Characterization of a novel tetravalent botulism antitoxin based on receptor-binding domain of BoNTs.

Botulinum neurotoxin (BoNTs; serotypes A, B, E, and F) cause botulism disease in humans, which could be effectively treated using antitoxins. Herein, we established a novel receptor-binding domain (RBD)-based antitoxin using recombinant C terminal heavy chain (Hc) domains of BoNTs as immunogens. Immunization of horses with these recombinant Hc domains allowed the purification and digestion of IgGs from hyper-immune sera to produce high-quality and high-efficiency monovalent botulism antitoxin F(ab')2 against each BoNT (M-BATs). However, these M-BATs could not bind or neutralize other serotypes of BoNTs, and that there were no cross-protective effects among these M-BATs. This suggested the need to prepare tetravalent antitoxins to neutralize the four BoNTs simultaneously. Thus, these M-BATs were formulated into a novel tetravalent botulism antitoxin (T-BAT), in which a 10-ml volume contained 10000 IU of BoNT/A and 5000 IU of BoNT/B, BoNT/E, and BoNT/F antitoxins. The novel antitoxin preparation could prevent and treat the four mixed botulinum neurotoxins simultaneously in vivo, representing strong efficacy in an animal poisoning model. Moreover, these antibodies in T-BAT could bind the RBD, whereas conventional antitoxins based on inactivated toxins mainly bind the light chain or heavy chain translocation domain (HN) and weakly bind the important RBD in current experimental conditions. The high levels of RBD-specific novel antitoxins can efficiently bind the RBD and neutralize natural or recombinant toxins containing this RBD. The findings of the present study experimentally support the use of RBD-specific antitoxins to treat BoNT serotype A, B, E, and F-mediated botulism. This study demonstrated the concept of developing potent novel multivalent antitoxins against all BoNTs or other toxins, using the RBD of these toxins as an alternative antigen to inactivated toxins. KEY POINTS: • Antitoxins based on the receptor-binding domains of botulinum neurotoxins were made. • Novel antitoxin binds RBD; traditional antitoxin mainly binds light chain or HN domain. • A tetravalent antitoxin could prevent and treat the four mixed neurotoxins in vivo.

Development and evaluation of a tetravalent botulinum vaccine.

Botulinum neurotoxins (BoNTs) are the most toxic known proteins. Naturally occurring botulism in humans is caused by botulinum serotypes A, B, E, and F. Vaccination is an effective strategy to prevent botulism. In this study, a tetravalent botulinum vaccine (TBV) that can prevent serotypes A, B, E, and F was developed using the C-terminal receptor-binding domain of BoNT (Hc) as an antigen. To develop a suitable vaccine formulation, in vitro binding experiments of antigens and aluminum adjuvant in different buffers, and in vivo experiments of TBV at different antigen concentrations, were conducted. Our results showed that the optimal vaccine formulation buffer was a pH 6.0 phosphate buffer, and the suitable antigen concentration was 40 or 80 µg/ml of each antigen. A pilot-scale TBV was then prepared and evaluated for immunogenicity and stability. The results showed that TBV could elicit strong protective efficacy against each BoNT in mice, and remain effective after two years of storage at 4ºC, indicating that the preparation was stable and highly effective. Adsorption experiments also showed that the antigens could be well adsorbed by the aluminum adjuvant after 2 years of storage. Our results provide valuable experimental data supporting the development of a tetravalent botulinum vaccine, which is a promising candidate for the prevention of botulinum serotypes A, B, E, and F.

Measurable Residual Disease Detected by Multiparameter Flow Cytometry and Sequencing Improves Prediction of Relapse and Survival in Acute Myeloid Leukemia.

The clinically ideal time point and optimal approach for the assessment of measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) are still inconclusive. We investigated the clinical value of multiparameter flow cytometry-based MRD (MFC MRD) after induction (n = 492) and two cycles of consolidation (n = 421). The latter time point was proved as a superior indicator with independent prognostic significance for both relapse-free survival (RFS, HR = 3.635, 95% CI: 2.433-5.431, P <0.001) and overall survival (OS: HR = 3.511, 95% CI: 2.191-5.626, P <0.001). Furthermore, several representative molecular MRD markers were compared with the MFC MRD. Both approaches can establish prognostic value in patients with NPM1 mutations, and FLT3, C-KIT, or N-RAS mutations involved in kinase-related signaling pathways, while the combination of both techniques further refined the risk stratification. The detection of RUNX1-RUNX1T1 fusion transcripts achieved a considerable net reclassification improvement in predicting the prognosis. Conversely, for patients with biallelic CEBPA or DNMT3A mutations, only the MFC method was recommended due to the poor prognostic discriminability in tracking mutant transcripts. In conclusion, this study demonstrated that the MFC MRD after two consolidation cycles independently predicted clinical outcomes, and the integration of MFC and molecular MRD should depend on different types of AML-related genetic lesions.

