Construction and anti-tumor efficacy of a peptide vaccine that targets calcium-binding protein S100A9 / 药学学报

Calcium-binding protein S100A9 is closely related to inflammation and tumor invasion, and is one of the specific markers of myeloid-derived suppressor cells (MDSC). In this study, a recombinant polypeptide vaccine CTB-S100A9 targeting mouse calcium-binding protein S100A9 was constructed by fusion cholera toxin B subunit (CTB) with S100A9 gene. The CTB-S100A9 fusion protein was expressed in E coli. and purified by Ni+ affinity chromatography. Vaccinate the purified recombinant CTB-S100A9 protein supplemented with aluminum hydroxide adjuvant can break the autoimmune tolerance and produce high titer of S100A9 antibody in mice. Moreover, the S100A9 antibody produced by CTB-S100A9 vaccination is more specific and does not cross-react with S100A8. In the mouse 4T1 breast cancer model, CTB-S100A9 vaccination not only has significant tumor prevention effects, but also has significant tumor therapeutic effects. In addition, CTB-S100A9 significantly inhibited lung metastasis in 4T1 mice breast cancer model. Further analysis by flow cytometry showed that CTB-S100A9 vaccination can significantly reduce the tumor induced Treg cells and granulocyte-derived MDSC in 4T1 mice model, and reverse the tumor immunosuppressive environment, thereby promote the anti-tumor efficacy. The animal experiments in this study were carried out under the animal care guidelines approved by the Animal Ethics Committee of the Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine. This study shows that CTB-S100A9 is a good recombinant vaccine that targets the tumor immune-suppression environment and has great potential for the future clinical application.

Construction and anti-tumor efficacy of a pentameric peptide vaccine that targets S100A8 / 药学学报

Myeloid-derived suppressor cells (MDSC) play critical roles in immune escape of tumor. We hypothesized that elimination of tumor-induced MDSCs might help to block tumor growth. Therefore, we constructed a cholera toxin B based peptide vaccine that targets a MDSC surface marker S100A8. Immunized BALB/c mice with CTB-S100A8 plus aluminum hydroxide induced high titers of anti-S100A8 antibodies and reduced tumor burden significantly in 4T1 mice model. We also found the vaccination led to significant reduction of tumor-induced monocytic MDSC (M-MDSC), with no effect on innate MDSCs, dendritic cell (DC) and macrophage (Mφ), demonstrating that targeting tumor-induced MDSC may be a promising approach in cancer immunotherapy.

Generation of transgenic corn-derived Actinobacillus pleuropneumoniae ApxIIA fused with the cholera toxin B subunit as a vaccine candidate

Corn, one of the most important forage crops worldwide, has proven to be a useful expression vehicle due to the availability of established transformation procedures for this well-studied plant. The exotoxin Apx, a major virulence factor, is recognized as a common antigen of Actinobacillus (A.) pleuropneumoniae, the causative agent of porcine pleuropneumonia. In this study, a cholera toxin B (CTB)-ApxIIA#5 fusion protein and full-size ApxIIA expressed in corn seed, as a subunit vaccine candidate, were observed to induce Apx-specific immune responses in mice. These results suggest that transgenic corn-derived ApxIIA and CTB-ApxIIA#5 proteins are potential vaccine candidates against A. pleuropneumoniae infection.

Effects of Peripheral Neuropathy on CTB-labeled Motor Neurons Following Ligation of the Tibial Nerve / 체질인류학회지

Changes in CTB labeled motor neurons of the spinal cord were observed after the induction of peripheral neuropathy by ligation of the tibial nerve. Rats were anesthetized and the tibial nerve was ligated with 3-0 silk. The rats were separated into three groups based on the length of time the tibial nerve was ligated (1, 2, or 4 weeks). After the ligation procedures were complete, the tibial nerve stumps were soaked in CTB solution. Tibial nerve segments and the spinal cord were then observed. In the control and experimental groups, CTB-labeled neurons formed a discrete population that was concentrated primarily at the L5 level, while the contributions from L4 and L6 were minor. According to the distributions, CTB-labeled neurons were divided into rostral and caudal groups. A selective decrease of CTB-labeled neurons was observed only in the caudal group, extending from the rostral L5 to one-half of the rostral L6. The total numbers of CTB-labeled motor neurons were 2,160+/-169.3, 1,002+/-245.1, 587.5+/-346.5, and 1,728+/-402.6 in the control group, 1 week group, 2 week group, and 4 week group, respectively. The selective decrease of CTB-labeled neurons in the caudal division was responsible for the decrease in the total number of labeled neurons in all groups. Following peripheral neuropathy caused by ligation of the tibial nerve, CTB-labeled neurons in the spinal cord decreased selectively. These results may provide important neuroanatomical data regarding the effects of peripheral neuropathy by ligation of the tibial nerve.

