Extremely preterm infants born outside a provincial tertiary perinatal center and transferred postnatally associated with poor outcomes: a real-world observational study.

Introduction: Extremely preterm infants (EPIs) have high morbidity and mortality, and are recommended to be born in a tertiary perinatal center (inborn). However, many EPIs in central China are born in lower-level hospitals and transferred postnatally, the outcomes of which remain to be investigated. Methods: EPIs admitted to the Department of Neonatology, Maternal and Child Health Hospital of Hubei Province from January 2013 to December 2022 were retrospectively recruited and divided into the control (inborn) and transfer groups (born in other hospitals). The neonatal and maternal characteristics, neonatal outcomes, and the treatment of survival EPIs were analyzed. Results: A total of 174 and 109 EPIs were recruited in the control and transfer groups, respectively. EPIs in the transfer group have a higher birth weight and a lower proportion of multiple pregnancies than the control group (all P < 0.05). The proportions of antenatal steroids, magnesium sulfate, cesarean delivery, premature rupture of membranes ≥18 h, gestational diabetes, and amniotic fluid abnormalities were lower in the transfer group (all P < 0.05). Survival rates (64.22% vs. 56.32%), proportions of severe periventricular-intraventricular hemorrhage (PIVH) (11.93% vs. 11.49%), severe bronchopulmonary dysplasia (sBPD) (21.05% vs. 20%), and severe retinopathy of prematurity (ROP) (24.77% vs. 20.11%) were similar in the transfer and control groups (all P > 0.05). However, the transfer group had higher proportions of severe birth asphyxia (34.86% vs. 13.22%, P < 0.001), PIVH (42.20% vs. 29.89%, P = 0.034), and extrauterine growth retardation (EUGR) (17.43% vs. 6.32%, P = 0.003). Less surfactant utilization was found in the transfer group among survival EPIs (70.00% vs. 93.88%, P < 0.001). Conclusion: EPIs born outside a tertiary perinatal center and transferred postnatally did not have significantly higher mortality and rates of severe complications (severe PIVH, severe ROP, and sBPD), but there may be an increased risk of severe asphyxia, PIVH and EUGR. This may be due to differences in maternal and neonatal characteristics and management. Further follow-up is needed to compare neurodevelopmental outcomes, and it is recommended to transfer the EPIs in utero to reduce the risk of poor physical and neurological development.

The outer membrane protein of Fusobacterium necrophorum, 43K OMP, stimulates inflammatory cytokine production through nuclear factor kappa B activation.

OBJECTIVE: Fusobacterium necrophorum causes bovine hepatic abscess, foot rot, mastitis, and endometritis. The 43 kDa outer membrane protein (43 K OMP) of F. necrophorum is a porin protein that plays an important role in infections by this bacterium, but the biological function and the pathogenesis of this protein are largely unknown. METHODS: In this study, we investigated the role of the 43 K OMP in bacterial infection of bovine mammary epithelial cells (MAC-T cells) by Tandem Mass Tag proteomic analysis. The RAW264.7 cells were incubated with recombinant 43 K OMP (12.5 µg/mL) for 2 h, 4 h, 6 h, and 12 h, and then the inflammatory related protein and inflammatory cytokine production were measured by Western blot analysis and ELISA, the mRNA expression levels of inflammatory cytokine were measured by Real-Time PCR. RESULTS: Proteomic analysis results demonstrated there were 224 differentially expressed proteins in the MAC-T cells stimulated with the 43 K OMP compared with control, and 118 proteins were upregulated and 106 proteins were downregulated. These differentially expressed proteins were mainly involved in NF-kappa B signaling, bacterial invasion of epithelial cells, cell adhesion, complement and coagulation cascades. The top six differentially expressed proteins were; MMP9, PLAU, STOM, PSMD13, PLAUR, and ITGAV, which were involved in a protein-protein interaction network. Furthermore, TLR/MyD88/NF-κB pathway related proteins and inflammatory cytokines (IL-6, TNF-α, and IL-1ß) were assessed by Western blot analysis and ELISA. Results showed the 43 K OMP to enhance the expression of TLR4 protein at 2 h (P < 0.01) and the MyD88 protein at 4 h (P < 0.05) post-stimulation, and to decrease IκBα expression at 4 h, 6 h and 12 h (P < 0.05) post-infection, as well as induce phosphorylation at Ser536 (P < 0.01). Levels of IL-6, IL-1ß, and TNF-α in the supernatants of mouse macrophages were increased (P < 0.05), as were mRNA expression levels of IL-6, IL-1ß, and TNF-α (P < 0.05), while IL-4 mRNA expression was decreased (P < 0.05). CONCLUSIONS: Taken together, these results suggested the important role for 43 K OMP in F. necrophorum infection, promoting the production of pro-inflammatory cytokines (IL-6 and TNF-α) by activation of the TLR/MyD88/NF-κB pathway. These findings provided a theoretical basis for a better understanding of the pathogenesis of F. necrophorum infection.

