Cohort profile: Japanese human milk study, a prospective birth cohort: baseline data for lactating women, infants and human milk macronutrients.

PURPOSE: The Japanese Human Milk Study, a longitudinal prospective cohort study, was set up to clarify how maternal health, nutritional status, lifestyle and sociodemographic and economic factors affect breastfeeding practices and human milk composition. This would eventually determine factors affecting the growth and development of infants and children. PARTICIPANTS: A total of 1210 Japanese lactating women who satisfied the inclusion criteria, were invited across the country at various participating sites, between 2014 and 2019. Finally a total of 1122 women were enrolled in this study. FINDINGS TO DATE: Among 1122 eligible participants, mean age at delivery was 31.2 (SD 4.4) years and mean prepregnancy BMI was 20.8 (SD 2.7). Among these women, 35% were previously nulliparous and 77.7% had college, university or higher education. The mean gestational period was 39.0 (SD 1.3) weeks. Caesarean section was reported among 11.9%; mean infant birth weight was 3082 (SD 360) g. Of the infants, 53.7% were male. Overall, our participants appeared to be healthier than the general population in Japan. Analyses of the 1079 eligible human milk samples obtained at the first and second months postpartum showed the following composition: carbohydrate, 8.13 (SD 0.32) g/100 mL; fat, 3.77 (SD 1.29) g/100 mL; and crude protein, 1.20 (SD 0.23) g/100 mL. We also analysed osteopontin, fatty acid, vitamin D and phospholipid levels in limited subcohorts of the samples. FUTURE PLANS: Follow-up surveys will be conducted to obtain milk samples every 2 months for 12 months and to investigate mother and child health until the children reach 5 years of age. These will be completed in 2024. We plan to longitudinally analyse the composition of macronutrients and various bioactive factors in human milk and investigate the lifestyle and environmental factors that influence breastfeeding practices, maternal and child health, and child development. TRIAL REGISTRATION NUMBER: UMIN000015494; pre-results.

Association of DHA Concentration in Human Breast Milk with Maternal Diet and Use of Supplements: A Cross-Sectional Analysis of Data from the Japanese Human Milk Study Cohort.

BACKGROUND: DHA (22:6n-3) is essential for neurodevelopment in children, and its concentration in human breast milk is historically high in Japan. Dietary patterns in Japan might affect the fatty acid (FA) composition among lactating mothers. OBJECTIVES: This study aimed to characterize the composition of milk FAs and to identify any dietary and sociodemographic factors associated with the variability of DHA concentration in breast milk in the Japanese population. METHODS: This cross-sectional study was performed as part of the Japanese Human Milk Study. Milk FAs were analyzed by GC at 1-6 mo postpartum, and maternal diet was estimated using an FFQ, including 11 types and cooking methods of seafoods, and the use of DHA supplements. The association of milk DHA with maternal diet and sociodemographic factors was investigated. RESULTS: Milk FA concentrations were measured in 78 mothers, including 24 who routinely used DHA supplements. The DHA concentration in milk (overall median: 0.62%; IQR: 0.47%-0.78%) was higher in women who took DHA supplements than in women who had never used DHA supplements (0.74%compared with 0.55%; P = 0.011). A linear regression model showed the association of milk DHA concentration with maternal dietary intake of grilled fish (ß ± SE: 0.006 ± 0.003; standardized ß: 0.234; r 2 = 0.232, P = 0.036) after adjustment for DHA supplementation status, maternal and infant age, maternal BMI, and infant birth weight. Other FA concentrations were consistent, whereas caproic acid (6:0), undecylic acid (11:0), pentadecylic acid (15:0), palmitoleic acid (16:1n-7), and vaccenic acid (18:1n-7) varied by DHA supplementation status. CONCLUSIONS: The DHA concentration in human milk may be influenced by maternal grilled fish consumption and frequent DHA supplementation in lactating Japanese women. Milk DHA concentrations may reflect a dietary habit in Japanese mothers.This trial was registered at www.umin.ac.jp/ctr as UMIN000015494.

Analysis of children with Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae respiratoryinfections by real-time PCR assay and serological tests.

We examined 73 children with respiratory infections for Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae using real-time PCR assay and serological tests. C. pneumoniae and M. pneumoniae infections were found in 11 (15.1%) and 6 (8.2%) cases, respectively. The sensitivities and specificities of real-time PCR versus definite diagnosis of acute infection were 63.6% and 100% for C. pneumoniae, and 100% and 100% for M. pneumoniae, respectively. C. pneumoniae PCR-negative results appeared to be due to poor growth of the organism. The sensitivity and specificity of ImmunoCard tests were 33.3% and 82.1%, respectively, indicating that the efficacy of rapid diagnosis was disputable. The present results suggest that real-time PCR is suitable for rapid diagnosis as a first screening test to determine first-line antibacterial agents to be used against these infectious diseases.

