Patterns of Human Milk Oligosaccharides in Mature Milk Are Associated with Certain Gut Microbiota in Infants.

Human milk oligosaccharides (HMOs) are complexes that play a crucial role in shaping the early-life gut microbiota. This study intends to explore whether HMO patterns are associated with the gut microbiota of infants. We included 96 Chinese breastfeeding mother-infant dyads. Breast milk and infant faecal samples were collected and tested. With milk 2'-fucosyllactose, difucosyllactose, and lacto-N-fucopentaose-I as biomarkers, we divided the mothers into secretor and non-secretor groups. HMO patterns were extracted using principal component analysis. The majority (70.7%) of mothers were categorised as secretor and five different HMO patterns were identified. After adjustment, the infants of secretor mothers exhibited a lower relative abundance of Bifidobacterium bifidum (ß = -0.245, 95%CI: -0.465~-0.025). An HMO pattern characterised by high levels of 3-fucosyllactose, lacto-N-fucopentaose-III, and lacto-N-neodifucohexaose-II was positively associated with the relative abundance of Bifidobacterium breve (p = 0.014), while the pattern characterised by lacto-N-neotetraose, 6'-sialyllactose, and sialyllacto-N-tetraose-b was negatively associated with Bifidobacterium breve (p = 0.027). The pattern characterised by high levels of monofucosyl-lacto-N-hexaose-III and monofucosyl-lacto-N-neohexaose was positively associated with Bifidobacterium dentium (p = 0.025) and Bifidobacterium bifidum (p < 0.001), respectively. This study suggests that HMO patterns from mature breast milk were associated with certain gut microbiota of breastfed infants.

The Concentration of Lactoferrin in Breast Milk Correlates with Maternal Body Mass Index and Parity.

Background: Lactoferrin (LF) is a multifunctional glycoprotein found in human milk and body fluids, which has been shown to play a vital role in regulating the immunity and supporting the intestinal health of infants. Aim: This study evaluated the association between maternal/parturient factors and LF concentration in the breast milk of Chinese mothers. Methods: 207 breast milk samples were collected from healthy mothers with in the first year of lactation. Maternal and parturient information was collected for these participants through questionnaires. The content of lactoferrin in breast milk was detected by liquid chromatography, and macronutrient concentration in breast milk was measured by human milk analyzer in only 109 samples. Results: Our findings demonstrated that the LF content was much higher within the first month of lactation than it was after that period (p < 0.05). When compared with normal and lean mothers, the LF content of obese mothers was considerably higher (p < 0.05). The parity and LF content showed a favorable correlation. The proportion of LF to total protein tended to decrease as lactation progressed. Protein, fat, dry matter, and energy content were significantly positively correlated with LF content (p < 0.001). Conclusion: Early breast milk tends to have a higher level of LF, and the change of LF concentration in breast milk is associated with the parity and body mass index of the mother.

Effect of Lacticaseibacillus paracasei N1115 on Immunomodulatory and Gut Microbial Composition in Young Children: A Randomized, Placebo-Controlled Study.

Lactobacillus paracasei N1115 (Lp N1115) was isolated from fermented milk products. The administration of Lp N1115 is safe and well tolerated in Chinese children, but its effectiveness among young Chinese children is still unclear. To investigate the efficacy of Lp N1115 as a probiotic to enhance gut development in Chinese infants and toddlers born by cesarean section, 109 healthy and cesarean-delivered infants aged 6-24 months were recruited for a 12-week randomized, placebo-controlled trial, with 101 finally completing the study. Saliva and stool samples were collected and detected at weeks 0, 4, 8, and 12 of the intervention. Statistical analyses were performed by using a per-protocol (PP) approach. After 12 weeks of intervention, the fecal pH in the control group increased (p = 0.003), while the fecal pH in the experimental group did not change. Salivary cortisol decreased from baseline in the experimental group (p = 0.023), while the control group showed little change. In addition, Lp N1115 increased the fecal sIgA content of infants aged 6-12 months (p = 0.044) but had no obvious effect on fecal calprotectin and saliva sIgA. At week 4, the increase in Lactobacillus relative to baseline was higher in the experimental group than in the control group (p = 0.019). Further analysis showed a trend toward a higher detection rate of Lactobacillus in the experimental group than in the control group (p = 0.039). In conclusion, Lp N1115 was able to enhance the content of Lactobacillus and maintain fecal pH levels. Its beneficial effects on gut development were more obvious in 6-12-month-old infants.

In vitro Intervention of Lactobacillus paracasei N1115 Can Alter Fecal Microbiota and Their SCFAs Metabolism of Pregnant Women with Constipation and Diarrhea.

