Association between Prehospital Visits and Poor Health Outcomes in Korean Acute Stroke Patients: A National Health Insurance Claims Data Study.

This study aimed to determine whether prehospital visits to other medical institutions before admission are associated with prolonged hospital stay, readmission, or mortality rates in acute stroke patients. Using the claims data from the Korean Health Insurance Service, a cross-sectional study was conducted on 58,418 newly diagnosed stroke patients aged ≥ 20 years from 1 January 2019 to 31 December 2019. Extended hospital stay (≥7 days; median value) following initial admission, readmission within 180 days after discharge, and all-cause mortality within 30 days were measured as health outcomes using multiple logistic regression analysis after adjusting for age, sex, income, residential area, and medical history. Stroke patients with a prehospital visit (10,992 patients, 18.8%) had a higher risk of long hospitalization (odds ratio = 1.06; 95% confidence interval = 1.02-1.10), readmission (1.19; 1.14-1.25), and mortality (1.23; 1.13-1.33) compared with patients without a prehospital visit. Female patients and those under 65 years of age had increased unfavorable outcomes (p < 0.05). Prehospital visits were associated with unfavorable health outcomes.

Neural regulation of energy and bone homeostasis by the synaptic adhesion molecule Calsyntenin-3.

Neuronal regulation of energy and bone metabolism is important for body homeostasis. Many studies have emphasized the importance of synaptic adhesion molecules in the formation of synapses, but their roles in physiology still await further characterization. Here, we found that the synaptic adhesion molecule Calsyntenin-3 (CLSTN3) regulates energy and bone homeostasis. Clstn3 global knockout mice show reduced body mass with improved leptin sensitivity and increased energy expenditure compared to their wild-type littermates. In addition, Clstn3 knockout mice show reduced marrow volume and cortical bone mass without alteration of trabecular bone microarchitecture. This reduced bone mass is not bone cell-autonomous because neither osteoblast- nor osteoclast-specific Clstn3 knockout mice show bone defects; similarly, in vitro cultures of both Clstn3 knockout osteoblasts and osteoclasts do not show any defects. These reduced body and bone mass phenotypes can be attributed instead to neuronal CLSTN3 because they are recapitulated by pan-neuronal but not sympathetic neuron-specific deletion of Clstn3. This study reveals novel physiological functions of neuronal Clstn3 as a key regulator of energy and bone homeostasis.

Immunization With Mycobacterium tuberculosis Antigens Encapsulated in Phosphatidylserine Liposomes Improves Protection Afforded by BCG.

Liposomes have been long considered as a vaccine delivery system but this technology remains to be fully utilized. Here, we describe a novel liposome-based subunit vaccine formulation for tuberculosis (TB) based on phosphatidylserine encapsulating two prominent TB antigens, Ag85B, and ESAT-6. We show that the resulting liposomes (Lipo-AE) are stable upon storage and can be readily taken up by antigen presenting cells and that their antigenic cargo is delivered and processed within endosomal cell compartments. The Lipo-AE vaccine formulation combined with the PolyIC adjuvant induced a mixed Th1/Th17-Th2 immune response to Ag85B but only a weak response to ESAT-6. An immunization regimen based on systemic delivery followed by mucosal boost with Lipo-AE resulted in the accumulation of resident memory T cells in the lungs. Most importantly though, when Lipo-AE vaccine candidate was administered to BCG-immunized mice subsequently challenged with low dose aerosol Mycobacterium tuberculosis, we observed a significant reduction of the bacterial load in the lungs and spleen compared to BCG alone. We therefore conclude that the immunization with mycobacterial antigens delivered by phosphatidylserine based liposomes in combination with Poly:IC adjuvant may represent a novel BCG boosting vaccination strategy.

Chest radiography surveillance for lung cancer: Results from a National Health Insurance database in South Korea.

