Summary Egg-based seasonal influenza vaccines are the major preventive countermeasure against influenza virus. However, their effectiveness can be compromised when antigenic changes arise from egg-adaptive mutations on influenza hemagglutinin (HA). The L194P mutation is commonly observed in egg-based H3N2 vaccine seed strains and significantly alters HA antigenicity. An approach to prevent L194P would therefore be beneficial. We show that emergence of L194P during egg passaging can be impeded by preexistence of a G186V mutation, revealing strong incompatibility between these mutations. X-ray structures illustrate that individual G186V and L194P mutations have opposing effects on the HA receptor-binding site (RBS), and when both G186V and L194P are present, the RBS is severely disrupted. Importantly, wild-type HA antigenicity is maintained with G186V, but not L194P. Our results demonstrate that these epistatic interactions can be used to prevent the emergence of mutations that adversely alter antigenicity during egg adaptation.

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Antisense oligonucleotides (ONs) have proven useful for selective inhibition of gene expression. However, their effective use is limited by inefficient cellular uptake and lack of cellular targeting. In this paper, we report a drug targeting system which utilizes mannose receptor-mediated endocytosis to enhance cellular uptake of ONs in alveolar macrophages (AMs). The system employs a molecular complex consisting of partially substituted mannosylated poly(L-lysine) (MPL), electrostatically linked to a 5' fluorescently labeled ON. Upon recognition by the macrophage mannose receptors, the MPL was internalized by the receptor-mediated pathway, co-transporting the ON. Our results indicate that the AMs treated with the MPL:ON complex exhibited a significant increase in ON uptake (up to 17-fold) over free ON-treated controls. Effective ON uptake was shown to require the recognition of the mannose moiety since unmodified polylysine was much less effective in promoting ON uptake. Specific internalization of the ON:MPL complex by the mannose receptor pathway was verified by competitive inhibition using mannosylated albumin. Under this condition, the ON complex uptake was inhibited. The requirement of mannose receptors for complex uptake was further demonstrated using a macrophage cell line, J774.1, which expresses a low level of mannose receptors. When treated with the complex, these cells showed no susceptibility to ON uptake, thus suggesting the targeting ability of the carrier system to the AMs. Following cellular internalization, the ON complex appeared largely accumulated in endocytic vesicles. Enhanced endosomal exit of the ON was achieved using a fusogenic peptide derived from the amino terminal sequence of influenza virus hemagglutinin HA2. Cytotoxicity studies showed that at the concentrations effectively enhancing ON uptake, both MPL and the fusogenic peptide caused no toxic effects to the cells, thereby suggesting their potential safety and utilization in vivo.

The follicular helper T cell (Tfh) and IL-21 have been shown to play an important role in many autoimmune diseases. However, less is known about their role in Graves' disease (GD). This study aimed to investigate the expression of Tfhs and related factors (IL-21, IL-21R, CXCR5, and CXCL13) in GD thyroid tissues and to explore the effect of IL-21 on thyroid follicular cells (TFCs). The expression of Tfh-related factors in GD and normal thyroid tissues was validated using immunohistochemistry, real-time polymerase chain reaction and Western blotting. Confocal microscopy confirmed the presence of Tfh and IL-21R on CD4(+)T-/CD19(+)B cell in GD thyroid tissues. Furthermore, the effect of IL-21 on cAMP production in TFCs upon thyroid-stimulating antibody (TSAb) stimulation was also examined by an in vitro bioassay. The increased expression of Tfh-related factors was observed in GD thyroid tissues compared to control subjects. Confocal microscopy further confirmed the presence of Tfhs and the expression of IL-21R on CD4(+)T cells and CD19(+)B cells in GD thyroid tissues. Moreover, the expression of IL-21mRNA in GD thyroid tissues was correlated with the levels of thyroid autoantibodies. Additionally, IL-21 could indirectly promote cAMP production upon TSAb stimulation in TFCs when cooperating with lymphocytes, and GD TFCs were more sensitive to IL-21 stimulation than normal TFCs. There is increased expression of Tfhs and related factors (IL-21, IL-21R, CXCR5, and CXCL13) in GD thyroid tissues, and the expression of IL-21mRNA in GD thyroid tissues was found to correlate with the serum levels of thyroid autoantibodies and thyroid hormones. Moreover, IL-21 could indirectly enhance the biological activity of TFCs upon TSAb stimulation when cooperating with lymphocytes in vitro, particularly in GD TFCs, suggesting that Tfh and IL-21 might be involved in the pathogenesis of GD.

