Hiromichi Shoji

Bifidobacterium breve enhances transforming growth factor beta1 signaling by regulating Smad7 expression in preterm infants.

Objectives: Transforming growth factor (TGF) &bgr;1 displays a broad spectrum of activities in mucosal regulation, including induction of oral tolerance, potent anti-inflammatory effects, mucosal IgA expression and effects on epithelial cell proliferation and differentiation. The present study examined the effect of probiotics on the immunologic system of preterm infants in relation to TGF-&bgr; signaling. Methods: Subjects comprised 19 preterm infants divided into 2 groups: receiving Bifidobacterium breve supplementation (B. breve group) and without supplementation (controls). Blood samples were collected from both groups on days 0, 14 and 28 after birth. Serum cytokine levels were measured using enzyme-linked immunosorbent assay, and expression levels of the TGF-&bgr; signaling molecule, Smad, were examined using semiquantitative reverse transcriptase-polymerase chain reaction. Results: Serum TGF-&bgr;1 level was elevated on day 14 and remained elevated on day 28 in the B. breve group. Level of messenger RNA expression was enhanced for Smad3 and reduced for Smad7 (antagonistic Smad) after B. breve administration relative to levels in controls on day 28. Conclusions: These results demonstrated that the administration of B. breve to preterm infants can up-regulate TGF-&bgr;1 signaling and may possibly be beneficial in attenuating inflammatory and allergic reactions in these infants.

The pilot study: Sphingomyelin-fortified milk has a positive association with the neurobehavioural development of very low birth weight infants during infancy, randomized control trial

AIM This study was a randomised control trial to examine the effects of sphingomyelin (SM), on the mental, motor and behavioural development of premature infants. PATIENTS AND METHODS Randomised, double-blind controlled trial, enroling infants born with a birth weight of less than 1500 g between January 2004 and October 2007 at Juntendo University Hospital, with follow-up to 18 months. Twenty-four preterm babies were randomly assigned; 12 were assigned to a test group and fed SM-fortified milk (SM 20% of all phospholipids in milk) and 12 were assigned to a control group (SM 13% of all phospholipids in milk). We analysed the composition of the plasma phospholipids and red-cell-membrane fatty acids, after which VEP, Fagan, BSID-II, attention and memory tests were performed. RESULTS The percentage of SM in the total phospholipids was significantly higher in the trial group than in the control group at 4, 6 and 8 weeks. The Behaviour Rating Scale of the BSID-II, the Fagan test scores, the latency of VEP, and sustained attention test scores at 18 months were all significantly better in the trial group than in the control group. CONCLUSION This study is the first to report that nutritional intervention via administration of SM-fortified milk has a positive association with the neurobehavioural development of low-birth-weight infants. However, detailed studies on the effects of SM on longer-term development are required.

Effects of oral administration of bifidobacterium breve on fecal lactic acid and short-chain fatty acids in low birth weight infants.

Objectives:Short-chain fatty acids (SCFAs) are known to provide energy to colonocytes, whereas overproduction of SCFAs can cause mucosal injury in premature infants. Our objective was to investigate the effects of the oral administration of Bifidobacterium breve M-16V (B breve) on fecal lactic acid and SCFAs in low birth weight (LBW) infants. Patients and Methods:Fecal lactic, acetic, propionic, and butyric acids from 66 premature infants were analyzed by high-performance liquid chromatography at 0, 2, and 4 weeks after birth. The subjects included 22 extremely LBW (ELBW, <1000 g), 22 very LBW (VLBW, <1500 g), and 22 LBW (<2500 g) infants. The infants were divided into two groups: those with and those without B. breve supplementation. Results:In the control groups, fecal acetic acid and total SCFA concentrations were significantly increased at 2 weeks in the VLBW and LBW infants (P < 0.05) and at 4 weeks in the ELBW, VLBW, and LBW infants (P < 0.01 for each) compared with those at week 0. Fecal lactic acid concentrations showed a similar pattern during follow-up, but the differences were not significant. Four weeks after B breve administration, the fecal butyric acid concentrations were significantly decreased in the ELBW and VLBW infants (P < 0.05 each), and the ratio of the acetic acid concentrations to the total SCFAs was significantly increased compared with those of the control groups in the ELBW (P < 0.05), VLBW (P < 0.05), and LBW infants (P < 0.01). Conclusions:Oral administration of B breve reduces the production of butyric acid, which may be helpful in protecting LBW infants from digestive diseases such as necrotizing enterocolitis.

