective study, specimens from 60 ARM patients (27 males, 33 females) treated between 2008 and 2019 were evaluated. RESULTS: Epithelium type and alignment sequence as well as ganglionic distribution were similar in the distal rectal end and in a normal anal canal. Stratified columnar epithelium (anal transitional zone, ATZ) was seen in 49/60 (81.7%) cases and in all ARM types, including the no-fistula type. Anal crypts were identified in the stratified columnar epithelium (ATZ) of 46/49 (93.9%) patients. Regarding distal rectal end-resecting anorectoplasty, in 90% of patients, resection was performed distal to the Herrmann line. Ganglion cell distribution was exclusively proximal to the Herrmann line. CONCLUSION: Epithelial and ganglionic distribution was similar in the distal rectal end of ARMs and in a normal anal canal. The ATZ is the epithelial boundary between the rectum and skin in a normal anal canal. ATZ preservation could reproduce anal canal structure in ARM reconstruction.

Inhibitory Effects of P2Y12 Receptor Antagonist on PAR1- and PAR4-AP-Induced Platelet Aggregation in Patients with Stroke or TIA.

OBJECTIVES: The inhibitory effects of P2Y12 receptor antagonist on PAR1- and PAR4-activating peptide (AP)-induced platelet aggregation have not been fully elucidated. The present study aimed to investigate the inhibitory effects of P2Y12 receptor antagonist on PAR1- and PAR4-AP-induced platelet aggregation using platelet-rich plasma (PRP) from individuals including patients with stroke or transient ischemic attack (TIA). MATERIALS AND METHODS: PRP was given to 10 healthy individuals pretreated in vitro with cangrelor, then stimulated with adenosine diphosphate (ADP), PAR4-AP, or PAR1-AP. Moreover, 20 patients were enrolled from 148 consecutive patients with acute ischemic stroke or TIA admitted to our institute between December 2017 and April 2019. PRP obtained from each patient before and >7 days after initiation of clopidogrel was similarly stimulated with these agonists. Platelet aggregation was measured using an automatic coagulation analyzer in all participants. RESULTS: In healthy individuals, ADP- and PAR4-AP-induced platelet aggregations were significantly inhibited depending on the cangrelor concentration in vitro, while PAR1-AP-induced platelet aggregation was slightly inhibited. In patients with stroke or TIA, clopidogrel inhibited ADP-induced platelet aggregation at all concentrations, and significantly inhibited PAR4-AP-induced platelet aggregation at 50 µmol/L of PAR4-AP (p<0.05), especially in 5 patients who showed high reactivity to PAR4-AP. PAR1-AP-induced platelet aggregation was also slightly inhibited. CONCLUSIONS: We showed significant inhibitory effects on PAR4-AP-induced platelet aggregation by clopidogrel in patients with stroke or TIA who had high reactivity to PAR4-AP.

A case of a parthenogenetic 46,XX/46,XY chimera presenting ambiguous genitalia.

Sex-chromosome discordant chimerism (XX/XY chimerism) is a rare chromosomal disorder in humans. We report a boy with ambiguous genitalia and hypospadias, showing 46,XY[26]/46,XX[4] in peripheral blood cells. To clarify the mechanism of how this chimerism took place, we carried out whole-genome genotyping using a SNP array and microsatellite analysis. The B-allele frequency of the SNP array showed a mixture of three and five allele combinations, which excluded mosaicism but not chimerism, and suggested the fusion of two embryos or a shared parental haplotype between the two parental cells. All microsatellite markers showed a single maternal allele. From these results, we concluded that this XX/XY chimera is composed of two different paternal alleles and a single duplicated maternal genome. This XX/XY chimera likely arose from a diploid maternal cell that was formed via endoduplication of the maternal genome just before fertilization, being fertilized with both X and Y sperm.

Thrombin-induced platelet aggregation -effect of dabigatran using automated platelet aggregometry.

Dabigatran, a direct oral thrombin inhibitor, has two therapeutic effects: anticoagulation; and antiplatelet activity. In the clinical field, evaluation of the effect of dabigatran on thrombin-induced platelet aggregation is difficult because of fibrin clot formation and platelet aggregation. The aim of this study was to establish a new platelet aggregation method and to investigate the effects of dabigatran on thrombin-induced platelet aggregation. Platelet aggregation with thrombin was performed with automated light transmission aggregometry (CS2400; Sysmex, Kobe, Japan) in 40 healthy subjects. Thrombin-induced platelet aggregation was performed using thrombin and platelet-rich plasma (PRP), and thrombin-induced fibrin polymerization was inhibited by adding the peptide Gly-Pro-Arg-Pro (GPRP). The effect of dabigatran was then evaluated using the above method. Thrombin at < 0.2 U/mL did not induce platelet aggregation in most normal subjects. Median maximum aggregation percent (MA%) (25th-75th percentile) with 0.5 and 1.0 U/mL of thrombin was 87.0% (79.3-90.8%), and 90.2% (86.5-92.2%), respectively. The anti-platelet effects of dabigatran were then evaluated with these concentrations of thrombin. Dabigatran (final concentration, 2.5-1000 nM) inhibited platelet aggregation by 0.2-1.0 U/mL of thrombin in a concentration-dependent manner in vitro. Dabigatran showed potent inhibitory effects against platelet aggregation induced by 0.5 and 1.0 U/mL thrombin with half maximal inhibitory concentrations of 10.5 and 40.4 nM, respectively. A standard for thrombin-induced platelet aggregation was developed using the CS2400 in healthy subjects, and dabigatran was confirmed to inhibit thrombin-induced platelet aggregation in vitro with PRP.