BACKGROUND: Lung cancer screening with low-dose computed tomography reduced mortality in selected high risk patients. However, the use of chest radiography for lung cancer screening in Asian populations is still controversial. We investigated the effectiveness of chest radiographic surveillance using a nationwide health service data in South Korea. METHODS: Data from the Korean National Health Insurance Service examinee cohort of 2004 to 2013 were examined, and 63,228 patients with lung cancer were identified, 38,494 (57%) of whom underwent chest radiography screening. The others did not undergo lung cancer screening and were considered as a control group. Clinical data including age, smoking, screening intervals, lung cancer stages, treatments, and survival were collected. Survival gain from surveillance after adjustment for lead-time bias based on the sojourn time was calculated. Cox-proportional hazard analyses were performed to evaluate the effectiveness of screening and to determine the appropriate screening interval for chest radiography surveillance. RESULTS: Early lung cancer was found in 38% of patients receiving chest radiography versus 26% of those without surveillance. A patient age of more than 65 years (hazard ratio [HR], 1.53; 95% confidence limits [CL], 1.50-1.56), male (HR, 1.66; 95% CL, 1.62-1.70), and high lung cancer stages at the time of diagnosis were independent factors associated with mortality (each, P < 0.001). Chest radiography surveillance was a factor for decreasing mortality in female (HR, 0.81; 95% CL, 0.77-0.84, P < 0.001), with mortality reduction of 10% at the 3- and 5-year survival time-points. In female patients, chest radiography surveillance at intervals of less than 3 years was an independent predictor of improved survival. CONCLUSIONS: Surveillance chest radiography increased survival in a female screened population in South Korea. Chest radiography intervals of less than 3 years may help to detect lung cancer in female patients.

Turo (Qi Dance) Program for Parkinson's Disease Patients: Randomized, Assessor Blind, Waiting-List Control, Partial Crossover Study.

CONTEXT: Qigong, Tai-chi and dancing have all been proven effective for Parkinson's disease (PD); however, no study has yet assessed the efficacy of Turo, a hybrid qigong dancing program developed to relieve symptoms in PD patients. OBJECTIVE: To determine whether Turo may provide benefit in addressing the symptoms of PD patients. DESIGN: Randomized, assessor blind, waiting-list control, partial crossover study. SETTING: Kyung Hee University Korean Medicine Hospital, Seoul, Republic of Korea. PARTICIPANTS: A total of 32 PD patients (mean age 65.7 ± 6.8). INTERVENTION: Participants were assigned to the Turo group or the waiting-list control group. The Turo group participated in an 8-week Turo training program (60-minute sessions twice a week). The waiting-list control group received no additional treatment during the same period; then underwent the same 8-week Turo training. OUTCOME MEASURES: The primary outcome was a score on the Unified Parkinson's Disease Rating Scale (UPDRS), and the secondary outcomes included the perceived health status assessed using the Parkinson's disease Quality of Life questionnaire (PDQL), balance function as assessed by the Berg Balance Scale (BBS) and the results of the Beck Depression Inventory (BDI). RESULTS: The Turo group showed statistically significant improvements in the UPDRS (P < 0.01) and PDQL (P < 0.05) as compared to the control group. The changes in BBS scores displayed a tendency toward improvement, but was not statistically significant (P = 0.051). CONCLUSION: These findings suggest that Turo PD training might improve the symptoms of PD patients.

Virtual Reality Rehabilitation With Functional Electrical Stimulation Improves Upper Extremity Function in Patients With Chronic Stroke: A Pilot Randomized Controlled Study.