Development and validation of a prognostic model for adult patients with acute myeloid leukaemia.

BACKGROUND: The high heterogeneity of acute myeloid leukaemia (AML) reflected in the patient- and disease-related factors accounts for the unsatisfactory prognosis despite the introduction of novel therapeutic approaches and drugs in recent years. METHODS: In the development set (n = 412), parameters including age, hematopoietic cell transplantation-comorbidity index, white blood cell count, hemoglobin, biallelic CEBPA mutations, DNMT3A mutations, FLT3-ITD/NPM1 status, and ELN cytogenetic risk status were identified as independent prognostic factors for overall survival (OS) in the multivariable Cox regression analysis. A nomogram combining these predictors for individual risk estimation was established thereby. FINDINGS: The prognostic model demonstrated promising performance in the development cohort. The calibration plot, C-index (0.74), along with the 1-, 2- and 3-year area under the receiver operating characteristic curve (AUC, 0.76, 0.79, and 0.74, respectively) in the validation set (n = 238) substantiated the robustness of the model. In addition to stratifying young (age ≤ 60 years) and elderly patients (age > 60 years) into three and two risk groups with significant distinct outcomes, the prognostic model succeeded in distinguishing eligible candidates for hematopoietic stem cell transplantation. INTERPRETATION: The prognostic model is capable of survival prediction, risk stratification and helping with therapeutic decision-making with the use of easily acquired variables in daily clinical routine. FUNDING: This work was supported in part by grants from the National Natural Science Foundation of China (81770141), the National Key R&D Program of China (2016YFE0202800), and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20161406).

Evaluation of a recombinant tetanus toxin subunit vaccine.

Tetanus is an acute, fatal disease caused by exotoxin produced by Clostridium tetani. The current vaccine against tetanus is based on inactivated tetanus toxin (TeNT). To develop a recombinant TeNT vaccine suitable for replacement of full-length tetanus toxoid (TT) vaccine for use in humans, a recombinant non-tagged isoform of the Hc domain of the tetanus toxin (THc) was expressed in Escherichia coli and purified by sequential chromatography steps. The immunogenicity and protective effect of the THc antigen were explored and compared with those of TT in Balb/c mice. The THc-based subunit vaccine provided complete protection against TeNT challenge following a high dosage as a toxoid vaccine. While the anti-THc and neutralising antibody titres were higher for the THc-based vaccine than the TT vaccine because protective epitopes are located on the THc domain. Frequency- and dose-dependent immunoprotection were also observed in THc-immunised mice. Mice immunised with one injection of 1 µg or 4 µg THc antigen were completely protected against 102 or 103 50% mouse lethal dose (LD50) of TeNT, respectively. Furthermore, the THc protein was found to recognise and bind to ganglioside GT1b in a dose-dependent manner, and anti-THc sera antibodies also inhibited binding between THc and GT1b. Antigen on the form of recombinant non-tagged THc domain expressed in E. coli achieved strong immunoprotective potency, suggesting that it could be developed into a candidate subunit vaccine against tetanus as an alternative to the current TT vaccine.

Immunological characterisation and immunoprotective efficacy of functional domain antigens of botulinum neurotoxin serotype A.

