Knockdown of the glutamate-gated chloride channel gene decreases emamectin benzoate susceptibility in the fall armyworm, Spodoptera frugiperda.

Emamectin benzoate (EB), a derivative of avermectin, is the primary insecticide used to control the fall armyworm (FAW) in China. However, the specific molecular targets of EB against FAW remain unclear. In this study, we cloned the glutamate-gated chloride channel (GluCl) gene, which is known to be a primary molecular target for avermectin. We first investigated the transcript levels of SfGluCl in FAW and found that the expression level of SfGluCl in the head and nerve cord was significantly higher than that in other tissues. Furthermore, we found that the expression level of SfGluCl was significantly higher in eggs than that in other developmental stages, including larvae, pupae, and adults. Additionally, we identified three variable splice forms of SfGluCl in exons 3 and 9 and found that their splice frequencies remained unaffected by treatment with the LC50 of EB. RNAi mediated knockdown of SfGluCl showed a significant reduction of 42% and 65% after 48 and 72 h of dsRNA feeding, respectively. Importantly, knockdown of SfGluCl sifgnificantly reduced LC50 and LC90 EB treatment induced mortality of FAW larvae by 15% and 44%, respectively, compared to the control group feeding by dsEGFP. In contrast, there were no significant changes in the mortality of FAW larvae treated with the control insecticides chlorantraniliprole and spinetoram. Finally, molecular docking simulations revealed that EB bound to the large amino-terminal extracellular domain of SfGluCl by forming five hydrogen bonds, two alkyl hydrophobic interactions and one salt bridge. These findings strongly suggest that GluCl may serve as one of the molecular targets of EB in FAW, shedding light on the mode of action of this important insecticide.

Comparison of Posterior Pedicle Screw Fixation and Lateral Fixation in the Extreme Lateral Interbody Fusion in Lumbar Degenerative Disease Patients with Osteopenia or Osteoporosis.

OBJECTIVE: Nowadays, with the increasing proportion of osteoporosis in patients with lumbar degenerative diseases, doctors are facing the choice of intraoperative internal fixation methods. The purpose of this study was to compare and assess the clinical results of posterior bilateral pedicle screw fixation and lateral fixation in the extreme lateral interbody fusion (XLIF) in patients with osteopenia or osteoporosis. METHODS: The retrospective review was performed on 67 degenerative lumbar diseases patients with osteopenia or osteoporosis who underwent XLIF in our hospital from January 2018 to July 2021. Patients in this study were classified into lateral screw (LS) group, lateral self-locking plate (LP) group, and bilateral pedicle screw (BPS) group. The functional evaluation factors containing Japanese Orthopaedic Association (JOA) score, visual analogue scale (VAS) of leg pain, and VAS of low back pain, radiological factors such as disc height (DH), lumbar lordotic (LL) angle, segmental lordotic (SL) angle, cage subsidence degree and interbody fusion degree were compared. RESULTS: Primary outcomes: no differences were observed with regards to the incidence of complications among LS, LP and BS group (P < 0.05). The JOA and leg pain VAS were significantly improved after operation (P < 0.05) and all groups demonstrated similar improvements in the leg pain VAS and JOA score (P > 0.05). When comparing VAS of leg pain and JOA scores, no differences were identified among LS, LP and BPS groups (P > 0.05). There are four thigh sensory complaint, one hip flexor weakness and one thigh pain occurred and no death was observed. There were significantly better DH, LL angle, SL angle, cage subsidence degree and interbody fusion degree in the BPS group than in LS and LP groups 1 year after surgery (P < 0.05). The DH loss ratio, LL angle loss ratio, SL angle loss ratio in the BPS group was significantly lower than in the LP and LS groups (P < 0.05). The 12-month SL angle improvement rate in the BPS group was significantly higher than in the LP and LS groups (20.20 ± 14.69, 0.73 ± 4.68, 6.20 ± 12.31, P < 0.05). SECONDARY OUTCOMES: the BPS patients had significantly worse intraoperative blood loss and operation time than LS and LP patients (P < 0.05). CONCLUSION: In lumbar diseases patients with osteopenia or osteoporosis, the bilateral pedicle screw fixation has better orthopedic effect than lateral internal fixation, and can better maintain the stability of the spine in the long-term follow-up, which is a better choice in XLIF surgery.