In vitro fermentation was used to evaluate the possible effects of intervention with Lactobacillus paracasei N1115 (LP N1115) on gut microbiota and metabolite shortchain fatty acids (SCFAs) in pregnant women with constipation and diarrhea. Feces were collected from pregnant women and fermented by YCFA medium to profile the changes in the gut microbiota before and after intervention with LP N1115 using 16SrRNA sequencing. At the same time, the changes in several specific bacteria were detected using quantitative real-time PCR (qPCR) and the SCFAs in fermentation were detected using gas chromatography (GC) for each subject to determine the effect of the intervention. In vitro intervention with LP N1115 significantly increased the relative abundances of Lactobacillus, Faecalibacterium prausnitzii, and Bifidobacterium in constipated pregnant women and reduced the contents of acetic acid, propanoic acid. Moreover, 16S rRNA gene analysis showed that LP N1115 also reduced the relative abundance of Clostridium_XI. The results of this study suggest that LP N1115 might increase the content of beneficial bacteria and reduce the relative abundance of pathogenic bacteria, which might be beneficial to gut health in pregnant women.

A randomized, double blind, parallel, placebo-controlled study to investigate the efficacy of Lactobacillus paracasei N1115 in gut development of young children.

In this clinical trial, the safety and effectiveness of Lactobacillus paracasei N1115 (LP N1115) were investigated as a potential probiotic to enhance gut development in young children born by caesarean section. Infants and young children between the ages of 6 months and 3 years were administered with a probiotic consisting of LP N1115 strain (n = 30) or placebo supplement (n = 30) over an 8 weeks intervention. And the stool consistency, bowel habits, salivary cortisol, fecal microbiota, and short-chain fatty acid metabolism were investigated. Efficacy data were obtained from 58 participants who completed the study. Overall, the placebo functioned similarly to LP N1115 group in relation to stool consistency, gastrointestinal symptoms, salivary cortisol, and short-chain fatty acids. However, the scoring data relating to the 6-18 months subgroup receiving LP N1115 remained stable over 8 weeks in comparison to placebo. Analysis of the fecal microbiota using 16S rRNA amplicon sequencing revealed that the phyla Firmicutes represented 62% of the microbial relative abundance in the feces of the subjects during the intervening period. No significant changes in alpha- or beta-diversity were noted between the placebo and LP N1115 groups overtime and at each time point. Differential abundance analysis indicated an increase in Lactobacillus in LP N1115 group at weeks 4 (p < .05) and 8 (p < .05) in comparison to the placebo group. These results suggest that probiotic supplementation with LP N1115 was well tolerated by the young children and subtle changes in the microbiome were noted throughout the intervention period.

Genome Sequence of Lactobacillus plantarum JMCC0013, Isolated from Traditional Chinese Fermented Milk.

Fermented food products have been consumed for thousands of years in China, so fermented Chinese foods may contain huge lactic acid bacterial resources. Here, we report the draft genome sequence of a Lactobacillus plantarum isolate, JMCC0013, collected from traditional Chinese fermented milk, which provides a precious resource for the genomic analysis of Lactobacillus strains.

Identification of Moesin as a Novel Autoantigen in Patients with Sjögren's Syndrome.

BACKGROUND: SjOgren's Syndrome (SS) is a systemic and chronic autoimmune disorder that affects the exocrine glands with massive autoantibody production. Although the pathogenesis of the disorder is incompletely understood, but some studies have reported that anti-moesin antibodies have been detected in autoimmune diseases with which SS is closely associated. Here, we have investigated moesin's potential involvement in SS. OBJECTIVE: This study aims to verify whether moesin is a specific autoantigen involved in Chinese Hans SS patients. METHODS: First, recombinant human moesin was expressed and purified. Next, the protein was verified as antigen by Western blotting and immunoprecipitation. The positive protein band in the immunoprecipitation was identified by (MALDI-TOF/TOF). Finally, an optimized ELISA (Enzyme- Linked Immunosorbent Assay) kit was developed to measure the titer concentration of anti-moesin antibody-positive patients in a large cohort of clinical subjects. RESULTS: Univariate analysis revealed that the proportion of individuals positive for serum IgG against recombinant human moesin was 42 % in a group of Chinese Hans SS patients (21 of 50), 22 % in systemic lupus erythematosus patients and (11 of 50), compared to only 4 % in healthy controls (2 of 50). CONCLUSION: An association between anti-moesin antibodies and SS manifestation have been found which may be considered a suspected serum biomarker for the development of SS.