Botulinum neurotoxins (BoNTs) are highly toxic proteins that mediate their effects by binding to neuronal receptors and block the neutralizing ability of therapeutic antibodies. Vaccination is currently the most effective strategy to prevent botulism. In this study, a series of recombinant functional domain antigens of BoNT/A were prepared and identified, and their immunoprotective efficacies were explored and compared. Our results showed that all antigens produced strong humoral immune responses, although their protective effects against the toxin were different. Only the Hc and HN-L antigens produced strong protective effects and afforded complete immunoprotection. In addition, the combined vaccine groups showed that there was no synergistic effect on immune responses after antigen combination, suggesting that the integrity of the toxin antigen or domain is crucial to the immune effects. Studies of the dose-dependent immunoprotective effects further confirmed that the Hc domain antigen afforded more effective protective potency than the HN-L antigen, equivalent to the immune effect of the full-length toxin (Hc + HN-L combination group). Overall, our results demonstrated that the Hc domain elicited a strong protective immune response and also provided basic data and theoretical support for the development of Hc-based BoNT/A subunit vaccine. Therefore, the receptor binding domain Hc is implicated as a promising target antigen of the BoNT/A recombinant subunit vaccine as an alternative to the toxoid vaccine.

Development and evaluation of candidate subunit vaccine and novel antitoxin against botulinum neurotoxin serotype E.

Botulinum neurotoxins (BoNTs) are among the most toxic proteins. Vaccination is an effective strategy to prevent botulism. To generate a vaccine suitable for human use, a recombinant non-His-tagged isoform of the Hc domain of botulinum neurotoxin serotype E (rEHc) was expressed in Escherichia coli and purified by sequential chromatography. The immunogenicity of rEHc was evaluated in mice and dose- and time-dependent immune responses were observed in both antibody titers and protective potency. Then, the pilot-scale expression and purification of rEHc were performed, and its immunological activity was characterized. Our results showed rEHc has good immunogenicity and can elicit strong protective potency against botulinum neurotoxin serotype E (BoNT/E) in mice, indicating that rEHc is an effective botulism vaccine candidate. Further, we developed a novel antitoxin against BoNT/E by purifying F(ab')2 from pepsin-digested serum IgG of rEHc-inoculated horses. The protective effect of the F(ab')2 antitoxin was determined in vitro and in vivo. The results showed that our F(ab')2 antitoxin can prevent botulism in BoNT/E-challenged mice and effectively alleviate the progression of paralysis caused by BoNT/E to achieve therapeutic effects. Therefore, our results provide valuable experimental data for the production of a novel antitoxin, which is a promising candidate for the treatment of BoNT/E-induced botulism.

Genetic alteration patterns and clinical outcomes of elderly and secondary acute myeloid leukemia.

To illustrate the clinical and genetic features of elderly and secondary acute myeloid leukemia (AML) patients, we compared 145 elderly AML (e-AML) and 55 secondary AML (s-AML) patients with 451 young de novo AML patients. Both e-AML and s-AML patients showed lower white blood cell (WBC) and bone marrow (BM) blasts at diagnosis. NPM1, DNMT3A, and IDH2 mutations were more common while biallelic CEBPA and IDH1 mutations were less seen in e-AML patients. s-AML patients carried a higher frequency of KMT2A-AF9. In treatment response and survival, e/s-AML conferred a lower complete remission (CR) rate and shorter duration of event-free survival (EFS) and overall survival (OS) compared with young patients. In multivariate analysis, s-AML was an independent risk factor for OS but not EFS in the whole cohort. Importantly, intensive therapy tended to improve the survival of e/s-AML patients without increasing the risk of early death, and hematopoietic stem cell transplantation (HSCT) could rescue the prognosis of s-AML, which should be recommended for the treatment of fit patients.

MiR-335 Regulates Hif-1α to Reduce Cell Death in Both Mouse Cell Line and Rat Ischemic Models.

Hypoxia inducible factor-1α facilitates cellular adaptation to hypoxic conditions. Hence its tight regulation is crucial in hypoxia related diseases such as cerebral ischemia. Changes in hypoxia inducible factor-1α expression upon cerebral ischemia influence the expression of its downstream genes which eventually determines the extent of cellular damage. MicroRNAs are endogenous regulators of gene expression that have rapidly emerged as promising therapeutic targets in several diseases. In this study, we have identified miR-335 as a direct regulator of hypoxia inducible factor-1α and as a potential therapeutic target in cerebral ischemia. MiR-335 and hypoxia inducible factor-1α mRNA showed an inverse expression profile, both in vivo and in vitro ischemic conditions. Given the biphasic nature of hypoxia inducible factor-1α expression during cerebral ischemia, miR-335 mimic was found to reduce infarct volume in the early time (immediately after middle cerebral artery occlusion) of embolic stroke animal models while the miR-335 inhibitor appears to be beneficial at the late time of stroke (24 hrs after middle cerebral artery occlusion). Modulation of hypoxia inducible factor-1α expression by miR-335 also influenced the expression of crucial genes implicated in neurovascular permeability, cell death and maintenance of the blood brain barrier. These concerted effects, resulting in a reduction in infarct volume bring about a beneficial outcome in ischemic stroke.