Comparison of multifidus degeneration between scoliosis and lumbar disc herniation.

OBJECTIVE: To assess and compare the pathological and radiological outcomes of multifidus degeneration in scoliosis and lumbar disc herniation patients. METHODS: We performed a retrospective review on 24 patients with scoliosis and 26 patients with lumbar disc herniation (LDH) in the Third Hospital of Hebei Medical University from January 2017 to March2021. The patients were divided into scoliosis group and LDH group according to the treatment. The MRI fatty infiltration rate (FIR) of multifidus and strength of back muscle were calculated to evaluate muscle condition. Multifidus biopsy samples were obtained during surgery in the affected side at L4 or L5 segment in LDH group and on the concavity side of apical vertebrae in scoliosis group. The biopsy fatty infiltration degree (FID) and FIR in two groups, the FIR of affected and unaffected side in LDH group, and the FIR of concavity and convexity side in scoliosis group were compared. The correlation between concavity-convexity FIR difference and cobb angle in scoliosis group, back muscle strength and FIR in LDH group, FID and FIR in both groups was calculated respectively. RESULTS: The FIR was higher in scoliosis group than in LDH group, higher in concavity side than convexity side in scoliosis group (both P < 0.05). The FID was higher in scoliosis group than in LDH group (P < 0.05). No significant difference was found between affected and unaffected side in LDH group (P > 0.05). There was a positive correlation between concavity-convexity FIR difference and cobb angle, FIR and FID (both P < 0.01). There was a negative correlation between back muscle strength and FIR (P < 0.01). The biopsy staining results showed that both two groups were found the existence of rimmed vacuoles, nuclear aggregation, and abnormal enzyme activity, indicating that the scoliosis and LDH may be associated with myogenic diseases. CONCLUSION: The scoliosis patients showed more serious fatty infiltration than LDH patients and rare pathological findings were found in both diseases.

A Comparative Study of a New Retractor-Assisted WILTSE TLIF, MIS-TLIF, and Traditional PLIF for Treatment of Single-Level Lumbar Degenerative Diseases.

OBJECTIVES: To compare the clinical efficacy of a new retractor-assisted Wiltse transforaminal lumbar interbody fusion (TLIF), minimally invasive TLIF (MIS-TLIF), and traditional posterior lumbar interbody fusion (PLIF) in treating single-level lumbar degenerative diseases. METHODS: A retrospective study was conducted by analyzing the clinical and imaging data of consecutive patients with single-level lumbar degenerative diseases who underwent the new retractor-assisted Wiltse TLIF, MIS-TLIF, or traditional PLIF. This study enrolled 87 concurrent patients between June 2016 and December 2019 (Wiltse TLIF 29 cases; MIS-TLIF 28 cases; PLIF 30 cases). The three groups were compared for perioperative indicators (including intraoperative blood loss, postoperative drainage volume, operation time, intraoperative fluoroscopy time, bedridden time), creatine kinase (CK), visual analog score (VAS), Oswestry disability index (ODI), Japanese Orthopaedic Association (JOA) score, intervertebral fusion rate, muscle atrophy, and fatty infiltration (including ratio of multifidus atrophy and ratio of lean-to-total cross-sectional area [CSA]). RESULTS: Intraoperative blood loss (F = 62.628, p < 0.001), postoperative drainage volume (F = 72.048, p < 0.001), and bedridden time (χ2  = 62.289, p < 0.001) were significantly lower in the MIS-TLIF and Wiltse groups than in the PLIF group. The operative and intraoperative radiation times of the MIS-TLIF group were significantly longer than those of the Wiltse and PLIF groups. The CK concentration in the Wiltse and MIS-TLIF groups were significantly lower than those in the PLIF group 1 day (F = 9.331, p < 0.001) and 3 days after surgery (F = 15.967, p < 0.001). The PLIF group's back pain VAS score was higher than those of the Wiltse and MIS-TLIF groups. The PLIF group had a higher ODI 6 months (F = 3.282, p = 0.042) and 12 months (F = 5.316, p = 0.007) after surgery and a lower JOA score than the Wiltse and MIS-TLIF groups 6 months (F = 3.234, p = 0.044) and 12 months (F = 3.874, p = 0.025) after surgery. The ratio of multifidus atrophy in the PLIF group (41.70 ± 8.84%) was significantly higher than those of the Wiltse group (24.13 ± 6.82%) and the MIS-TLIF group (22.35 ± 5.03%). The ratio of lean-to-total CSA in the PLIF group was lower than those of the Wiltse and MIS-TLIF groups after surgery (F = 8.852, p < 0.001). MIS-TLIF group showed longer operation time (169.11 ± 29.38 min) and intraoperative fluoroscopy time (87.61 ± 3.13 s) than the Wiltse group. CONCLUSION: Wiltse TLIF assisted by the new retractor is a more convenient and minimally invasive surgical method than the traditional PLIF and MIS-TLIF methods, which are linked to a long learning curve and long operation and fluoroscopy time.

