Cytokine profiles in an extremely preterm infant with congenital syphilis.

We report the cytokine profiles of an infant with congenital syphilis as a first case. This female infant was born by vaginal delivery at a gestational age of 27 weeks during her mother's treatment for syphilis. Elevation of T helper (Th)-1 cytokines (interleukin (IL)-2, IL-12) and IL-17, which supports immunological mechanisms of Th-1, was similar to that in cases of syphilis in adults. IL-6 and granulocyte colony-stimulating factor (G-CSF), the synergistic effects of which cause the leukemoid reaction, were also elevated. The levels of interferon-γ and IL-17 in cerebral spinal fluid, which are elevated in neurosyphilis in adults, were slightly elevated.

Urinary and serum levels of high mobility group box 1.

Background: High mobility group box 1 (HMGB-1) has been extensively studied in adults and to a certain extent in neonates as well. Clinical examination of neonates, especially unwell neonates soon after birth, should be minimally invasive. Objective: This study aimed to investigate whether the urinary HMGB-1 level is comparable to the serum HMGB-1 level in neonates. Methods: In all, 87 neonates (37.5 ± 2.9 weeks of gestation and a mean birth weight of 2588 ± 649 g) were enrolled. Of these, 53 were males and 34 were females. The umbilical cord blood and the first or second spontaneous voiding urine samples were stored, and the HMGB-1 level in the samples was measured. Results: HMGB-1 was detected in all urinary samples. In these samples, we found acetylated HMGB-1 and may be devoid of nine residues at the N-terminal amino acid sequence. There was a significant correlation between the serum HMGB-1 level and urinary HMGB-1 level (r = 0.73, p < 0.001). Urinary HMGB1 levels in fetal neonatal asphyxia were significantly higher than those in healthy controls (p = 0.09). Conclusion: Urinary excretion may be one of the metabolic pathways of HMGB-1. The urinary HMGB-1 level may be comparable to the serum HMGB-1 level in the early neonatal period.

Simple chitin-based cell culture platform for production of biopharmaceuticals.

OBJECTIVES: Gene therapy using viral vectors and antibody-based therapies continue to expand within the pharmaceutical market. We evaluated whether Cellhesion® VP, a chitin-based material, can be used as 3D culture platform for cell lines used for the production of antibodies and viral vectors. RESULTS: The results of Cell Counting Kit-8 assay and LDH assay revealed that Cellhesion® VP had no adverse effect to Human Embryonic Kidney (HEK) 293, A549 and Chinese hamster ovary (CHO) DG44-Interferon-ß (IFN) cells. Cell growth analyses showed that Cellhesion® VP supported the 3D culture of HEK293, A549 and CHO DG44- IFN-ß cells with a spherical morphology. Importantly, subculture of these cell lines on Cellhesion® VP was easily performed without trypsinization because cells readily transferred to newly added scaffold. Our data also suggest that CHO DG44-IFNß, cultured on Cellhesion® VP secreted IFNß stably and continuously during the culture period. CONCLUSIONS: Cellhesion® VP provides a simple and streamlined expansion culture system for the production of biopharmaceuticals.

Reversible suspension culture of human vascular smooth muscle cells using the functional biopolymer FP003.

Human vascular smooth muscle cells (SMCs) are adherent cells, and they cannot survive without scaffolds in suspension culture. Here, we aimed to establish a suspension culture of SMCs using the functional biopolymer FP003 and to investigate the proliferation status of the cells. When SMCs were suspension cultured with FP003, their proliferation was inhibited with a viability of 75% until day 15. When SMCs were re-plated on plastic plates after suspension culture with FP003 for 48 h, the SMCs proliferated as in a normal plate culture. The SMCs cultured in suspension with FP003 showed a relatively low phosphorylation of retinoblastoma protein, low expression of cyclin D1, high proportion of G0/G1 phase cells, low proportion of S phase cells, and no obvious signs of apoptosis, indicating that this culture system inhibited progression from the G1 to S phase. This growth arrest was a reversible property that showed no significant changes in the expressions of the marker proteins α-smooth muscle actin and smooth muscle myosin heavy chain. These results suggest that human SMCs can be stably cultured in suspension with FP003 without losing their characteristics when they are cultured on plastic plates again.