Circulating microRNAs as biomarkers of acute stroke.

MicroRNAs have been identified as key regulators of gene expression and thus their potential in disease diagnostics, prognosis and therapy is being actively pursued. Deregulation of microRNAs in cerebral pathogenesis has been reported to a limited extent in both animal models and human. Due to the complexity of the pathology, identifying stroke specific microRNAs has been a challenge. This study shows that microRNA profiles reflect not only the temporal progression of stroke but also the specific etiologies. A panel of 32 microRNAs, which could differentiate stroke etiologies during acute phase was identified and verified using a customized TaqMan Low Density Array (TLDA). Furthermore we also found 5 microRNAs, miR-125b-2*, -27a*, -422a, -488 and -627 to be consistently altered in acute stroke irrespective of age or severity or confounding metabolic complications. Differential expression of these 5 microRNAs was also observed in rat stroke models. Hence, their specificity to the stroke pathology emphasizes the possibility of developing these microRNAs into accurate and useful tools for diagnosis of stroke.

Effects of hyperbaric oxygen preconditioning on ischemia-reperfusion inflammation and skin flap survival.

BACKGROUND: Hyperbaric oxygen preconditioning (HBO) is a new method of ischemia preconditioning. In this study, we examined its effects on skin flap survival and the mechanisms involved. METHODS: Thirty-six rats were divided into three groups: HBO preconditioning, control, and sham groups. An extended epigastric adipocutaneous flap based on the right superficial epigastric artery and vein was raised. A 3-hour period of flap ischemia was induced by clamping the pedicle vessels with a microvascular clamp. At the end of ischemia induction, the clamp was removed and the flap was resutured. Rats in the HBO preconditioning group were treated with HBO four times before surgery. Microcirculation in the skin flap was measured on postoperative days 1, 3 and 5. The size of the flap was measured on postoperative day 5, before the animals were sacrificed. Samples of the skin flap were prepared and stained with hematoxylin and eosin. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in the flap samples were measured. RESULTS: Surviving flap size was significantly higher in the HBO preconditioning group compared with controls, with a reduced inflammatory response and increased perfusion. IL-1, TNF-α, and IL-6 levels in the HBO preconditioning group were lower than in controls. CONCLUSIONS: HBO preconditioning improved flap survival in this ischemia-reperfusion rat model. The mechanisms responsible for this effect may relate to attenuation of the inflammatory response and increased flap perfusion following HBO preconditioning.

microRNAs Involved in Regulating Spontaneous Recovery in Embolic Stroke Model.

To date, miRNA expression studies on cerebral ischemia in both human and animal models have focused mainly on acute phase of ischemic stroke. In this study, we present the roles played by microRNAs in the spontaneous recovery phases in cerebral ischemia using rodent stroke models. Brain tissues were harvested at different reperfusion time points ranging from 0-168 hrs after middle cerebral artery occlusion using homologous emboli. MiRNA and mRNA expression profiles were investigated by microarray followed by multiple statistical analysis. Candidate transcripts were also validated by quantitative RT-PCR. Three specific groups of miRNAs were observed among a total of 346 differentially expressed miRNAs. miRNAs, miR-21, -142-3p, -142-5p, and -146a displayed significant upregulation during stroke recovery (48 hrs to 168 hrs) compared with those during acute phases (0 hrs to 24 hrs). On the other hand, an opposite trend was observed in the expression of miR-196a/b/c, -224 and -324-3p. Interestingly, miR-206, -290, -291a-5p and -30c-1*, positively correlated with the infarct sizes, with an initial increase up to 24hrs followed by a gradual decrease from 48 hrs to 168 hrs (R = 0.95). Taken together with the expression levels of corresponding mRNA targets, we have also found that Hedgehog, Notch, Wnt and TGF-ß signaling pathways could play significant roles in stroke recovery and especially in neuronal repair.