Insights Gained Into the Treatment of COVID19 by Pulmonary Surfactant and Its Components.

Pulmonary surfactant constitutes an important barrier that pathogens must cross to gain access to the rest of the organism via the respiratory surface. The presence of pulmonary surfactant prevents the dissemination of pathogens, modulates immune responses, and optimizes lung biophysical activity. Thus, the application of pulmonary surfactant for the treatment of respiratory diseases provides an effective strategy. Currently, several clinical trials are investigating the use of surfactant preparations to treat patients with coronavirus disease 2019 (COVID-19). Some factors have been considered in the application of pulmonary surfactant for the treatment COVID-19, such as mechanical ventilation strategy, timing of treatment, dose delivered, method of delivery, and preparation utilized. This review supplements this list with two additional factors: accurate measurement of surfactants in patients and proper selection of pulmonary surfactant components. This review provides a reference for ongoing exogenous surfactant trials involving patients with COVID-19 and provides insight for the development of surfactant preparations for the treatment of viral respiratory infections.

Comparison of Anterior Cervical Discectomy and Fusion with Cervical Laminectomy and Fusion in the Treatment of 4-Level Cervical Spondylotic Myelopathy.

OBJECTIVE: To assess and compare the therapeutic effects of Anterior Cervical Discectomy and Fusion (ACDF) and Cervical Laminectomy and Fusion (CLF) in the treatment of 4-level cervical. METHODS: We performed a retrospective review on 39 patients with 4-level CSM who underwent ACDF or CLF in the Third Hospital of Hebei Medical University from January 2010 to December 2018. The patients were divided into ACDF group and CLF group according to the treatment. The operative index was evaluated based on intraoperative blood loss and operation time. The functional outcomes including Japanese Orthopedic Association (JOA) score and visual analogue scale (VAS) of axial pain were compared. The Cobb angle, Cobb angle improvement rate, range of motion (ROM) and ROM loss ratio were measured for radiographic evaluation. RESULTS: No major complications or deaths occurred. The average age at baseline was 55 years. There was no significant difference between the ACDF and CLF group in follow-up time (26.29 months, 25.39 months, P > 0.05). The intraoperative blood loss was higher in the CLF group than in the ACDF group (692.67 ± 38.68 vs 392.14 ± 128.06, P < 0.05). The operation time was longer in the CLF group than in the ACDF group (206.60 ± 49.37 vs 172.64 ± 31.96, P < 0.05). Significant improvements in the VAS and JOA scores were observed in both groups (P < 0.05). No significant difference in VAS was found between the ACDF and CLF groups (P < 0.05). There was a significantly larger improvement rate of JOA score in the ACDF group than in the CLF group (60.9% ± 9.57% vs 31.5% ± 15.70%, P < 0.05). There were two (9.6%) cases with complications In the ACDF group, including one (4.8%) case of dysphagia and one (4.8%) case of pharyngodynia. In the CLF group, two patients (11.1%) developed C5 palsy. No significant difference in the incidence of complications, ROM loss ratio and Cobb angle improvement rate was found between group ACDF and group CLF (all P < 0.05). CONCLUSION: Both ACDF and CLF were effective in the treatment of multi-level cervical spondylosis and ACDF is more suitable for patients with 4-level CSM.

