Characterizing the dynamics of BCR repertoire from repeated influenza vaccination.

Yearly epidemics of seasonal influenza cause an enormous disease burden around the globe. An understanding of the rules behind the immune response with repeated vaccination still presents a significant challenge, which would be helpful for optimizing the vaccination strategy. In this study, 34 healthy volunteers with 16 vaccinated were recruited, and the dynamics of the BCR repertoire for consecutive vaccinations in two seasons were tracked. In terms of diversity, length, network, V and J gene segments usage, somatic hypermutation (SHM) rate and isotype, it was found that the overall changes were stronger in the acute phase of the first vaccination than the second vaccination. However, the V gene segments of IGHV4-39, IGHV3-9, IGHV3-7 and IGHV1-69 were amplified in the acute phase of the first vaccination, with IGHV3-7 dominant. On the other hand, for the second vaccination, the changes were dominated by IGHV1-69, with potential for coding broad neutralizing antibody. Additional analysis indicates that the application of V gene segment for IGHV3-7 in the acute phase of the first vaccination was due to the elevated usage of isotypes IgM and IgG3. While for IGHV1-69 in the second vaccination, it was contributed by isotypes IgG1 and IgG2. Finally, 41 public BCR clusters were identified in the vaccine group, with both IGHV3-7 and IGHV1-69 were involved and representative complementarity determining region 3 (CDR3) motifs were characterized. This study provides insights into the immune response dynamics following repeated influenza vaccination in humans and can inform universal vaccine design and vaccine strategies in the future.

A biocleavable pullulan-based vector via ATRP for liver cell-targeting gene delivery.

Pullulan due to its specificity for liver has been widely exploited for biomedical applications. In this work, a tailor-made biocleavable pullulan-based gene vector (PuPGEA) with good hemocompatibility was successfully proposed via atom transfer radical polymerization (ATRP) for efficient liver cell-targeting gene delivery. A two-step method involving the reaction of hydroxyl groups of pullulan with cystamine was developed to introduce reduction-sensitive disulfide-linked initiation sites of ATRP onto pullulan. The poly(glycidyl methacrylate) (PGMA) side chains prepared subsequently via ATRP were functionalized with ethanolamine (EA) to produce the resultant biocleavable comb-shaped PuPGEA vectors consisting of nonionic pullulan backbones and disulfide-linked cationic EA-functionalized PGMA (PGEA) side chains with plentiful secondary amine and nonionic hydroxyl units. The cationic PGEA side chains can be readily cleavable from the pullulan backbones of PuPGEA under reducible conditions. Due to the liver targeting performance of pullulan backbones, such PuPGEA vectors exhibited much higher gene transfection efficiency and cellular uptake rates in HepG2 cell lines than in Hella cell lines. In addition, in vitro transfection efficiency and uptake mechanism of polyplex in HepG2 cells were evaluated in the presence of different endocytosis inhibitors, indicating that the asialoglycoprotein receptor was involved in transfection process of hepatocytes. More importantly, in comparison with gold standard polyethylenimine (PEI, ∼25 kDa), PuPGEA vectors possessed excellent hemocompatibility without causing undesirable hemolysis. Properly grafting short bioreducible PGEA polycation side chains from a liver cell-targeting pullulan backbone is an effective means to produce new hemocompatible polysaccharide-based gene delivery vectors.

Up-regulation of TNF-alpha in neurons of dorsal root ganglia and spinal cord during coronary artery occlusion in rats.

Knowledge about the physiologic and pathophysiologic roles of tumor necrosis factor-alpha (TNF-alpha) in acute myocardial ischemia/infarction is still very limited. Evidence implies that TNF-alpha is involved in neural activity including nociception in peripheral and central nervous system. Current study was designed to examine the association of change in TNF-alpha and its mRNA in upper thoracic dorsal root ganglia and spinal cord (T1-T5) during acute myocardial ischemia/infarction induced by coronary artery occlusion (CAO) in rats. The experiment was performed using immunohistochemistry, enzyme immunoassay, in situ hybridization and real time reverse transcription-polymerase chain reaction techniques. At 0.5h, 1h, 3h and 6h of acute myocardial ischemia/infarction, TNF-alpha was mainly up-regulated in a sub-population of small and medium neurons and satellite cells in the dorsal root ganglia (DRG) and spinal neurons, mainly in laminae I, II and V, VI of the spinal dorsal horn of upper thoracic segments. The up-regulation of TNF-alpha mRNA was observed at 30min of CAO, which was statistically significant, compared with the control and the sham surgery groups (P<0.01). The TNF-alpha mRNA was located in the satellite cells and afferent neurons of the DRG and spinal neurons, located mainly in laminae II-VI. The findings indicate an association of up-regulation of TNF-alpha in DRG and spinal cord with acute myocardial ischemia/infarction, suggesting that TNF-alpha may be associated with the nociception initiated by acute myocardial ischemia/infarction, while the pathophysiological role needs to be studied.

[Expression of tumor necrosis factor-alpha in myocardium and dorsal root ganglion of coronary artery occlusion in rats].

OBJECTIVE: To observe the temporal expression of tumor necrosis factor-alpha (TNF-alpha) in myocardium and dorsal root ganglion (DRG) following ligation of the left anterior descending branch of coronary artery in the rats, and to explore the potential role of TNF-alpha in the neurogenic mechanisms induced by myocardial ischemia injury and the potential neurogenic mechanisms of acute local myocardial ischemia provoking global injuries of the heart. METHODS: Healthy adult male Sprague-Dawley rats weighing (275+/-15) g were randomly divided into three groups: Control group (n = 6), the samples were harvested from normal healthy rats directly after anaesthesia; Sham surgery group (group Sham, n = 24), with the same scheme of surgery except the procedure of coronary artery occlusion (CAO); CAO group (group CAO, n = 24), with ligation of the left anterior descending branch of coronary artery. Then group Sham and group CAO were further divided into four subgroups (each n = 6): group CAO 0.5 hour, group CAO 1 hour, CAO 3 hours, CAO 6 hours, after CAO for 0.5, 1, 3 and 6 hours respectively. Then rats were sacrificed as scheduled, and the samples were collected for immunohistochemistry (IHC), enzyme linked immunosorbent assay (ELISA) and real time reverse transcription-polymerase chain reaction (RT-PCR) tests to determine the changes in TNF-alpha and its mRNA in myocardium and DRG. RESULTS: (1)The expression of TNF-alpha and TNF-alpha mRNA in ischemic myocardium and non-ischemic myocardium increased significantly after CAO as compared with the baseline in the control group and that of Sham group ( P<0.05 or P<0.01), and they peaked at CAO 1 hour, but there was no significant difference in the level of expression between CAO 1 hour and CAO 0.5 hour groups (all P>0.05). The level of expression of TNF-alpha and TNF-alpha mRNA in ischemic myocardium were significantly higher than those in non-ischemic myocardium after CAO (all P<0.05). (2) The expression of TNF-alpha and TNF-alpha mRNA in DRG increased significantly after CAO as compared with the baseline in the control group and that of sham surgery control group, and they peaked at CAO 0.5 hour (P<0.05 or P<0.01). CONCLUSION: CAO can provoke a significant increase in TNF-alpha and TNF-alpha mRNA in the myocardium (ischemic and non-ischemic) and DRG of rats. It demonstrates that acute myocardial ischemia, as a nociceptive stimulus, may activate the neurogenic reactions in body, and TNF-alpha might play an important role in the whole process, while the neurogenic mechanisms may contribute to the development of myocardial injury.