Effect of Nasal Administration of Compound of Cholera Toxin B Subunit and Nerve Growth Factor on Spatial Learning and Memory Abilities in the Amnesic Mice / 中国康复理论与实践

@#ObjectiveTo explore the effects of the conjugate prepared from the cholera toxin B subunit(CB) and nerve growth factor(NGF) on the spatial learning and memory abilities and cholinergic function.MethodsThe conjugate of CB-NGF was prepared by the improved sodium metaperiodate method and nasally administrated to the β-amyloid protein(Aβ25-35) induced amnesic mice for 7 days with 2 dosage (7-5 μg/d、15 μg/d). Spatial learning and memory abilities were evaluated by Morris water maze and cholinergic function was assessed with the choline acetyl transferase (ChAT) immunohistochemical methods.ResultsMorris water maze test showed that the escape latency in Aβ25-35-treated mice prolonged and the staying time reduced in the crossed first quadrant where the platform had been located, compared with the control mice (P<0-01). In addition, the number of ChAT positive neuron declined in the model mice(P<0-001). CB-NGF nasal administration significantly shortened the escape latency and elevated the staying time and number of ChAT positive neuron(P<0-01).ConclusionCB-NGF treatment can improve the spatial and memory performance which may involve the neuroprotection to cholinergic system.

The effect of meningococcal serogroup A polysaccharide-protein conjugate vaccine prepared with TT and rCTB on mucosal immune responses / 中华微生物学和免疫学杂志

Objective To analyze the feasibility of the recombinant cholera toxin B subunit (rCTB) as a carrier protein candidate for the preparing of polysaccharide-protein conjugate, and to discuss the immune effects of tetanus toxoid (TT) as the carrier protein in mucosal delivery vaccine. Methods The refolded pentrumer protein, rCTB was obtained by genetic engineering methods. Then conjugated the refold-ed protein with group A meningococcal polysaccharide (GAMP) using the chemical method(ADH) ,the pol-ysaccharide-protein conjugates(GAMP-rCTB) were prepared. BALB/c mice were immunized either intraper-itoneally ( i. p. ) or intranasally ( i. n. ) with GAMP-rCTB. Moreover, GAMP-TT vaccine that TT as carrier proteins was i.n. immunized to the mice. The evaluation of immunology is performed. Results The conju-gates of polysaccharide-potein with the rCTB and TT as protein carrier both are able to elicit high level of GAMP specific IgG antibody in serum after i.n. immunization, and the conjugates can also elicit specific IgA antibody in lung lavage and intestinal mucosa. Conclusion rCTB and TT can both as the protein carri-er for polysaccharide-protein conjugate as mucosal vaccine. The route of intranasal may be more ways for im-mune function than i.p. immunization when rCTB is used as the carrier of the polysaccharide-protein conju-gates.

Clone and express ctb-stx2 b fusion gene in Enterohemrrhagic escherichia coli O157:H7 Shigeai toxin 2B subunit and V cholera toxin B subunit and the detection of their immunogenicity / 中华流行病学杂志

Objective To clone and express the fusion gene encoding Enterohemrrhagic escherichia coli O157:H7(EHEC O157:H7)Shigela toxin 2B subunit(Stx2B)and vibrio cholera toxin B subunit (CTB)as well as to detect the immunogenicity and GM1-binding ability of fusion protein.Methods To design a primer to amplify stx2b gene and ctb-stx2b fusion gene encoding Stx2B and CTB-Stx2B respectively and to clone the genes into express plasmid pET30a(+)C in order to construct pET30a-ctb-stx2b after T-A sequencing was varified,then to transform constructed plasmid into E.coliBL21(DE3)induced by IPTG and purified by a purify kit and to detect molecular weight and immunogenicity by SDSPAGE and Western-blot.Results The amplified ctb-stx2b fragments appeared to he 750 bp and gene sequence was identical to designed sequence.The prokaryotic expression system pET30a-ctb-stx2b/BL21 could express protein weight about Mr20×103and the expressed protein could react to CTB monoclone anti-body.The fusion protein CTB-Stx2B could bind GM1.Conclusion CTB-Stx2B had successfully been expressed in prokaryotic while the expressed protein had good immunogenicity and GM1-Binding ability.This study provided information on further EHEC O157:H7 vaccine research.

Activation of Vestibular Neurons Projecting to Autonomic Brain Stem Nuclei Following Acute Hypotension in Rats

The purpose of the present study was to elucidate the possible involvement of the medial vestibular nucleus (MVN) and inferior vestibular nucleus (IVN) following acute hypotension in the vestibulo- autonomic reflex through vestibulosolitary or vestibuloventrolateral projections. Acute hypotension- induced cFos expression was assessed in combination with retrograde cholera toxin B subunit (CTb) tract tracing. After injection of CTb into the solitary region, CTb-labeled neurons were located prominently around the lateral borders of the caudal MVN and medial border of the IVN. The superior vestibular nucleus also had a scattered distribution of CTb-labeled neurons. After injection of CTb toxin into the unilateral VLM, the distributions of CTb-labeled neurons in the MVN and IVN were similar to that observed after injection into the solitary region, although there were fewer CTb-labeled neurons. In the caudal MVN, about 38% and 13% of CTb-labeled neurons were double-labeled for cFos after injection of CTb into the solitary region and the VLM, respectively. In the IVN, 14% and 7% of CTb-labeled neurons were double-labeled for cFos after injection of CTb into the solitary region and the VLM, respectively. Therefore, the present study suggests that acute arterial hypotension may result in activation of vestibulosolitary pathways that mediate behavioral and visceral reflexes, and vestibuloventrolateral medullary pathways that indirectly mediate vestibulosympathetic responses.