Huanglong Antitussive Granule Relieves Acute Asthma Through Regulating Pulmonary Lipid Homeostasis.

Background: Asthma is a respiratory disease with chronic airway inflammatory, and individuals with asthma exacerbations is one of the most frequent causes of hospitalization. Huanglong antitussive granule (HL Granule), a Chinese proprietary herbal medicine, has been proved to be effective in the clinical treatment of pulmonary disease. This study is devoted to the pharmacodynamics of HL Granule in acute asthma and the possible mechanism from the perspective of lipidomics. Methods: Mice were divided into four groups, control group, acute asthma model group, HL Granule treatment and montelukast sodium treatment group. Acute asthma was induced by ovalbumin (OVA). Histopathology, pulmonary function and enzyme linked immunosorbent assay (ELISA) were used to validated model and effect of HL Granule. Lipids were detected by ultra-high-performance liquid chromatography coupled to hybrid Quadrupole-Exactive Orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) and identified by MS-DAIL and built-in Lipidblast database. Differentially expressed lipids recalled in HL Granule treatment group were extracted for heatmap, enrichment analysis and correlation analysis. Results: HL Granule was effective in decreasing airway hyperresponsiveness (AHR), airway inflammatory and the levels of IL-4 and IL-5. A total of 304 and 167 lipids were identified in positive and negative ion mode, respectively. Among these, 104 and 73 lipids were reserved in HL Granule group (FDR < 0.05), including acylcarnitine (ACar), fatty acid (FA), lysophosphatidylcholine (LPC), phosphatidylcholine (PC), lysophosphatidylethanolamine (LPE), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), diglyceride (DG), triglyceride (TG), sphingomyelin (SM) and ceramide (Cer). Furthermore, 118 and 273 correlations among 47 and 96 lipids in the positive and negative were observed, with ether-linked phosphatidylethanolamine (PEe) and phosphatidylcholine (PCe) (FDR < 0.001, Spearman correlation coefficient r 2 > 0.75). Conclusion: HL Granule might improve pulmonary lipid homeostasis and could be used as an alternative or supplementary therapy in clinical for the treatment of asthma.

Research Progress on the Mechanism of Lumbarmultifidus Injury and Degeneration.

This review summarizes recent research progress in the clinical features, image manifestations, and pathological mechanism of multifidus injury. After a brief introduction to the fiber classification, innervation, blood supply, and multifidus function, some factors of multifidus injury, consisting of denervation, intraoperative incision selection and traction, and lumbar degenerative disease are overviewed. In addition, the clinical index of multifidus injury including myoglobin, creatine kinase, IL-6, C-reactive protein, the cross-sectional area of multifidus, the degree of fat infiltration, and intraoperative biopsy are summarized. Furthermore, we recommend that patients with chronic low back pain should take the long-term exercise of lumbodorsal muscles. Finally, some remaining issues, including external fixation and the imaging quantitative evaluation criteria of multifidus, need to be further explored in the future.

Elevated monocytic myeloid-derived suppressor cells positively correlate with infection frequency in children with RRTIs.

In children with recurrent respiratory tract infections (RRTIs), the percentages and numbers of monocytic myeloid-derived suppressor cells (MDSCs) were elevated. The elevated MDSCs positively correlate with respiratory tract infection frequency in RRTIs children. The elevated MDSCs can inhibit CD8+ T-cells proliferation in RRTIs children.

Novel Implementation of Extreme Lateral Interbody Fusion to Avoid Intraoperative Lumbar Plexus Injury: Technical Note and Preliminary Results.