Circulation autoantibody against Lamin A/C in patients with Sjögren's syndrome.

Lamin A/C proteins are major components of nuclear laminae and were encoded by the LMNA gene. Recent studies have found that in addition to provides nuclear-membrane strength; it also regulates the gene expression. Lamin A/C has been confirmed as an autoantigen in RA, SLE and vasculitis. Anti-Lamin A/C antibodies also have been found by indirect immunofluorescence method. In this study, we used various research methods to confirm Lamin A/C is an autoantigen in Han Chinese patients with confirmed Sjögren's syndrome (SS). To further investigate the relationship between the autoimmune disease antigens, we compared the amino acid sequence of Lamin A/C epitope and several common antigens' antigenic determinant. As a result, we found that Lamin A/C has similar epitopes with U1RNP. It means that the potential relationship exist between Lamin A/C and U1RNP. Clinical data we collected also showed that anti-Lamin A/C and anti-U1RNP antibodies always appear in same serum sample. Therefore, we speculated that cross-reaction may take place between antigen and potential antigen, which have similar epitope. Then, by epitope spreading, the potential antigen can be a new autoantigen. Our study provided a new thinking for further research about the relationship between autoantigens and their development mechanism in autoimmune diseases.

Prohibitin as a novel autoantigen in rheumatoid arthritis.

THE AIM OF THIS STUDY: The aim of this study was to verify whether prohibitin is a novel autoantigen in rheumatoid arthritis. MATERIAL AND METHODS: First, recombinant human prohibitin (rhPHB) protein was cloned, expressed, and purified. Then the anti-prohibitin autoantibodies were detected by western blotting by using rhPHB protein to incubate sera from patients with rheumatoid arthritis (RA). Next, immunoprecipitation was employed to further illustrate whether anti-prohibitin antibodies exist in RA patients. And finally, autoantibodies against the rhPHB protein were investigated using a homemade ELISA kit through the assessment of 258 real clinical samples. RESULTS: It was revealed that anti-prohibitin antibodies existed in the sera of patients with RA. Reactivity of serum IgG against rhPHB was detected in 26 of 86 RA patients (30.3%), 7 of 86 systemic lupus erythematosus (SLE) patients (8.1%), and 1 of 86 apparently healthy donors (HC) (1.2%). CONCLUSIONS: Prohibitin was proved to be a novel autoantigen and the corresponding anti-prohibitin autoantibodies were present in the RA patients' blood circulation.

Prohibitin Is Involved in Patients with IgG4 Related Disease.

OBJECTIVE: IgG4-related disease (IgG4-RD) is a chronic systemic disease involved in many organs and tissues. As only limited autoantigens have been found since the beginning of this century, the aim of this study was to reveal new candidate autoantigens of IgG4-RD. METHODS: Multiple cell lines including HT-29, EA.hy926, HEK 293 and HepG2 were used to test the binding ability of circulating autoantibodies from IgG4-RD sera. The amino-acid sequence was then analyzed by matrix-assisted laser desorption/ionization time-of-flight tandem (MALDI-TOF/TOF) mass spectrometry. After the cloning and expression of recombinant putative autoantigen in a bacterial expression system, the corresponding immuno assay was set up and utilized to observe the prevalence of serum autoantibodies in a large set of confirmed clinical samples. RESULTS: One positive autoantigen was identified as prohibitin. ELISA analysis showed that a majority of patients with IgG4-RD have antibodies against prohibitin. Anti-prohibitin antibodies were present in the sera of patients with definite autoimmune pancreatitis (25/34; 73.5%), Mikulicz's disease (8/15; 53.3%), retroperitoneal fibrosis (6/11; 54.5%), other probable IgG4-RD (26/29; 89.7%) and Sjögren's syndrome (4/30; 13.3%) but not in apparently healthy donors (1/70; 1.4%). CONCLUSIONS: An association between prohibitin and patients with some IgG4-RD was observed, although the results were quite heterogeneous among different individuals within autoimmune pancreatitis, Mikulicz's disease and retroperitoneal fibrosis.

Screening hybridomas for anabolic androgenic steroids by steroid analog antigen microarray.

BACKGROUND: Currently, dozens of anabolic androgenic steroids (AAS) are forbidden in the World Anti-Doping Agency Prohibited List, however, despite extensive investigation, there are still lots of AAS without corresponding monoclonal antibodies. RESULTS: A steroid analog antigen microarray made up of ten AAS was fabricated to screen the hybridoma and it was found an original unsuccessful clone turned out to be a candidate anti-boldenone antibody, without any cross-reactions with endogenous AAS or 44 different AAS standard reference materials tested. CONCLUSION: Our findings suggested that steroid analog antigen microarray could be a promising tool to screen and characterize new applications of antibodies for structure analogs, and this also exhibits the potential to fast identify effective epitopes of hybridomas in a single assay.