Effects of iron-unsaturated human lactoferrin on hydrogen peroxide-induced oxidative damage in intestinal epithelial cells.

Human milk (HM) contains various bioactive antioxidants. Lactoferrin (Lf) has been assumed to be one of the major antioxidants in HM. We examined the antioxidative properties of iron-unsaturated human Lf (apo-hLf, the major form of Lf in HM) in two intestinal epithelial cell lines: (1) An intestinal epithelial cell line (IEC-6) were preincubated for 24 h with either 50 μg/mL of apo-hLf, iron-saturated human Lf (holo-hLf), iron-unsaturated bovine transferrin (apo-bTf), or 800 ng/mL of the iron-chelating compound deferoxamine (DFX), followed by hydrogen peroxide (H2O2) challenge to induce oxidative stress. Survival rates were significantly higher in the cells preincubated with apo-hLf and DFX than those preincubated with holo-hLf. (2) Caco-2 cells were preincubated with or without apo-hLf for 24 h, followed by an H2O2 challenge. Intracellular oxidative stress was assessed by a fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). Fluorescent intensity of cell images and cell homogenates was significantly lower in the cells preincubated with apo-hLF than those preincubated without apo-hLF. Our study indicates that apo-hLf alleviates H2O2-induced oxidative damage in intestinal cells due to the iron-chelating capacity. Therefore, Lf in HM may act as an antioxidant in the gastrointestinal tract (GIT).

Neonatal transient eosinophilic colitis causes lower gastrointestinal bleeding in early infancy.

Background: Lower gastrointestinal bleeding (LGB), particularly in newborns, is of serious concern and requires urgent investigation and hospital care. Whereas allergic proctocolitis caused by food protein is a significant cause of LGB in infants with eosinophilia, there are several cases of diseases with symptoms similar to those of allergic proctocolitis but without an apparent allergic reaction influence. Patients and Methods: We examined 2 neonates using rectosigmoidoscopy who showed eosinophilia and experienced fresh LGB soon after birth and before their first feedings. Serum eosinic cationic protein (ECP) and platelet activating factor (PAF) levels were also examined in the second case to confirm the involvement of eosinophils for its pathogenesis. Results: Both patients were in a clinically stable condition, and their abdomens were soft. The results of their blood analyses, abdominal radiographs, and stool cultures were normal, but they had gross eosinophilia: the eosinophil counts were 9014/mm3 (patient 1) and 1955/mm3 (patient 2). Rectosigmoidoscopy with colonic mucosal biopsy revealed nodular lymphoid hyperplasia with a pale mucosal surface and massive oozing with diffuse eosinophilic infiltration in the lamina propria. In patient 2 the serum ECP and PAF levels were elevated to 123 μg/L (normal, <14.7) and 13.1 μmol/L/min (normal, <6). A few days after intravenous hydration therapy, LGB was no longer detected, and the serum ECP and PAF levels returned to normal. Conclusions: Inasmuch as these infants had LGB similar to allergic proctocolitis without any allergic reactions, we suggest that infiltrated eosinophils in the colonic mucosa could be involved in the pathogenesis of LGB in early infancy.

Effect of human breast milk on urinary 8-hydroxy-2'-deoxyguanosine excretion in infants

During the perinatal period, oxidative stress is intimately involved in pathologic processes of serious diseases. Although breast milk contains many antioxidants, it is not clear whether breast milk can act as an antioxidant in infants in vivo. We compared the oxidative stress levels in total of 41 healthy 1-mo-old infants by measuring urinary 8-hydroxy-2′-deoxyguanosine, which is one of the biomarkers of oxidative DNA damage. These infants were divided into four groups according to the type of feeding. Urinary 8-hydroxy-2′-deoxyguanosine excretion of the breast-fed group was significantly lower than those of the artificial milk dominant mixed-fed group or the bottle-fed group. Our data suggest that breast milk, not artificial formula, acts as an antioxidant during infancy.

IGF-I, leptin and active ghrelin levels in very low birth weight infants during the first 8 weeks of life

Aim:  We investigated the relationship between plasma insulin‐like growth factor I (IGF‐I), leptin, active ghrelin levels, and postnatal growth in very low birth weight (VLBW) infants.