BACKGROUND: This study modified the traditional extreme lateral lumbar interbody fusion (XLIF) surgery and was intended to reduce the approach related to lumbar plexus injury. METHODS: The patients receiving a new modified XLIF for treatment of lumbar degenerative diseases since September 2017 in our hospital were retrospectively collected. Postoperative additional symptoms of leg numbness, pain, or weakness were recorded as lumbar plexus nerve injury. Intraoperative electromyographic monitoring was recorded during surgery to evaluate the safety of the modified entry point. The visual analog scale and Oswestry Disability Index were adopted to evaluate the postoperative clinical efficacy. Modified MacNab criteria were introduced to evaluate the patients' satisfaction 12 months after surgery. The preoperative and postoperative intervertebral height, foraminal height, and lumbar lordotic angle were measured. Repeated measurement variance analysis was used for comparison of clinical and imaging indexes in various periods. P < 0.05 indicated statistical difference. RESULTS: Fifty-nine patients were finally included in the retrospective study. The intraoperative average blood loss and operation time were 70 mL (40-130 mL) and 77.90 ± 13.65 minutes. The average follow-up time was 18 months. Postoperative visual analog scale and Oswestry Disability Index were significantly decreased compared with those before the operation. The intervertebral height and foraminal height were dramatically higher than those before surgery. No lumbar plexus injury occurred. CONCLUSIONS: The initial result was optimistic in reducing lumbar plexus injury and obtaining good clinical efficacy. We need to further expand the sample size and carry out a comparative study to observe the advantages and disadvantages of modified XLIF.

Safety Analysis of Two Anterior Lateral Lumbar Interbody Fusions at the Initial Stage of Learning Curve.

OBJECTIVE: Until now, there were few studies on the safety analysis of oblique lumbar interbody fusion (OLIF) and extreme lateral interbody fusion (XLIF) in the initial stage of learning curve. The purpose of this study was to find out the safety differences between the 2 minimally invasive fusion methods in the initial stage of learning curve and to provide reference for beginners. METHODS: We retrospectively collected the first 30 cases of lumbar degenerative disease with OLIF or XLIF in our center since June 2014. Patients were divided into group OLIF and group XLIF according to different operative methods. The clinical efficacy and complications of the 2 groups were compared. A P <0.05 was statistically significant. RESULTS: Group XLIF were aged 37-74 years (mean 58.4 years) and group OLIF were aged 39-71 years (mean 56.1 years). There were no significant differences between the 2 groups in age, sex, operation time, intraoperative bleeding volume, operation segment, and follow-up time. The incidence of complications in group XLIF was significantly lower than that in group OLIF (10% vs. 33.3%; P = 0.028). CONCLUSIONS: OLIF has a higher risk of neurovascular injury in the initial stage of learning. By contrast, the XLIF approach is simple and the incidence of complications is relatively low. Therefore, we believed that XLIF is more acceptable in the initial stage of anterolateral lumbar interbody fusion.

Percutaneous transforaminal endoscopic discectomy in the treatment of senior patients with lumbar degenerative disc disease.

The aim of the current study was to analyze the efficacy of percutaneous transforaminal endoscopic discectomy (PTED) in the treatment of lumbar degenerative disc disease for senior patients. The clinical and follow-up data of senior patients were retrospectively reviewed. Patients were divided into a PTED group and an open surgery group. Parameters were analyzed, including surgery time, intraoperative fluoroscopy time, intraoperative blood loss, postoperative complications, visual analog scale (VAS) and Japan Orthopedic Association (JOA) scores. Compared with the open surgery group, the surgery time and intraoperative blood loss were decreased, while the intraoperative fluoroscopy time was increased, in the PTED group (P<0.001). Significant improvements in VAS and JOA scores were identified within both groups from preoperative to 12 months following surgery (P<0.001). VAS and JOA scores were significantly improved in the PTEN group compared with the open surgery group at 1 week after surgery (P<0.001), but there was no significant difference between groups prior to and at 12 months following surgery. The incidence of venous thrombosis of the lower extremities in the PTED group was decreased compared with the open surgery group (P<0.05). In the open surgery group, patients suffered from multiple postoperative complications, including constipation, urinary system infection, wound infection, gastrointestinal hemorrhagic stress ulcer, pneumonia, pulmonary embolism, mortality following myocardial infarction, mortality following cerebral infarction, and hemiplegia following cerebral hemorrhage. By contrast, patients in the PTED group did not experience any of these complications. In conclusion, PTED resulted in reduced trauma and a lower incidence of severe complications compared with open surgery, which suggests that PTED is a safe and effective minimally invasive surgery for senior patients with lumbar degenerative disc disease.