OBJECTIVE: To compare virtual reality (VR) combined with functional electrical stimulation (FES) with cyclic FES for improving upper extremity function and health-related quality of life in patients with chronic stroke. DESIGN: A pilot, randomized, single-blind, controlled trial. SETTING: Stroke rehabilitation inpatient unit. PARTICIPANTS: Participants (N=48) with hemiplegia secondary to a unilateral stroke for >3 months and with a hemiplegic wrist extensor Medical Research Council scale score ranging from 1 to 3. INTERVENTIONS: FES was applied to the wrist extensors and finger extensors. A VR-based wearable rehabilitation device was used combined with FES and virtual activity-based training for the intervention group. The control group received cyclic FES only. Both groups completed 20 sessions over a 4-week period. MAIN OUTCOME MEASURES: Primary outcome measures were changes in Fugl-Meyer Assessment-Upper Extremity and Wolf Motor Function Test scores. Secondary outcome measures were changes in Box and Block Test, Jebsen-Taylor Hand Function Test, and Stroke Impact Scale scores. Assessments were performed at baseline (t0) and at 2 weeks (t1), 4 weeks (t4), and 8 weeks (t8). Between-group comparisons were evaluated using a repeated-measures analysis of variance. RESULTS: Forty-one participants were included in the analysis. Compared with FES alone, VR-FES produced a substantial increase in Fugl-Meyer Assessment-distal score (P=.011) and marginal improvement in Jebsen-Taylor Hand Function Test-gross score (P=.057). VR-FES produced greater, although nonsignificant, improvements in all other outcome measures, except in the Stroke Impact Scale-activities of daily living/instrumental activities of daily living score. CONCLUSIONS: FES with VR-based rehabilitation may be more effective than cyclic FES in improving distal upper extremity gross motor performance poststroke.

Adjunctive Breast-Specific Gamma Imaging for Detecting Cancer in Women with Calcifications at Mammography.

BACKGROUND: Mammography detects calcium deposits sensitively, but the specificity for differentiating malignancy from benign calcifications is low. Thus, we investigated whether adjunctive breast-specific gamma imaging (BSGI) has incremental value for detecting cancer in women with suspicious calcifications detected by mammography, and compared BSGI with adjunctive ultrasonography (US). METHODS: The medical records of women without a personal history of breast cancer who underwent mammography for breast evaluation from 2009 to 2014 were reviewed retrospectively. Patients who had calcifications detected by mammography, with a result of Breast Imaging Reporting and Data System (BI-RADS) categories 3-5, underwent adjunctive US and BSGI and were included in this study. A total of 302 breast lesions in 266 women (mean age ± standard deviation 49 ± 9 years) were selected for this study. RESULTS: For detecting breast cancer using mammography plus BSGI, the sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiver operating curve with 95% confidence intervals were 94% (91-96), 90% (86-93), 91% (87-94), 94% (90-96), and 0.92 (0.89-0.95), respectively. For mammography plus US, the respective values were 97% (94-98), 51% (46-57), 68% (63-73), 94% (90-96), and 0.74 (0.70-0.78). CONCLUSIONS: Adjunctive BSGI had higher specificity than adjunctive US without loss of sensitivity. This finding suggests that adjunctive BSGI may be a useful complementary initial imaging method to improve the detection of breast cancer in women who have calcifications with suspicious morphology at mammography.

Expression and assembly of cholera toxin B subunit and domain III of dengue virus 2 envelope fusion protein in transgenic potatoes.

The rates of mosquito-transmitted dengue virus infection in humans have increased in tropical and sub-tropical areas. Domain III of dengue envelope protein (EDIII) is involved in cellular receptor binding and induces serotype-specific neutralizing antibodies. EDIII fused to the B subunit of Vibrio cholera (CTB-EDIII) was expressed in potatoes to develop a plant-based vaccine against dengue virus type 2. CTB-EDIII fused to an endoplasmic reticulum (ER) retention signal, SEKDEL, was introduced into potatoes by A. tumefaciens-mediated gene transformation. The integration of the CTB-EDIII fusion gene into the nuclear genome of transgenic plants was confirmed by genomic DNA polymerase chain reaction (PCR), and mRNA transcripts of CTB-EDIII were detected. CTB-EDIII fusion protein was expressed in potato tubers and assembled into a pentameric form capable of binding monosialotetrahexosylganglioside (GM1). The level of expression was determined to be ∼0.005% of total soluble protein in potato tubers. These results suggest that dengue virus antigen could be produced in potatoes, raising the possibility that edible plants are employed in mucosal vaccines for protection against dengue infection.

The ω-3 polyunsaturated fatty acids prevented colitis-associated carcinogenesis through blocking dissociation of ß-catenin complex, inhibiting COX-2 through repressing NF-κB, and inducing 15-prostaglandin dehydrogenase.