Investigation of biomarkers in a rare case of fulminant necrotizing enterocolitis in a preterm infant.

We encountered a very rare case of fulminant necrotizing enterocolitis (F-NEC) in a preterm male baby. The course of NEC and sepsis in this case was clearly different from the usual course. After onset at 14 days of life, catheter-related bloodstream infection was first assumed, and antibiotics and γ-globulin administration were started. However, 12 hours after onset, the baby's abdominal distension increased remarkably, and his entire abdominal wall turned red to purple. Escherichia coli were isolated from the blood culture, but the catheter tip culture was negative. Exchange transfusion was performed 32 hours after onset, but no significant changes were observed in the baby's general condition, and he died 46 hours after onset. The acute phase reactants of CRP and α1-acid glycoprotein increased, but haptoglobin did not. Although IL-1ß and TNFα increased as expected with sepsis, IL-6, IL-8, IL-10, and G-CSF however increased to a greater extent than expected. From the above, we diagnosed the development of intestinal necrosis as a result of widespread intestinal ischemia, and that sepsis was associated with this poor condition.

A neonatal case of an elongated soft palate.

The development of stridor and periodic desaturation in a 2-day-old neonate born at term lead to the suspicion of upper airway obstruction. The patient underwent flexible fiberoptic laryngo-tracheo-bronchoscopy and was diagnosed as having an elongated soft palate and secondary mild pharyngomalacia. Early intervention with high PEEP therapy using nasal CPAP led to improvement in the patient condition.

Peripheral platelet phagocytosis in an extremely low birth weight infant:a case report.

A 768 g female neonate, born at 25 weeks' gestation, developed sepsis due to methicillin-resistant Staphylococcus epidermidis on day 14. Severe thrombocytopenia was observed, and hemophagocytic macrophages were identified in her peripheral blood smear. Cytokine profiles at the time of onset suggested that an inflammatory cytokine storm had activated lymphocytes and macrophages, leading to platelet phagocytosis. After administration of vancomycin for 14 days and immunoglobulin therapy, she improved without any complications. Considering the results of cytokine profiles, early intervention for infection may have prevented progression to hemophagocytic lymphohistiocytosis and reduced the severity of clinical symptoms.

A Novel 3D Culture System Using a Chitin-Based Polysaccharide Material Produces High-Quality Allogeneic Human UCMSCs with Dispersed Sphere Morphology.

Mesenchymal stem cell (MSC) transplantation, in particular allogeneic transplantation, is a promising therapy for a variety of diseases. However, before performing allograft treatment it is necessary to find suitable donors, establish culture methods that maintain cell quality, and reduce cell production costs. Here, we present a new method of producing allogeneic MSCs combining human umbilical cord-derived mesenchymal stem cells (UCMSCs) and chitin-based polysaccharide fibers (Cellhesion® MS). UCMSC numbers significantly increased, and cells grew as dispersed spheres on Cellhesion® MS. Subsequent biological analyses showed that the expression levels of stemness-related and migration-related genes were significantly upregulated, including octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), and C-X-C chemokine receptor type 4 (CXCR4). The secretion levels of paracrine factors such as prostaglandin E2 (PGE2), TNFα-stimulating gene (TSG)-6, fibroblast growth factor 2 (bFGF), and Angiogenin (Ang) from UCMSCs using Cellhesion® MS were significantly higher than with microcarrier and U-bottom plate culture. In addition, culture supernatant from UCMSCs with Cellhesion® MS had better angiogenic potential than that from monolayer cultured UCMSCs. Furthermore, we succeeded in a scaled-up culture of UCMSCs with Cellhesion® MS using a closed culture bag. Therefore, Cellhesion® MS is a key material for producing high-quality UCMSCs in a three-dimensional (3D) culture system.