Clearance of HBeAg and HBsAg of HBV in mice model by a recombinant HBV vaccine combined with GM-CSF and IFN-α as an effective therapeutic vaccine adjuvant.

Chronic hepatitis B virus (CHB) infection is a significant public threat. Current interferon-α (IFN-α) based therapies and anti-viral drugs have failed to clear the infection in the majority of CHB patients and animal models. In our previous study, we established a combined protocol that employed a 3-day pretreatment with granulocyte-macrophage colony stimulating factor (GM-CSF) prior to a standard HBV vaccine. It achieved a 90% reduction of HBsAg level in the HBsAg transgenic mouse model. This protocol, while effective, remains too complex for clinical use. In this study, we formulated a new regimen by combining GM-CSF, IFN-α and a recombinant HBV vaccine (GM-CSF/IFN-α/VACCINE) into a single preparation and tested its efficacy in a HBV infection model. After four vaccinations, both serum HBeAg and HBsAg were cleared, accompanied by a 95% reduction of HBV+ hepatocytes and the presence of a large number of infiltrating CD8+ T cells in the liver. Mechanistically these robust responses were initiated by a vaccine-induced conversion of CCR2-dependent CD11b+Ly6Chi monocytes into CD11b+CD11c+ DCs. This finding sheds light on the potential mechanism of action of the GM-CSF-based vaccine adjuvant and provides definable markers for clinical assessment during future testing of such highly potent vaccine protocols in HBV patients.

Enrichment of Ly6Chi monocytes by multiple GM-CSF injections with HBV vaccine contributes to viral clearance in a HBV mouse model.

Adjuvants are considered a necessary component for HBV therapeutic vaccines but few are licensed in clinical practice due to concerns about safety or efficiency. In our recent study, we established that a combination protocol of 3-day pretreatments with GM-CSF before a vaccination (3 × GM-CSF+VACCINE) into the same injection site could break immune tolerance and cause over 90% reduction of HBsAg level in the HBsAg transgenic mouse model. Herein, we further investigated the therapeutic potential of the combination in AAV8-1.3HBV-infected mice. After 4 vaccinations, both serum HBeAg and HBsAg were cleared and there was a 95% reduction of HBV-positive hepatocytes, in addition to the presence of large number of infiltrating CD8+ T cells in the livers. Mechanistically, the HBV-specific T-cell responses were elicited via a 3 × GM-CSF+VACCINE-induced conversion of CCR2-dependent CD11b+ Ly6Chi monocytes into CD11b+CD11c+ DCs. Experimental depletion of Ly6Chi monocytes resulted in a defective HBV-specific immune response thereby abrogating HBV eradication. This vaccination strategy could lead to development of an effective therapeutic protocol against chronic HBV in infected patients.

Analysis of immunological mechanisms exerted by HBsAg-HBIG therapeutic vaccine combined with Adefovir in chronic hepatitis B patients.

An HBsAg-HBIG therapeutic vaccine (Yeast-derived Immune Complexes, YIC) for chronic hepatitis B (CHB) patients has undergone a series of clinical trials. The HBeAg sero-conversion rate of YIC varied from 21.9% to 14% depending on the immunization protocols from 6 to 12 injections. To analyze the immunological mechanisms exerted by 6 injections of YIC, 44 CHB patients were separately immunized with YIC, alum as adjuvant control or normal saline as blank control, with add on of antiviral drug Adefovir in all groups. Kinetic increase in Th1 and Th2 cells CD4+ T cell sub-populations with association in decrease in Treg cells and increase of Tc1 and Tc17 cells in CD8+ T cells were observed in YIC immunized group. No such changes were found in the other groups. By multifunctional analysis of cytokine profiles, significant increase of IL-2 levels was observed, both in CD4+ and CD8+ T cells in the YIC immunized group, accompanied by increase in IFN-gamma and decrease of inhibitory factors (IL-10, TGF-ß and Foxp3) in CD4+ T cells. In the alum immunized group, slight increase of IL-10, TGF-ß and Foxp3 in CD4+ T cells was found after the second injection, but decreased after more injections, suggesting that alum induced early inflammatory responses to a certain extent. Similar patterns of responses of IL-17A and TNF-α in CD8+T cells were shown between YIC and the saline group. Results indicate that add on of Adefovir, did not affect host specific immune responses.