Numerous studies have demonstrated that diets containing an increased ratio of ω-6 : ω-3 polyunsaturated fatty acids (PUFAs) are a risk factor for colon cancer and might affect tumorigenesis. Therefore, dietary ω-3 PUFA administration may be a preventive strategy against colon cancer. Until now, the exact molecular mechanisms and required dietary doses of ω-3 PUFAs for cancer prevention were unknown. In this study, we explored the anti-tumorigenic mechanisms of ω-3 PUFAs against a colitis-associated cancer (CAC) model. Through in vitro cell models involving docosahexaenoic acid (DHA) administration, down-regulation of survivin and Bcl-2, and up-regulation of Bax, accompanied by blockage of ß-catenin complex dissociation, the main mechanisms responsible for DHA-induced apoptosis in HCT116 cells were determined. Results included significant reduction in azoxymethane-initiated, dextran sodium sulfate-promoted CACs, as well as significant preservation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and significant inhibition of Cyclooxyganase-2 (COX-2) and Prostaglandin E2(P < 0.01). Additional mechanisms and significant induction of apoptosis in both tumor and non-tumor tissues were also noted in fat-1 transgenic (TG) mice. The lipid profiles of colon tissues measured in all specimens revealed that intake greater than 3 g ω-3 PUFA/60 kg of body weight showed tissue levels similar to those seen in fat-1 TG mice, preventing cancer. Our study concluded that COX-2 inhibition, 15-PGDH preservation, apoptosis induction, and blockage of ß-catenin complex dissociation contributed to the anti-tumorigenesis effect of ω-3 PUFAs, and an intake higher than 3g ω-3 PUFAs/60 kg of body weight can assist in CAC prevention.

Protective effect of dietary chitosan on cadmium accumulation in rats.

BACKGROUND/OBJECTIVES: Cadmium is a toxic metal that is an occupational and environmental concern especially because of its human carcinogenicity; it induces serious adverse effects in various organs and tissues. Even low levels of exposure to cadmium could be harmful owing to its extremely long half-life in the body. Cadmium intoxication may be prevented by the consumption of dietary components that potentially reduce its accumulation in the body. Dietary chitosan is a polysaccharide derived from animal sources; it has been known for its ability to bind to divalent cations including cadmium, in addition to other beneficial effects including hypocholesterolemic and anticancer effects. Therefore, we aimed to investigate the role of dietary chitosan in reducing cadmium accumulation using an in vivo system. MATERIALS/METHODS: Cadmium was administered orally at 2 mg (three times per week) to three groups of Sprague-Dawley rats: control, low-dose, and high-dose (0, 3, and 5%, respectively) chitosan diet groups for eight weeks. Cadmium accumulation, as well as tissue functional and histological changes, was determined. RESULTS: Compared to the control group, rats fed the chitosan diet showed significantly lower levels of cadmium in blood and tissues including the kidneys, liver, and femur. Biochemical analysis of liver function including the determination of aspartate aminotransferase and total bilirubin levels showed that dietary chitosan reduced hepatic tissue damage caused by cadmium intoxication and prevented the associated bone disorder. CONCLUSIONS: These results suggest that dietary chitosan has the potential to reduce cadmium accumulation in the body as well as protect liver function and bone health against cadmium intoxication.

Is complementary and alternative therapy effective for women in the climacteric period?

Vasomotor symptoms start about 2 years prior to menopause in women who are approaching menopause, and early menopause symptoms appear including emotional disturbance and anxiety, followed by physical changes such as vaginal dryness, urinary incontinence and skin wrinkles. As time progresses, osteoporosis, cardiovascular diseases, and dementia occur consecutively. Hormone therapy is primarily considered for the relief of menopause symptoms in postmenopausal women. However, as hormone replacement has emerged as a therapy that increases the potential risk of thrombosis, cerebral infarction and breast cancer, complementary and alternative medicine has drawn much attention. This study aimed to examine the types and effects of evidence-based complementary and alternative therapies that are currently used.

Novel vaccination approach for dengue infection based on recombinant immune complex universal platform.