Interaction of 43K OMP of Fusobacterium necrophorum with fibronectin mediates adhesion to bovine epithelial cells.

Fusobacterium necrophorum, a Gram-negative anaerobe, is an important bovine pathogen that causes hepatic abscesses, foot rot, mastitis and endometritis. We have previously shown that the 43 kDa outer membrane protein (43 K OMP) of F. necrophorum is a porin protein that plays an important role in bacterial infections; however, the molecular mechanisms by which this protein mediates adhesion remain unclear. In this study, we investigated the role of 43 K OMP in F. necrophorum adhesion to bovine epithelial cells using 43 K OMP-deficient mutants, and identified the protein that interacts with 43 K OMP by immunoprecipitation-mass spectrometry. Our results indicated that the native 43 K OMP and recombinant 43 K OMP could bind to the cell membrane of MAC-T or bovine endometrial epithelial cells (BEECs). When F. necrophorum was preincubated with antibodies against the recombinant 43 K OMP or bovine epithelial cells were preincubated with 43 K OMP, the adhesion of F. necrophorum to MAC-T or BEECs decreased significantly (P<0.01). We successfully constructed a 43 K OMP-deficient strain (A25Δ43 K OMP) and bacterial attachment to MAC-T or BEECs was significantly higher with the F. necrophorum A25 strain than with mutant strain A25Δ43 K OMP (P<0.01). The deficiency of 43 K OMP reduced the binding of F. necrophorum to bovine epithelial cells by 90.5 %-94.9 %. Among the 39 potential differential proteins, fibronectin, collagen and myosin were selected as the target proteins, and direct interaction between 43 K OMP of F. necrophorum and fibronectin was demonstrated. Taken together, these results suggest that 43 K OMP plays a key role in adhesion of F. necrophorum to bovine epithelial cells through its interaction with fibronectin. These findings provide a theoretical basis for the pathogenic mechanism of F. necrophorum.

Liver X receptor α participates in LPS-induced reduction of triglyceride synthesis in bovine mammary epithelial cells.

Lipopolysaccharides (LPS) could induce milk fat depression via regulating the body and blood fat metabolism. However, it is not completely clear how LPS might regulate triglyceride synthesis in dairy cow mammary epithelial cells (DCMECs). DCMECs were isolated and purified from dairy cow mammary tissue and treated with LPS. The level of triglyceride synthesis, the expression and activity of the liver X receptor α (LXRα), enzymes related to de novo fatty acid synthesis, and the expression of the fatty acid transporters were investigated. We found that LPS decreased the level of triglyceride synthesis via a down-regulation of the transcription, translation, and nuclear translocation level of the LXRα. The results also indicated that the transcription level of the LXRα target genes, sterol regulatory element binding protein 1 (SREBP1), fatty acid synthetase (FAS), acetyl-CoA carboxylase-1 (ACC1), were significantly down-regulated in DCMECs after LPS treatment. Our data may provide new insight into the mechanisms of milk fat depression caused by LPS.