Efficacy of bacterial ribosomal RNA-targeted reverse transcription-quantitative PCR for detecting neonatal sepsis: a case control study

BackgroundNeonatal sepsis is difficult to diagnose and pathogens cannot be detected from blood cultures in many cases. Development of a rapid and accurate method for detecting pathogens is thus essential. The main purpose of this study was to identify etiological agents in clinically diagnosed neonatal sepsis using bacterial ribosomal RNA-targeted reverse transcription-quantitative PCR (BrRNA-RT-qPCR) and to conduct comparisons with the results of conventional blood culture. Since BrRNA-RT-qPCR targets bacterial ribosomal RNA, detection rates using this approach may exceed those using conventional PCR.MethodsSubjects comprised 36 patients with 39 episodes of suspected neonatal sepsis who underwent BrRNA-RT-qPCR and conventional blood culture to diagnose sepsis. Blood samples were collected aseptically for BrRNA-RT-qPCR and blood culture at the time of initial sepsis evaluation by arterial puncture. BrRNA-RT-qPCR and blood culture were undertaken using identical blood samples, and BrRNA-RT-qPCR was performed using 12 primer sets.ResultsPositive rate was significantly higher for BrRNA-RT-qPCR (15/39, 38.5%) than for blood culture (6/39, 15.4%; p = 0.0039). BrRNA-RT-qPCR was able to identify all pathogens detected by blood culture. Furthermore, this method detected pathogens from neonates with clinical sepsis in whom pathogens was not detected by culture methods.ConclusionsThis RT-PCR technique is useful for sensitive detection of pathogens causing neonatal sepsis, even in cases with negative results by blood culture.

Immunological development of preterm infants in early infancy

To evaluate the immunological development of preterm infants, especially in early infancy, we examined the serum cytokine levels and the expression of Th2 and Th1 chemokine receptors, CCR4 and CCR5, on days 0, 14 and 28 in 16 low birth weight infants (1720·38 ± 502·80 g) born at less than 37 (33·63 ± 3·29) weeks of gestation. Using an enzyme‐linked immunosorbent assay (ELISA), serum interleukin (IL)‐4 levels exhibited an increase on day 14, but decreased to the initial level on day 28 (P < 0·05). The significant elevation of serum transforming growth factor (TGF)‐β levels was confirmed on day 14 (P < 0·05) but decreased to the initial level on day 28 (P < 0·05). The expression of CCR4 and CCR5 were examined using reverse transcription‐polymerase chain reaction (RT‐PCR) and flow cytometric analysis. The RT‐PCR confirmed the expression of CCR5‐mRNA soon after birth, while there was no expression of CCR4‐mRNA. Thereafter, the expression of CCR4‐mRNA increased significantly and reached the level of CCR5‐mRNA expression on day 28 (P < 0·05). Flow cytometric analysis, however, revealed that the expression levels of both CCR4 and CCR5 were low at birth. Thus, CCR4+ CD4+ cells were significantly increased from days 0–28 (P < 0·05), while CCR5+ CD4+ cells were not. Increased IL‐4 and TGF‐β synthesis as well as increased CCR4+ CD4+ cells suggest that, under extra‐maternal circumstances, there is a shift in bias toward Th2 responses even in preterm infants soon after delivery, while they may be capable of developing Th1 mediated responses soon after birth.

Effects of human milk and spermine on hydrogen peroxide-induced oxidative damage in IEC-6 cells

Objective: Oxidative stress is intimately involved in the pathologic processes of serious diseases in the perinatal period. Human milk (HM) contains various bioactive substances, some of which are known as antioxidants, including polyamines such as spermine (SPM). We examined the antioxidative properties of HM and SPM in an intestinal epithelial cell line. Method: Confluent Intestinal Epithelial Cells-6 (IEC-6) cells were preincubated with 100-fold dilutions of defatted HM, bovine milk, or three artificial milks for 24 hours, followed by hydrogen peroxide (H2O2) challenge (0.5 mM, 30 min) for oxidative stress. Cells were preincubated with either HM or increasing concentrations (within the range of HM) of SPM for 24 hours followed by an H2O2 challenge (0.25 mM, 30 min). Results: HM-treated cells showed the highest survival rate (50%) compared with no pretreatment (27%), bovine milk-treated (6%), or artificial formula-treated (13-16%) cells. Significantly higher survival rates were observed in the cells treated with HM (44.0%) and in those treated with 0.5, 1, or 5 μM of SPM (12.6, 13.1, or 22.2%, respectively) in comparison with the nontreated cells (7.0%). Conclusion: Our results demonstrated that HM and SPM alleviated H2O2-induced oxidative damage in IEC-6 cells, whereas bovine milk and artificial formula did not show any antioxidative capacity. These results suggest that HM acts as an antioxidant in the gastrointestinal tract of infants and that SPM plays an important role in the antioxidative properties of HM.