Dengue infection is on the rise in many endemic areas of the tropics. Vaccination remains the most realistic strategy for prevention of this potentially fatal viral disease but there is currently no effective vaccine that could protect against all four known serotypes of the dengue virus. This study describes the generation and testing of a novel vaccination approach against dengue based on recombinant immune complexes (RIC). We modelled the dengue RIC on the existing Ebola RIC (Phoolcharoen, et al. Proc Natl Acad Sci USA 2011;108(Dec (51)):20695) but with a key modification that allowed formation of a universal RIC platform that can be easily adapted for use for other pathogens. This was achieved by retaining only the binding epitope of the 6D8 ant-Ebola mAb, which was then fused to the consensus dengue E3 domain (cEDIII), resulting in a hybrid dengue-Ebola RIC (DERIC). We expressed human and mouse versions of these molecules in tobacco plants using a geminivirus-based expression system. Following purification from the plant extracts by protein G affinity chromatography, DERIC bound to C1q component of complement, thus confirming functionality. Importantly, following immunization of mice, DERIC induced a potent, virus-neutralizing anti-cEDIII humoral immune response without exogenous adjuvants. We conclude that these self-adjuvanting immunogens have the potential to be developed as a novel vaccine candidate for dengue infection, and provide the basis for a universal RIC platform for use with other antigens.

Intake of hot water-extracted apple protects against myocardial injury by inhibiting apoptosis in an ischemia/reperfusion rat model.

Intakes of apple and its products are shown to reduce the risk of coronary heart disease by delaying occlusion of coronary arteries. In our previous study, we showed that apple pectin protected against myocardial injury by prohibiting apoptotic cascades in a rat model of ischemia/reperfusion. Thus, we hypothesized that water-extracted apple, into which apple pectin was released from the cell wall, might exhibit the same efficacy as apple pectin. To test this hypothesis, we fed rats either cold water- (400 mg kg(-1) d(-1)) or hot water-extracted apples (HWEA; 40, 100, and 400 mg kg(-1) d(-1)). Three days later, the rats were subjected to myocardial injuries by ligating the left anterior descending coronary artery (30 minutes), and subsequently, the heart (3 hours) reperfused by releasing the ligation. Only the rats that were supplemented with HWEA (400 mg kg(-1) d(-1)) showed significant reductions in infarct size, which was 28.5% smaller than that of the control group. This infarct size reduction could be partly attributed to the prevention of steps leading to apoptosis. These steps are manifested by a higher Bcl-2/Bax ratio, lower procaspase-3 conversion to caspase-3, and inhibition of DNA nick generation, which reflects the extent of apoptosis. The findings indicate that HWEA supplementation reduces myocardial injury by inhibiting apoptosis under ischemia/reperfusion conditions. In conclusion, this study suggests that apple intake, specifically boiled apple, might reduce the risk of coronary heart disease by inhibiting postocclusion steps, such as myocardial injury after artery occlusion, as well as preocclusion steps, such as atherosclerotic plaque formation.

Protective effects of Pogostemon cablin Bentham water extract on inflammatory cytokine expression in TNBS-induced colitis in rats.

In inflammatory bowel disease (IBD), colon epithelial cells express a variety of inflammatory mediators, including chemokines, which perpetuate inflammatory response. In the current study, we report that water extract of Pogostemon cablin Bentham aerial parts (PCW), which has traditionally been used for treatment of the common cold and infectious disease, suppressed colon inflammation. Treatment with PCW resulted in effective inhibition of tumor necrosis factor (TNF)-α-induced adhesion of monocytes to HT-29 human colonic epithelial cells. In a trinitrobenzene sulfonic acid (TNBS)-induced rat model of IBD, PCW suppressed clinical signs of colitis, including weight loss, colon tissue myeloperoxidase activity, a marker for inflammatory cell infiltration, and cyclooxygenase-2 expression in a dose-dependent manner. In addition, PCW suppressed TNBS-induced mRNA expression of IL-8, MCP-1, and IL-6 in rat colon. The nuclear level of NF-κB in TNBS-treated rat colon and NF-κB luciferase reporter gene activity in TNF-α-treated HT-29 cells were significantly inhibited by PCW. Taken together, the results of this study suggest that PCW suppressed colon inflammation via suppression of NF-κB-dependent expression of pro-inflammatory cytokines.