Electron Transfer Flavoprotein Subunit Beta Is a Candidate Endothelial Cell Autoantigen in Behçet's Disease.

Behçet's disease (BD) is a chronic inflammatory disease with multisystem involvement, and it is listed as a rare disease in the United States but is common in the Middle East, China, and Japan. The aim of this study was to identify novel autoantigens in Chinese patients with BD. First, the candidate autoantigens were screened by Western blotting, and the sequences of putative antigens were identified by LC-MALDI-TOF/TOF mass spectrometry. Next, the screened protein was cloned, expressed and purified. Then, an optimized ELISA was developed, and the serological criteria were evaluated using a large number of confirmed patients. One antigen with a molecular weight of approximately 28 kDa was identified as electron transfer flavoprotein subunit beta (ETFB). Positive reactivity was detected in recombinant human ETFB sera from 38 of 92 BD patients (41 %) and 1 of 90 healthy controls (1 %).

Annexin A2 as a target endothelial cell membrane autoantigen in Behçet's disease.

Cell membrane proteins are believed to play a critical role in the pathogenesis of autoimmune diseases. However, few membrane autoantigens have been linked with Behçet's disease. Here, a cell-chip was performed to identify autoantibody target cells, and the suspected autoantigens were detected using immunoblotting. The amino acid sequences of the detected proteins were determined using LC-MALDI-TOF/TOF. Putative proteins were recombinantly expressed and purified, and a corresponding ELISA was developed and clinically validated using real clinical samples. It was found that a 36-kDa membrane protein--annexin A2--was detected in approximately one-third of the patients' blood circulation. The immunohistochemistry results showed that annexin A2 was highly expressed in vascular endothelial cells. Moreover, vascular involvement was significantly higher in the anti-annexin A2 antibody-positive group versus the anti-annexin A2 antibody-negative group among all the clinical samples analyzed, indicating that annexin A2 is a novel endothelial cell membrane antigen involved in Behçet's disease.

Identification of heat shock protein 27 as a novel autoantigen of Behçet's disease.

OBJECTIVE: The aim of this study was to identify candidate pathogenic autoantigens of Behçet's disease (BD) in pathogen-stimulated target cells. METHODS: First, three cell lines were used as target cells to screen autoantibody. Second, selected target cells were simulated with pathogens. Third, western blotting was used for detecting the auto-antigens in cell extracts. Next, immunoprecipitation was performed and the amino-acid sequences of target antigens were analyzed by LC-MALDI-TOF/TOF. Then, the potential target antigen was expressed, purified, and immunologically confirmed. And finally, an ELISA kit was developed and clinically validated through the assessments of 456 clinical samples with BD. RESULTS: One antigen with a molecular weight of approximately 27-kDa was identified as heat shock protein 27 (HSP27). The reactivity of serum IgG against recombinant human HSP27 was detected in 52 of 91 BD patients (57%), 66 of 92 rheumatoid arthritis (RA) patients (72%), 32 of 90 Sjogren syndrome (SS) patients (36%), 22 of 92 systemic lupus erythematosus (SLE) patients (24%) and 0 of 91 healthy controls (HC). The reactivity of BD serum IgG antibodies against HSP27 was significantly higher than SLE (P<0.0001) SS (P<0.0001) and HC (P<0.0001). CONCLUSIONS: This study identified HSP27 as a candidate endothelial cell autoantigen of BD, which is interesting and probably worth further exploration.

Identification of prohibitin as an antigen in Behcet's disease.

OBJECTIVE: This study is intended to screen potential antigen for Behcet's disease (BD) by using human microvascular endothelial cells (HUVEC). METHODS: Following cell-based indirect immunofluorescence assay with sera from BD patients, proteins extracted from HUVEC were separated and detected by Western blotting. Then the target protein was identified by LC-MALDI-TOF/TOF, the recombinant target protein was expressed, purified and then used as coating antigen to test the prevalence of autoantibodies in patient's sera. RESULTS: The Western blotting result showed that some patients' sera could react with a protein band with about 30 kDa of molecular weight, which was further identified as prohibitin by mass spectrometry. The prevalence of serum antibodies against recombinant human prohibitin was detected in 16 of 58 BD patients (28%) but none in healthy controls.

Multiple correlations of mRNA expression and protein abundance in human cytokine profile.