Vasculitis induced by immunization with Bacillus Calmette-Guérin followed by atypical mycobacterium antigen: a new mouse model for Kawasaki disease.

Kawasaki disease causes systemic vasculitis. The development of skin lesions at the vaccination site with Bacillus Calmette-Guérin (BCG) is an important diagnostic symptom. We hypothesized that infection with ubiquitous microorganisms immunogenically related to BCG might induce an immunopathologic reaction leading to the development of Kawasaki disease. Mice were first inoculated with BCG, and then secondarily inoculated 4 weeks later with crude extract from Mycobacterium intracellulare (cMI), an abundant atypical mycobacterium. Animals inoculated with BCG followed by cMI developed coronary arteritis with infiltration of inflammatory cells, whereas control animals inoculated with only cMI or BCG did not, suggesting that the immune response to the mycobacteria induced autoimmunity to the vascular wall. Intravenous injection with antibodies to peroxiredoxin II, a modulator of vascular remodeling and a suggested target for autoimmune vasculitis, also resulted in coronary arteritis, but only after prior inoculation with BCG. Tumor necrosis factor-alpha, MCP1 and interferon-gamma production were significantly higher in the animals inoculated with BCG than in the control groups (P<0.05). BCG immunization was required for the development of coronary arteritis, suggesting that these cytokines might play important roles. The results indicate that BCG induces primary autoimmunity and stimulates cytokine induction, and that atypical mycobacterial infection boosts the autoimmunity resulting in coronary arteritis.

The fragments of bovine high molecular weight kininogen promote osteoblast proliferation in vitro.

High molecular weight (HMW) kininogen is known to be a large plasma protein and cleaved by plasma proteinase kallikrein, then it generates four fragments in the blood coagulation cascade: heavy chain, bradykinin, fragment 1.2, and light chain. The fragment 1.2 has also been found in the basic protein fraction of bovine milk as a bioactive protein which promotes osteoblast proliferation. The milk basic protein has been shown to be a multi functional edible protein which promotes bone formation and inhibits bone resorption. In the present study, we purified the fragment 1.2 from bovine plasma and assessed it could promote osteoblast proliferation and posses the activity after pepsin digestion. Purified plasma HMW kininogen did not promote the proliferation, however, the kallikrein-cleaved HMW kininogen promoted the proliferation. The fragment 1.2, purified from the proteolysate, also promoted the proliferation. The pepsin digestion was performed according to the method of the assessment of allergenesity of genetically modified crops. After pepsin digestion, the fragment 1.2 generated resistant fragments and showed the promoting activity of osteoblast proliferation. These results suggest that the enzymatically-digested fragments of bovine HMW kininogen are able to be a naturally occurred active protein that promotes the bone formation by oral administration.

Regulated transgene delivery by ganciclovir in the brain without physiological alterations by a live attenuated herpes simplex virus vector.

The distribution of a live attenuated herpes simplex virus (betaH1)-mediated gene delivery into the central nervous system (CNS) was regulated by growth inhibition with ganciclovir (GCV) and the effect of this transgene expression system on the physiologic response was characterized by the acoustic startle response and its prepulse inhibition. We inoculated betaH1 expressing beta-galactosidase (beta-gal) driven by the latency associated transcripts promoter into the right caudate putamen of rats. Histochemical analysis demonstrated that the inoculation of betaH1 in the right caudate putamen resulted in a high level of beta-gal expression in the neurons of the area projecting to the inoculation site. On 14 days after inoculation without GCV-treatment, beta-gal activity localized in the anterior olfactory nucleus, frontal, insular, orbital, parietal, perirhinal, piriform cortices and the temporal region including the amygdala. In contrast, the distribution of beta-gal activity was regulated by the interval between virus inoculation and GCV-treatment and maintained after its cessation without significant alteration. The whole process of transgene expression did not influence the emotional behavior, indicating that this vector system is a suitable model for analyzing the transgene function or applying the gene therapy for the CNS diseases.

A natural inter-genotypic (2b/1b) recombinant of hepatitis C virus in the Philippines.