Overcoming HBV immune tolerance to eliminate HBsAg-positive hepatocytes via pre-administration of GM-CSF as a novel adjuvant for a hepatitis B vaccine in HBV transgenic mice.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to be a potential vaccine adjuvant despite contradictory results from animal and human studies. The discrepancies may be due to the different doses and regimens of GM-CSF that were used, given that either mature or immature dendritic cells (DCs) could be induced under different conditions. To test the hypothesis that GM-CSF can be used as a novel adjuvant for a hepatitis B virus (HBV) therapeutic vaccine, we administered GM-CSF once per day for three days prior to vaccination with recombinant HBV vaccine (rHBVvac) in mice. We observed greater DC maturation in these pre-treated animals at day 3 as compared to day 1 or day 2 of daily GM-CSF administration. This strategy was further investigated for its ability to break the immune tolerance established in hepatitis B surface antigen-transgenic (HBsAg-Tg) animals. We found that the levels of induced anti-HBsAg antibodies were significantly higher in animals following three days of GM-CSF pre-treatment before rHBV vaccination after the third immunization. In addition to the increase in anti-HBsAg antibody levels, cell-mediated anti-HBsAg responses, including delayed-type hypersensitivity, T-cell proliferation, interferon-γ production, and cytotoxic T lymphocytes, were dramatically enhanced in the three-day GM-CSF pre-treated group. After adoptive transfers of CD8+ T cells from immunized animals, antigen-specific CD8+ T cells were observed in the livers of recipient HBsAg-Tg animals. Moreover, the three-day pre-treatments with GM-CSF prior to rHBVvac vaccination could significantly eliminate HBsAg-positive hepatocytes, suggesting beneficial therapeutic effects. Therefore, this protocol utilizing GM-CSF as an adjuvant in combination with the rHBVvac vaccine has the potential to become a novel immunotherapy for chronic hepatitis B patients.

[Study on the expression and signification of TLR4/NO pathway in cervical tumorigenesis with high risk HPV infection].

OBJECTIVE: To explore the role of toll-like receptor (TLR)/ nitric oxide (NO) pathway in cervical tumor with high risk human papillomavirus (hrHPV) infection. METHODS: (1)Study was based on 36 women with nonmalignantcervical tissue as control group and 36 women with squamous cell cervical cancer (SCC), all with hrHPV infection which were assessed by using 14 types hrHPV E6/E7 mRNA real-time PCR kit. The amount of NO was detected by Griess reaction, the expression of inducible nitric oxide synthase(iNOS) was detected by immunohistochemistry (IHC). The mRNA expression of TLR3, TLR4, TLR7, TLR8, TLR9, nuclear factor-κB (NF-κB) p65 and iNOS in control and SCC epithelium which was captured by laser capture microdissection (LCM) were determined.(2)The expressions of TLR4 in CaSki, HeLa and C33a were detected by cell immunofluorescence method. The mRNA and protein expression of TLR/NO pathway transduction molecules including TLR4,NF-κBp65 and iNOS in CaSki, HeLa and C33a cell lines were detected by real-time PCR and western blot. RESULTS: (1)The level of NO was much higher in SCC group than that in control group [(42.92±0.36)µmol/L vs(15.49±0.24)µmol/L; P < 0.05 ]. iNOS was detected in 75% (27 cases ) of patients with squamous cervical carcinoma, while only 6% (2 cases) of normal controls were confirmed with positve result (P < 0.05). TLR/NO pathway maybe activated in SCC, for the mRNA levels of TLR3, TLR4, TLR7, TLR8, NF-κBp65 and iNOS increased significantly when compared to control group (all P < 0.05), and the greatest change in the expression level of TLR in SCC was spotted on TLR4(7.41±0.39 vs 1.86±0.21). (2)The results of immunofluorescence showed that TLR4 was located at plasma membrance of hrHPV positive HeLa and CaSki cells, while the integral optical density of TLR4 in HeLa cells (3 599±427) or CaSki cells (2 080±456) were higher than that in C33a cells (730± 96; P < 0.05). The mRNA and protein level of TLR4, NF-κBp65 and iNOS in HeLa and CaSki cells were higher than those of C33a cells (P < 0.05). CONCLUSION: TLR4/NO pathway is highly expressed in cervical cancer with hrHPV infection, while the pathway may be involved in cervical tumorigenesis with hrHPV infection.