Potential role of the PD-L1 expression and tumor-infiltrating lymphocytes on neuroblastoma.

PURPOSE: The programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway has garnered much attention for its roles in clinical oncology. The aim of this study was to examine the clinical impact of the PD-L1 expression and tumor-infiltrating lymphocytes (TILs) on neuroblastoma. METHODS: We evaluated the PD-L1 expression and TIL status in 31 patients with neuroblastoma who underwent a biopsy or resection by an immunohistochemical analysis. Furthermore, we performed the serial analysis of the PD-L1 status before and after chemotherapy in 15 patients. RESULTS: Among the 31 cases, 11 (35%) showed a positive PD-L1 expression. The survival analysis showed a trend toward an association between PD-L1 positivity and a decreased overall survival. PD-L1 positivity tended to be associated with higher levels of tumor markers. In the serial analysis of the PD-L1 status, positivity was noted in 8 of 15 patients before chemotherapy and 6 after chemotherapy. Notably, all four patients with a positive PD-L1 status both before and after chemotherapy had recurrence, and 3 of them died during the follow-up period. CONCLUSION: Our findings suggest that the PD-L1 tumor expression might be a good biomarker for the treatment of neuroblastoma patients, especially for advanced neuroblastoma.

Presacral malignant teratoid neoplasm in association with pathogenic DICER1 variation.

We report two malignant sacrococcygeal tumors in infants that were associated with pathogenic DICER1 variation. These tumors were composed of primitive neuroepithelium, embryonal rhabdomyosarcoma, and cartilage and initially diagnosed as immature teratomas. One child developed intracranial metastasis and died. The second child underwent surgery and chemotherapy and achieved complete remission. This child subsequently developed five additional DICER1-associated neoplasms by age nine. Genetic analysis revealed that both tumors harbored biallelic pathogenic DICER1 variation. We believe these cases represent another novel subtype of DICER1-associated tumor. This new entity, which we propose to call DICER1-associated presacral malignant teratoid neoplasm, may be difficult initially to distinguish from immature teratoma, but recognizing it as an entity can prompt appropriate classification as an aggressive malignancy and facilitate appropriate genetic counseling, DICER1 germline variant testing, screening, and education.

Collaboration of cancer-associated fibroblasts and tumour-associated macrophages for neuroblastoma development.

Neuroblastoma is the most common extracranial solid tumour in children and is histologically classified by its Schwannian stromal cells. Although having fewer Schwannian stromal cells is generally associated with more aggressive phenotypes, the exact roles of other stromal cells (mainly macrophages and fibroblasts) are unclear. Here, we examined 41 cases of neuroblastoma using immunohistochemistry for the tumour-associated macrophage (TAM) markers CD68, CD163, and CD204, and a cancer-associated fibroblast (CAF) marker, alpha smooth muscle actin (αSMA). Each case was assigned to low/high groups on the basis of the number of TAMs or three groups on the basis of the αSMA-staining area for CAFs. Both the number of TAMs and the area of CAFs were significantly correlated with clinical stage, MYCN amplification, bone marrow metastasis, histological classification, histological type, and risk classification. Furthermore, TAM settled in the vicinity of the CAF area, suggesting their close interaction within the tumour microenvironment. We next determined the effects of conditioned medium of a neuroblastoma cell line (NBCM) on bone marrow-derived mesenchymal stem cells (BM-MSCs) and peripheral blood mononuclear cell (PBMC)-derived macrophages in vitro. The TAM markers CD163 and CD204 were significantly up-regulated in PBMC-derived macrophages treated with NBCM. The expression of αSMA by BM-MSCs was increased in NBCM-treated cells. Co-culturing with CAF-like BM-MSCs did not enhance the invasive ability but supported the proliferation of tumour cells, whereas tumour cells co-cultured with TAM-like macrophages had the opposite effect. Intriguingly, TAM-like macrophages enhanced not only the invasive abilities of tumour cells and BM-MSCs but also the proliferation of BM-MSCs. CXCL2 secreted from TAM-like macrophages plays an important role in tumour invasiveness. Taken together, these results indicate that PBMC-derived macrophages and BM-MSCs are recruited to a tumour site and activated into TAMs and CAFs, respectively, followed by the formation of favourable environments for neuroblastoma progression. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Medulloblastoma with suprasellar solitary massive metastasis: Case report.

It is extremely rare for metastasised medulloblastoma to form a large tumour in the suprasellar region. We present a case of medulloblastoma with large suprasellar metastasis at initial presentation. A 3-year and 5-month-old boy presented with a 1-month history of vomiting and loss of appetite, and body weight. Computed tomography and magnetic resonance imaging revealed a 20 mm × 20 mm mass in the suprasellar region and a 30 mm × 30 mm mass in the fourth cerebral ventricle. We performed endoscopic biopsy of the suprasellar tumour, and subsequently totally removed the vermian tumour through a suboccipital craniotomy. The histopathological findings revealed that both the suprasellar and vermian tumours were classic type and non SHH/WNT type medulloblastoma. The postoperative course was uneventful. The patient showed complete remission after chemotherapy. The tumour in the suprasellar region was most likely metastatic from the vermis. Endoscopic biopsy of the tumour in the suprasellar region and total removal of the tumour in the vermis in a one-stage operation followed by intensive chemotherapy with reduced dose radiotherapy may provide a satisfactory outcome.