The prevalence study and the characterization of hepatitis C virus (HCV) was carried out in the Philippines and the sequence determination of the 5'-untranslated region (5'-UTR)-Core and the NS5B regions of HCV was carried out in this study. An HCV strain (SE-03-07-1689) collected in Metro Manila, Philippines, belonged to discordant subtypes, 2b and 1b in 5'-UTR-Core and NS5B regions, respectively. The 9.3 kb sequence of this strain including the entire open reading frame was compared with those of the reference strains retrieved from the HCV sequences database (GenBank/EMBL/DDBJ) and indicated a recombination event. The computation of the sequence similarity mapped a crossover point within the NS3 region. This is the second report on the inter-genotype recombinant of HCV and the third when an intra-genotype recombinant is included. This recombinant strain, SE-03-07-1689, is designated tentatively as RF3_2b/1b according to the suggestions used for the other two HCV recombinants.

Superiority of varicella skin test antigen over purified varicella-zoster virus glycoproteins in monitoring booster response to Oka varicella vaccine.

Varicella skin test antigen has been developed based on the induction of delayed-type hypersensitivity (DTH) to varicella-zoster virus (VZV). The booster immune response to Oka varicella vaccine was assessed by cutaneous reactivity to purified VZV glycoprotein complexes, gB, gE:gI, gH:gL, and varicella skin test antigen. Skin tests with these antigens significantly augmented antibody production to glycoproteins and VZV antigen resulting in no further augmentation by the subsequent vaccination. All glycoprotein complexes induced the cutaneous reaction similarly to varicella skin test antigen. Cutaneous reaction to glycoproteins and varicella skin test antigen was boosted after vaccination. However, the magnitude of cutaneous reaction to each glycoprotein complex before and after vaccination was rich in variety. These results indicated that skin test with varicella skin test antigen is a more suitable indicator in monitoring cell-mediated immunity to VZV than that using purified glycoproteins.

Milk basic protein (MBP) increases radial bone mineral density in healthy adult women.

We studied the effects of daily intake of milk basic protein (MBP) on radial bone mineral density (BMD) in healthy adult women. Thirty-three healthy women were randomly assigned to a 6-month trial with either placebo or MBP (40 mg per day). The radial BMD of each volunteer was measured at the beginning of and at six months after the trial. The mean BMD value at the 6th month in the MBP group increased significantly at both 1/6 and 1/10 portion from the distal end of the radius, whereas that in the control group did not. The BMD gain of each volunteer in the MBP group was significantly higher than that in the placebo group. Thus a daily MBP supplementation of 40 mg in healthy adult women can significantly increase radial BMD.

Milk sphingomyelin accelerates enzymatic and morphological maturation of the intestine in artificially reared rats.

OBJECTIVES: Sphingomyelin (SPM) is the dominant phospholipid, comprising 38% of total human milk phospholipids. Although little is known about the nutritional importance of SPM during the neonatal period, SPM may affect the growth and development of tissues in the newborn infant through mechanisms regulating cell proliferation and differentiation. We evaluated the effect of sphingomyelin (SPM) in artificially reared rats as a suitable model of gut maturation in the suckling infant. METHODS: Seven-day-old Sprague-Dawley rat pups were cannulated intragastrically and reared artificially on milk containing 0.5% SPM or 0.5% phosphatidylcholine (PC) for 1 week. RESULTS: Intestinal lactase activity in the SPM group was significantly lower than that in the control or PC group. Upon histologic examination, intestinal villi were found to be occupied with vacuolated cells in the control and the PC group, whereas the vacuolated cells were restricted to the tip of villi in the SPM group. The Auerbach nerve plexus area of the ileum in the SPM group was significantly greater, possibly due to accelerated development, than that in the control group or PC group. CONCLUSIONS: The present results suggest that SPM, the dominant phospholipid in milk, plays an important role in neonatal gut maturation during the suckling period.

Cystatin C in milk basic protein (MBP) and its inhibitory effect on bone resorption in vitro.

A cystein protease inhibitor was identified in the basic fraction of bovine milk. We have reported in our previous study that the milk basic protein (MBP) fraction suppressed osteoclast-mediated bone resorption in vitro. Since osteoclasts secreted cystein protease to digest collagen in the bone matrix, we identified the cystein protease inhibitor in MBP. A 12-kDa inhibitor was purified from MBP by papain affinity gel chromatography and subsequent Hi-Load Superdex 75 gel filtration chromatography. The N-terminal sequence of the 18 amino acid residues of the inhibitor corresponded to bovine cystatin C. The 12-kDa cystein protease inhibitor in MBP therefore seemed to be cystatin C. Purified cystatin suppressed bone resorption with the use of isolated osteoclasts in vitro. Cystatin in MBP is suggested as one of the factors inhibiting bone resorption.