With the development of genomic study, researchers found that it is insufficient to predict protein expression from quantitative mRNA data in large scale, which is contrary to the traditional opinion that mRNA expression correlates with protein abundance at the single gene level. To try to solve the apparent conflicting views, here we set up a series of research models and chose soluble cytokines as targets. First, human peripheral blood mononuclear cell (PBMC) from one health donor was treated with 16 continuously changing conditions, the protein and mRNA profile were analyzed by multiplex Luminex and genomic microarray, respectively. Among the tested genes, around half mRNA correlated well with their corresponding proteins (ρ > 0.8), however if we put all the genes together, the correlation coefficient for the 16 conditions varied from 0.29 to 0.71. Second, PBMC from 14 healthy donors were stimulated with the same condition and it was found that the correlation coefficient went down (ρ < 0.6). Third, 28 rheumatoid arthritis (RA) patients were tested for their response to the same external stimuli and it turned out different individual displayed different protein expression pattern as expect. Lastly, autoimmune disease cohorts (8 diseases including RA, 103 patients in total) were assayed on the whole view. It was observed that there was still some similarity in the protein profile among patients from the single disease type although completely different patterns were displayed across different disease categories. This study built a good bridge between single gene analysis and the whole genome study and may give a reasonable explanation for the two conflicting views in current biological science.

Generation and evaluation of antibodies against human MGF E-peptide by reverse phase protein microarray and reverse competitive ELISA.

BACKGROUND: Since 2005, as one of prohibited substances on the Prohibited List of the World Anti-Doping Agency (WADA), the occurence of mechano growth factor (MGF) abuse in sport has likely increased. However, there is still no WADA-validated and -approved method for its detection. RESULTS: Four polyclonal antibodies (Ab-K01, Ab-B01, Ab-B02 and Ab-K02) against MGF C-terminal peptides were generated, purified and evaluated by western blot, ELISA and reverse-phase protein microarray, respectively. It was found that all the antibodies could identify their corresponding antigen in mouse serum by reverse-phase protein microarray, in particular, Ab-K01 showed the highest affinity among them and might be a potential tool for the detection of antibody affinity. Furthermore, Ab-B01 and Ab-K01 were successfully used for the determination of MGF-40 by reverse competitive ELISA. CONCLUSION: The quantitative measurement of MGF-40 has laid the foundation for doping detection of MGF and further biological research on MGF.

Immunological screening and characterization of highly specific monoclonal antibodies against 20 kDa hGH.

BACKGROUND: hGH has been widely abused as a doping agent in sports for many years. There are some important approaches for the detection of hGH doping, and the ratio of 22:20 kDa GH was considered one of the most suitable detection indicators of GH abuse. Currently, effective anti-GH antibodies and related reagents are needed to develop a detection method, in particular, highly specific anti-20 kDa hGH monoclonal antibodies are a prerequisite. Herein we constructed the expression vector of 20 kDa hGH and prepared the corresponding antibodies by the immunization of the recombinant human 20 kDa into mice. Positive clones that can specifically recognize 20 kDa hGH were screened and characterized by enzyme immunoassay, Dot-ELISA and surface plasmon resonance. In total, 14 specific monoclonal cell lines were screened out. RESULTS: By a series of characterization, it was found that the 6C8, 44H3, 12G7 and 33Y19 clones were showing much higher specificity and affinity to 20 kDa hGH, and P3H9 could recognize both 20 and 22 kDa hGH isoforms. 6C8 and 44H3 matched well with P3H9 in the surface plasmon resonance testing. The 12G7 clone had the best surface properties with an association constant of 3.4 × 10(9) M(-1) and a dissociation constant of 2.95 × 10(10) M. CONCLUSION: Highly specific monoclonal antibodies against 20 kDa hGH were generated, and also two paired antibodies (P3H9 and 6C8 or P3H9 and 44H3) were characterized, which can serve as the potential components for 22:20 kDa detection kit.

Circular IGFBP-3 level affected by the gene transfer of different growth hormone isoforms.

To evaluate whether through gene transfer, which growth hormone (GH) isoform can influence the circulation IGFBP-3 level, mice were injected three times with the human cytomegalovirus (CMV) mediated mammalian expression vector encoding different GH isoform gene. Then blood samples were drawn and the ELISA method was used to determine the circulation concentration of IGFBP-3. It was found that the 22 kDa GH gene could increase IGFBP-3 levels to 1.94 microg/ mL whereas 20 kDa GH, 17 kDa GH and 5 kDa GH genes did not play a role in the circulation expression of IGFBP-3.