Graves' disease (GD) is a common autoimmune disease mainly caused by thyroid-stimulating antibodies (TSAbs). Interleukin 21 (IL-21) has recently been reported to play a vital role in the production of pathogenic autoantibodies in several autoimmune diseases, but less is known about GD. This study aimed to investigate the serum levels of IL-21 in GD patients and to explore their association with disease activity. We performed a case-control association study of 152 patients with GD and 32 healthy controls. All patients were further classified into three subgroups: the GD-untreated group (n = 70), the GD-recurrence group (n = 41), and the GD-remission group (n = 41). Serum IL-21 levels were assayed with ELISA. TSAb activity was measured by an in vitro bioassay. Changes in serum IL-21 were also observed in 12 GD patients before and after treatment. Additionally, correlations among the serum IL-21 and free triiodothyronine (FT3), free thyroxine (FT4), thyrotropin (TSH), thyroperoxidase antibodies (TPOAb), thyroglobulin antibodies (TGAb), thyrotropin receptor antibody (TRAb), and TSAb were also analyzed. The serum IL-21 levels in all GD patients were significantly higher than those in the control group (P < 0.001), and specifically, both the GD-untreated and GD-recurrence groups had elevated serum IL-21 compared to the control group (P < 0.001). Moreover, serum IL-21 in newly diagnosed patients markedly decreased after treatment (P < 0.001). Additionally, the serum IL-21 levels in GD-goiter patients were higher than those of the GD-non-goiter patients (P < 0.001). However, no significant differences were found in the serum IL-21 levels in patients with or without Graves' ophthalmopathy. Importantly, serum IL-21 positively correlated with FT3, FT4, TPOAb, TGAb, and TRAb (r = 0.5053, r = 0.3266, r = 0.1792, r = 0.2445, and r = 0.4096, respectively; all P < 0.0001), and particularly with TSAb activity (r = 0.8171, P < 0.0001), negatively correlated with TSH (r = -0.2713, P < 0.0001). Serum IL-21 levels were significantly elevated in patients with GD and decreased after treatment; moreover, IL-21 may be associated with the clinical disease activity. These observations suggest that IL-21 may play an important role in the pathogenesis of GD.

Background Dengue virus (DENV) is transmitted by mosquito and has been circulating in Guangdong, China, for over 30 years. Dengue infection causes mild to severe disease symptoms in human. Cytokine profiles were suggested to be crucial especially during the acute stage in the dengue infection. Aim To determine the cytokine profiles at the acute stage in patients with primary or secondary dengue infection in Guangzhou city in the 2014 outbreak. Methods We investigated 23 inflammatory cytokines in serum collected from dengue-infected patients and analyzed their correlations with their clinical indexes. Results The concentrations of CXCL9, IP-10, CXCL11, IL-8, IL-10, and CCL2 in serum were significantly higher in the groups of DENV-infected patients during the first two weeks than those of control group while CCL17 and CXCL5 showed lower expression level in the patients. Among these cytokines, CXCL9, CCL17, and CXCL5 showed statistical difference between the groups of primary and secondary infections. The platelet count and lactate dehydrogenase were correlated with the level of CCL17 and MIP-1α/CXCL5, respectively, in the group of secondary infection. Conclusions We determined the cytokine profiles in serum of the patients during the 2014 dengue outbreak. The expression of specific cytokines was associated with the secondary infection.

For mission-critical applications of wireless sensor networks (WSNs) involving extensive battlefield surveillance, medical healthcare, etc., it is crucial to have low-power, new protocols, methodologies and structures for transferring data and information in a network with full sensing coverage capability for an extended working period. The upmost mission is to ensure that the network is fully functional providing reliable transmission of the sensed data without the risk of data loss. WSNs have been applied to various types of mission-critical applications. Coverage preservation is one of the most essential functions to guarantee quality of service (QoS) in WSNs. However, a tradeoff exists between sensing coverage and network lifetime due to the limited energy supplies of sensor nodes. In this study, we propose a routing protocol to accommodate both energy-balance and coverage-preservation for sensor nodes in WSNs. The energy consumption for radio transmissions and the residual energy over the network are taken into account when the proposed protocol determines an energy-efficient route for a packet. The simulation results demonstrate that the proposed protocol is able to increase the duration of the on-duty network and provide up to 98.3% and 85.7% of extra service time with 100% sensing coverage ratio comparing with LEACH and the LEACH-Coverage-U protocols, respectively.