Selenium deficiency induces splenic growth retardation by deactivating the IGF-1R/PI3K/Akt/mTOR pathway.

Selenium (Se) deficiency impairs the development and function of immune system in human beings and animals. We investigated the effect and molecular mechanism of Se deficiency on spleen development in chicken. The concentration of Se in blood and spleen, the spleen weight and splenocyte number, the histological characteristics of spleen, the concentration of growth factors in serum, the transcription level of growth factor receptor gene and the activity of growth and proliferation pathway in spleen were investigated. We found that the growth of the spleen and the splenocyte number were significantly lower in the chicken fed with Se-deficient diet for 21 and 35 days. The ELISA and qRT-PCR results showed that the serum IGF-I concentration and the transcription level of IGF1R gene in spleen were significantly lower in the SD group. The Western blotting and immunohistochemistry results showed that Se deficiency could deactivate the PI3K/Akt/mTOR pathway in spleen. In summary, the results indicated that Se deficiency decreases the growth rate of spleen and the number of splenic lymphocytes by deactivating the IGF-1R/PI3K/Akt/mTOR pathway.

LPS-induced reduction of triglyceride synthesis and secretion in dairy cow mammary epithelial cells via decreased SREBP1 expression and activity.

Sterol regulatory element binding protein 1 (SREBP1) has a central regulatory effect on milk fat synthesis. Lipopolysaccharides (LPS) can induce mastitis and cause milk fat depression in cows. SREBP1 is also known to be associated with inflammatory regulation. Thus, in the current study, we hypothesized that LPS-induced milk fat depression in dairy cow mammary epithelial cells (DCMECs) operates via decreased SREBP1 expression and activity. To examine the hypothesis, DCMECs were isolated and purified from dairy cow mammary tissue and treated with LPS (10 µg/ml). LPS treatment of DCMECs suppressed lipid-metabolism-related transcription factor SREBP1 mRNA expression, nuclear translocation and protein expression, leading to reduced triglyceride content. The transcription levels of acetyl-CoA carboxylase-1 and fatty acid synthetase were significantly down-regulated in DCMECs after LPS treatment, suggesting that acetyl-CoA carboxylase-1 and fatty acid synthetase involved in de novo milk fat synthesis was regulated by SREBP1. In summary, these results suggest that LPS induces milk fat depression in dairy cow mammary epithelial cells via decreased expression of SREBP1 in a time-dependent manner.

17ß-Estradiol and progesterone decrease MDP induced NOD2 expression in bovine mammary epithelial cells.

During the periparturient period, many neuroendocrine changes develop in cows. Periparturient hormone fluxes may adversely affect mammary gland immunity and mastitis susceptibility. 17ß-Estradiol (E2) and progesterone (P4) have been reported to function on immune regulation, and their concentration fluctuates dramatically during the perinatal period. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) mediate numerous aspects of innate immunity in humans and experimental animals. This study aimed to explore the effects of E2 and P4 on NOD2 expression in bovine mammary epithelial cells (BMECs). BMECs were isolated and purified from bovine mammary tissue and treated with E2/P4 and muramyl dipeptide (MDP). After these treatments, the mRNA levels of NOD2, receptor-interacting protein kinase (RIP) 2, interleukin (IL) 1ß, IL-6, IL-8 and tumor necrosis factor (TNF) α were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) respectively, and the protein levels of NOD2 were analyzed by western blotting. The results showed that E2 and P4 decreased MDP-induced transcriptional expression of NOD2 and the downstream molecules. Moreover, E2 reduced MDP-induced NOD2 protein expression levels. Our study suggests that down-regulation of NOD2 by E2 and P4 may be one of the reasons for mastitis susceptibility in periparturient dairy cows.