A male preterm infant with cow's milk allergy to human milk fortifier showing only severe respiratory symptoms.

We report a male infant with a birthweight of 1,400 g at 29 weeks 2 days gestation diagnosed as having cow's milk allergy (CMA) due to human milk fortifier, who developed severe respiratory symptoms. The infant had no gastrointestinal symptoms; rather, the initial symptoms were apnea attacks and wheezing with a prolonged expiratory phase that progressed to severe ventilatory insufficiency requiring mechanical ventilation. Aggravation of his general condition, which appeared to be due to sepsis, was improved by temporary starvation and respiratory care, but he relapsed on the resumption of enteral feeding of his mother's milk with a human milk fortifier. As a result, this event was interpreted as a positive oral food challenge test. The infant resumed complete breastfeeding without the fortifier and has not relapsed since. Examination of his serial cytokine profiles from residual serum revealed that although interleukin-5 was not increased, interferon (IFN)-γ was increased, suggesting some relation between the time course of IFN-γ and the infant's eosinophil count. These findings may indicate that the involvement of IFN-γ is one cause of the onset of this disease.

Lateralization of the Tibial Tubercle in Recurrent Patellar Dislocation: Verification Using Multiple Methods to Evaluate the Tibial Tubercle.

BACKGROUND: The tibial tubercle deviation associated with recurrent patellar dislocation (RPD) has not been studied sufficiently. New methods of evaluation were used to verify the extent of tubercle deviation in a group with patellar dislocation compared with that in a control group, the frequency of patients who demonstrated a cutoff value indicating that tubercle transfer was warranted on the basis of the control group distribution, and the validity of these methods of evaluation for diagnosing RPD. METHODS: Sixty-six patients with a history of patellar dislocation (single in 19 [SPD group] and recurrent in 47 [RPD group]) and 66 age and sex-matched controls were analyzed with the use of computed tomography (CT). The tibial tubercle-posterior cruciate ligament (TT-PCL) distance, TT-PCL ratio, and tibial tubercle lateralization (TTL) in the SPD and RPD groups were compared with those in the control group. Cutoff values to warrant 10 mm of transfer were based on either the minimum or -2SD (2 standard deviations below the mean) value in the control group, and the prevalences of patients in the RPD group with measurements above these cutoff values were calculated. The area under the curve (AUC) in receiver operating characteristic (ROC) curve analysis was used to assess the effectiveness of the measurements as predictors of RPD. RESULTS: The mean TT-PCL distance, TT-PCL ratio, and TTL were all significantly greater in the RPD group than in the control group. The numbers of patients in the RPD group who satisfied the cutoff criteria when they were based on the minimum TT-PCL distance, TT-PCL ratio, and TTL in the control group were 11 (23%), 7 (15%), and 6 (13%), respectively. When the cutoff values were based on the -2SD values in the control group, the numbers of patients were 8 (17%), 6 (13%), and 0, respectively. The AUC of the ROC curve for TT-PCL distance, TT-PCL ratio, and TTL was 0.66, 0.72, and 0.72, respectively. CONCLUSIONS: The extent of TTL in the RPD group was not substantial, and the percentages of patients for whom 10 mm of medial transfer was indicated were small. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Neonatal Leukemoid Reaction with Fetal Inflammatory Response Syndrome Is Associated with Elevated Serum Granulocyte Colony Stimulating Factor and Interleukin-6.