Use of praziquantel as an adjuvant enhances protection and Tc-17 responses to killed H5N1 virus vaccine in mice.

BACKGROUND: H5N1 is a highly pathogenic influenza A virus, which can cause severe illness or even death in humans. Although the widely used killed vaccines are able to provide some protection against infection via neutralizing antibodies, cytotoxic T-lymphocyte responses that are thought to eradicate viral infections are lacking. METHODOLOGY/PRINCIPAL FINDINGS: Aiming to promote cytotoxic responses against H5N1 infection, we extended our previous finding that praziquantel (PZQ) can act as an adjuvant to induce IL-17-producing CD8(+) T cells (Tc17). We found that a single immunization of 57BL/6 mice with killed viral vaccine plus PZQ induced antigen-specific Tc17 cells, some of which also secreted IFN-γ. The induced Tc17 had cytolytic activities. Induction of these cells was impaired in CD8 knockout (KO) or IFN-γ KO mice, and was even lower in IL-17 KO mice. Importantly, the inoculation of killed vaccine with PZQ significantly reduced virus loads in the lung tissues and prolonged survival. Protection against H5N1 virus infection was obtained by adoptively transferring PZQ-primed wild type CD8(+) T cells and this was more effective than transfer of activated IFN-γ KO or IL-17 KO CD8(+) T cells. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that adding PZQ to killed H5N1 vaccine could promote broad Tc17-mediated cytotoxic T lymphocyte activity, resulting in improved control of highly pathogenic avian influenza virus infection.

Praziquantel facilitates IFN-γ-producing CD8+ T cells (Tc1) and IL-17-producing CD8+ T cells (Tc17) responses to DNA vaccination in mice.

BACKGROUND: CD8(+) cytotoxic T lymphocytes (CTLs) are crucial for eliminating hepatitis B virus (HBV) infected cells. DNA vaccination, a novel therapeutic strategy for chronic virus infection, has been shown to induce CTL responses. However, accumulated data have shown that CTLs could not be effectively induced by HBV DNA vaccination. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that praziquantel (PZQ), an anti-schistoma drug, could act as an adjuvant to overcome the lack of potent CTL responses by HBV DNA vaccination in mice. PZQ in combination with HBV DNA vaccination augmented the induction of CD8(+) T cell-dependent and HBV-specific delayed hypersensitivity responses (DTH) in C57BL/6 mice. Furthermore, the induced CD8(+) T cells consisted of both Tc1 and Tc17 subtypes. By using IFN-γ knockout (KO) mice and IL-17 KO mice, both cytokines were found to be involved in the DTH. The relevance of these findings to HBV immunization was established in HBsAg transgenic mice, in which PZQ also augmented the induction of HBV-specific Tc1 and Tc17 cells and resulted in reduction of HBsAg positive hepatocytes. Adoptive transfer experiments further showed that PZQ-primed CD8(+) T cells from wild type mice, but not the counterpart from IFN-γ KO or IL-17 KO mice, resulted in elimination of HBsAg positive hepatocytes. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PZQ is an effective adjuvant to facilitate Tc1 and Tc17 responses to HBV DNA vaccination, inducing broad CD8(+) T cell-based immunotherapy that breaks tolerance to HBsAg.