Localization of Motor Neuronal Cell Bodies Innervating Heart in the Cat Using CTB as Neuronal Tracer / 체질인류학회지

The origin of motor neuronal cell bodies innervating heart in the cat was investigated using CTB (Cholera toxin B subunit) as neuronal tracer. The neural tracer was injected into subepicardial layer and myocardium of the right atrium, left atrium, right ventricle and left ventricle, respectively. Labeled parasympathetic motor neuronal cell bodies were found in DMV (dorsal motor nucleus of vagus nerve) and anterolateral part of NA (nucleus ambiguus) of brainstem. Heavier labeled neuronal cell bodies were found in the NA when the neural tracers were injected into left ventricle and DMV when injected into left atrium and left ventricle. These results may provide a neuroanatomical data on origin of motor neuronal cell bodies innervating the heart of the cat.

Localization of Nerves Innervating Lacrimal, Submandibular and Sublingual Gland in the Rat Brain Stem Using Cholera Toxin B Subunit / 대한해부학회지

In the rat brain stem, the origin of neurons and afferent fibers projecting to the lacrimal, submandibular or sublingual gland was investigated by means of retrograde transport of Cholera Toxin B Subunit (CTB), respectively. Injection of CTB into the lacrimal gland labeled their neurons in superior salivatory nucleus (SSN) and facial nucleus. Superior salivatory neurons innervating lacrimal gland were labeled more densely in the rostral and caudal part of SSN. In the facial nucleus, labeled cell bodys were seen in the posterolateral part of facial nucleus. Injection of CTB into the submandibular or sublingual gland labeled their neurons in SSN and their afferent fibers in nucleus tractus solitarius (NTS). The superior salivatory neurons innervating submandibular or sublingual gland were labeled densely in the middle part of SSN. In the middle part of SSN, neurons innervating submandibular gland were labeled diffusely in the medial part of facial nerve and neurons innervating sublingual gland were labeled in the anteromedial and posterior part of facial nerve. The labeled nerve fibers in NTS were seen in the middle part of NTS.

Localization of Nerves Innervating Sublingual Gland in the Rat Brain Stem Using Cholera Toxin B Subunit / 체질인류학회지

In the rat brain stem, the nerves innervating sublingual gland was studied with submandibular gland together. Cholera Toxin B subunit (CTB), neural tracer, is not yet used to study the sublingual gland. The purpose of this study is to investigate the origin of neurons and afferent fibers projecting to sublingual gland by means of retrograde transport of CTB. CTB was injected into the sublingual gland. In the rat brain stem, neurons were labeled with CTB in superior salivatory nucleus (SSN), inferior salivatory nucleus (ISN), facial nucleus and their afferent fibers in nucleus tractus solitarius. At the rostal level of SSN, the labeled cells were found in lateral aspect of pontine reticular formation. At the level of facial nerve that transverse the dorsal part of the spinal trigeminal tract, the labeled cells of SSN extended in the area of facial nerve fibers. Labeled cells were also seen at the level of internal genu of facial nerve. In ISN at the level of facial nerve that traverse the dorsal part of the spinal trigeminal tract, the labeled cells were seen in the anterolateral direction of lateral aspect of reticular formation. In the facial nucleus, the labeled cells were confined in central part of facial nucleus. The labeled nerve fibers in nucleus tractus solitarius were seen in the level at which the medial border of the nucleus tractus solitarius meets the 4th ventricle.

Localization of Nerves Innervating Submandibular Gland in the Rat Brain Stem Using Cholera Toxin B Subunit / 대한해부학회지

The purpose of this study is to investigate the origin of neurons and afferent fibers projecting to submandibular gland by means of retrograde transport of Cholera Toxin B Subunit (CTB). CTB was injected into the both side submandibular gland or left side submandibular gland. In the rat brain stem, neurons were labeled with CTB in superior salivatory nucleus (SSN), facial nucleus, caudal region of hypoglossal nucleus, lateral horn of spinal cervical segment and their afferent fibers in nucleus tractus solitarius. At the most rostal level of SSN, the labeled cells were seen in lateral aspect of pontine reticular formation. At the level of facial nerve that traverse the dorsal part of the spinal trigeminal tract, the labeled cells of SSN extended to the anterolateral direction of lateral aspect of reticular formation. At the level of facial nucleus, the labeled cells of SSN were seen in the area of caudal prologation of the same region of rostral ones, but decreased in cell number. In the facial nucleus, the labeled cells were confined in central part of facial nucleus. In the first and second spinal cervical segment, the labeled cells were seen in the intermediomedial nucleus of lateral horn. The labeled nerve fibers in nucleus tractus solitarius were seen at the level of the 4th ventricle which the medial border of the nucleus tractus solitarius meets. Injection of CTB into the left submandibular gland labeled their neurons on the left and right superior salivatory nucleus (SSN), but other labeled cells and fibers were localized only on the left side.