Leukemoid reaction (LR) is a reactive disease that exhibits abnormal blood values similar to leukemia, but not due to leukemia. One report showed that neonatal LR (NLR) was associated with elevated serum granulocyte colony stimulating factor (G-CSF) in only 30% of the study neonates. NLR is not always associated with the elevation of serum G-CSF. NLR was defined as a white blood cell count of ≥ 40 × 103/µL and/or blast cell concentration of > 2%. We have focused on NLR with fetal inflammatory response syndrome (FIRS), defined as a fetal systemic inflammatory reaction triggered by intrauterine infection. FIRS was diagnosed based on a cord serum interleukin-6 (IL-6) concentration ≥ 17.5 pg/mL and histopathological chorioamnionitis. Because NLR is highly associated with FIRS, we have hypothesized that NLR is associated with the elevation of both G-CSF and IL-6. This is the first report to measure multiple cytokines in NLR at the same time. The study comprised 19 preterm infants with FIRS: 8 with NLR (study group) and 11 without NLR (control group). Serum G-CSF and IL-6 concentrations were significantly higher in the study group than the control group. There was a positive correlation between G-CSF and IL-6 levels in the study group but not in the control group. These results suggest that elevated serum G-CSF and IL-6 may underlie NLR. Thus, G-CSF and IL-6 concentrations may be predictive of the onset of NLR. Measuring these cytokines is useful for judging the prognosis of preterm infants and for their post-natal clinical management.

Serial investigation of continuous glucose monitoring in a very low birth weight infant with transient late-onset hyperglycemia.

Transient late-onset hyperglycemia was detected in a very low birth weight (VLBW) infant (gestational age 28 weeks, birth weight 1,082 g) by routine point-of-care glucose monitoring.　The infant had no clinical symptom.　Serial continuous glucose monitoring (CGM) was conducted for 3 days at 31, 35, and 39 weeks' post conceptual age.　The difference values between the maximum and minimum blood glucose levels during the interval from one enteral feeding to the next enteral feeding were 32.3±14.3 mg/dL, 47.5±22.9 mg/dL, and 27.5±12.9 mg/dL for the 1st, 2nd, and 3rd CGM, respectively.　The serial change in the values was statistically significant (p<0.01).CGM is widely used as a routine clinical practice, which is true even in VLBW infants.　Hyperglycemic events detected by only once of CGM in otherwise healthy preterm infants have already been reported on larger numbers of patients.　To our knowledge, this is the first report that the change of glucose intolerance in a VLBW infant with transient late-onset hyperglycemia was investigated by serial CGM.

DOWN'S SYNDROME WITH NEONATAL ALLOIMMUNE THROMBOCYTOPENIA DUE TO HLA-A2 ANTIBODY.

Anti-HLA antibodies reportedly exist in the one third of pregnant women. But few occurrences of neonatal alloimmune thrombocytopenia (NAIT) caused by anti-HLA antibodies have been reported. Here a male baby, who was admitted for low birth weight with Down syndrome (DS), was suffered from thrombocytopenia without transient myeloproliferative disorder (TMD). Positive reactions of HLA-specific antibodies were detected in maternal serum. Cross-matching tests between maternal serum and paternal platelets and lymphocytes were strongly positive. It is most conceivable that the previous pregnancy of the mother induced the production of anti-HLA-A2 antibody, which crossed the placenta and subsequently caused an NAIT in the case presented. This is the first case of DS with NAIT due to anti-HLA antibodies.

Preparation of chitin nanofiber-reinforced carboxymethyl cellulose films.

In this study, we investigated the preparation of chitin nanofiber (CNF)-reinforced carboxymethyl cellulose (CMC) films by their electrostatic interaction. First, CMC films and self-assembled CNF dispersions with methanol were prepared by casting technique and regeneration from chitin ion gels with an ionic liquid, respectively. Then, the CMC films were immersed in the dispersions with the different CNF contents, followed by centrifugation to obtain the desired composite films. The amounts of the absorbed CNFs on the films were calculated by the weight increases after the above compatibilization procedure. The presence of CNFs on the films was also confirmed by the SEM and IR measurements. The mechanical properties of the composite films were evaluated by tensile testing, which suggested the reinforcing effect of CNFs present on the CMC films.

Preparation of polysaccharide supramolecular films by vine-twining polymerization approach.

In this study, we investigated the preparation of polysaccharide supramolecular films through the formation of inclusion complexes by amylose in vine-twining polymerization using carboxymethyl cellulose-graft-poly(ε-caprolactone) (CMC-g-PCL) as a new guest polymer. First, hydrogels were prepared by phosphorylase-catalyzed enzymatic polymerization in the presence of CMC-g-PCL according to the vine-twining polymerization manner. The XRD result of a powdered sample obtained by lyophilization of the resulting hydrogel indicated the presence of inclusion complexes of amylose with the PCL graft-chains between intermolecular (CMC-g-PCL)s, which acted as supramolecular cross-linking points for the hydrogelation. Then, the supramolecular films were obtained by adding water to the powdered samples, followed by drying. The mechanical properties of the selected films examined by tensile testing were superior to those of a CMC film. The effect of the supramolecular cross-linking structures on the mechanical properties of the films was evaluated further by several investigations.

Severe human herpesvirus 6-associated encephalopathy in three children: analysis of cytokine profiles and the carnitine palmitoyltransferase 2 gene.

Three children developed severe encephalopathy associated with human herpesvirus 6 infection. Magnetic resonance imaging of the brain showed either basal ganglia involvement or diffusion abnormalities in the cerebral white matter. Coagulopathy with hypercytokinemia was observed in 2 patients. One demonstrated thermolabile variation in carnitine palmitoyltransferase 2. These results suggest a heterogeneous pathogenic mechanism in encephalopathy associated with human herpesvirus 6 infection.

[Clinico-pathological investigation of two patients with dementia with motor neuron disease].

Dementia with motor neuron disease (D-MND) is characterized clinically by frontal and neurological signs, and pathologically by localized atrophy of the fronto-temporal lobes and neuronal ubiquitin(Ub)-positive inclusions. In this study, we compared the clinico-pathological findings of two patients with D-MND. Case 1 (55-year-old male): At the age of 51, he developed personality change and disinhibition, lacking neurological signs. Brain MRI exhibited localized atrophy of the frontal lobes. At the age of 54, he showed dysphagia and died after a disease duration of 4 years. Neuropathologically, the cerebrum showed localized atrophy of the dorsal area of the frontal lobes. The atrophied cerebral cortex demonstrated moderate neuronal loss with spongy change and gliosis in the superficial layers. The brainstem and spinal cord revealed moderate neuronal loss in the substantia nigra, severe neuronal loss with Bunina bodies in the hypoglossal nucleus, and moderate neuronal loss in the cervical anterior horn. There were some Ub-positive neuronal inclusions in the atrophied cortex and many in the dentate gyrus. Case 2 (68-year-old female): At the age of 64, she developed personality change, and then gait disturbance and dysarthria. Brain MRI exhibited localized atrophy of the fronto-temporal lobes. At the age of 67, she showed dysphagia with Babinski signs and died after a disease duration of 4 years. Neuropathologically, the cerebrum showed localized atrophy of the basal area of the temporal lobes, especially on the right side. The atrophied cerebral cortex demonstrated moderated neuronal loss with spongy change and gliosis in the superficial layers. The pre-central cortex revealed severe loss of Betz cells. The brainstem and spinal cord showed mild neuronal loss without Bunina bodies in the hypoglossal nucleus and cervical anterior horn, accompanied by severe degeneration of the bilateral pyramidal tracts. There were many Ub-positive neuronal inclusions with a few neurites in the atrophied cortex and some in the dentate gyrus. Cases 1 and 2 were clinically diagnosed as Pick's disease (PiD) and D-MND, respectively, although pathological diagnoses were both D-MND. Case 1 showed neuropathological findings typical to D-MND, whereas case 2 showed neuropathological findings common to atypical Pick's disease (aPiD). D-MND and aPiD are should be clinico-pathologically differentiated, although they are included in the frontotemporal lobar degeneration with motor neuron disease-type inclusions.