Effect of lithospermi radix on contact dermatitis induced by dinitrofluorobenzene in mice.

OBJECTIVE: The root of Lithospermum erythrorhizon Sieb. et Zucc. (Lithospermi Radix, LR) is a kind of heat clearing and blood cooling medicinal herbs. It can clear away heat and cool the blood, reduce toxins and disperse maculae. LR has long been used as efficacious therapy for inflammation, burns, frostbite and skin diseases such as eczema and psoriasis. METHODS: In the present study, we investigate anti-allergic and anti-inflammatory effects of LR by using the 1-fluoro-2, 4- dinitrofluorobenzene (DNFB)-induced contact dermatitis mouse model. RESULTS: Topical application of 10 mg/mL of LR effectively inhibited skin lesions induced by repeated paintings with DNFB. Topical application of LR also inhibited hyperplasia, edema, spongiosis and infiltrations of mononuclear cells. In addition, production levels of total immunoglobulin and IgG1 in serum were decreased by using LR in vivo. CONCLUSION: These data suggest that LR acts as an antiinflammatory agent, improving skin lesions in CD mice.

Effects of α-lipoic acid and L-carnosine supplementation on antioxidant activities and lipid profiles in rats.

α-Lipoic acid and L-carnosine are powerful antioxidants and are often used as a health supplement and as an ergogenic aid. The objective of this study was to investigate the effects of α-lipoic acid and/or L-carnosine supplementation on antioxidant activity in serum, skin, and liver of rats and blood lipid profiles for 6 weeks. Four treatment groups received diets containing regular rat chow diet (control, CON), 0.5% α-lipoic acid (ALA), 0.25% α-lipoic acid + 0.25% L-carnosine (ALA + LC), or 0.5% L-carnosine (LC). Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and lipid peroxidation products, malondialdehyde (MDA) concentrations, were analyzed in serum, skin, and liver. Blood lipid profiles were measured, including triglycerides (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C). Skin and liver SOD activities of the ALA and LC groups were higher than those of the CON group (P < 0.05), but serum SOD activity was higher only in the LC group compared to that in the CON group (P < 0.05). Additionally, only liver GSH-Px activity in the LC group was higher than that of the CON and the other groups. Serum and skin MDA levels in the ALA and LC groups were lower than those in the CON group (P < 0.05). Serum TG and TC in the ALA and ALA + LC groups were lower than those in the CON and LC groups (P < 0.05). The HDL-C level in the LC group was higher than that in any other group (P < 0.05). LDL-C level was lower in the ALA + LC and LC groups than that in the CON group (P < 0.05). Thus, α-lipoic acid and L-carnosine supplementation increased antioxidant activity, decreased lipid peroxidation in the serum, liver, and skin of rats and positively modified blood lipid profiles.

Anti-obesity and antioxidative effects of purple sweet potato extract in 3T3-L1 adipocytes in vitro.

The purpose of the current study was to determine the anti-obesity and anti-inflammatory effects of an extract of purple sweet potatoes (PSPs) on 3T3-L1 adipocytes. For this purpose, differentiated 3T3-L1 adipocytes were treated with a PSP extract at concentrations of 1,000, 2,000, and 3,000 µg/mL for 24 hours. Then, we measured the changes in the sizes of the adipocytes, the secretion of leptin, and the mRNA/protein expression of lipogenic, inflammatory, and lipolytic factors after the treatment with the PSP extract. The PSP extract diminished leptin secretion, indicating that growth of fat droplets was suppressed. The extract also suppressed the expression of mRNAs of lipogenic and inflammatory factors and promoted lipolytic action. The antioxidative activity of the PSP extract was also measured using three different in vitro methods: 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity, ferric reducing ability potential assay, and chelating activity of transition metal ions. Taken together, our study shows that PSP extract has antilipogenic, anti-inflammatory, and lipolytic effects on adipocytes and has radical scavenging and reducing activity.

Protective effect of components isolated from Lindera erythrocarpa against oxidative stress-induced apoptosis of H9c2 cardiomyocytes.

Eight compounds were isolated from the methanol fraction of Lindera erythrocarpa and assessed for their ability to protect H9c2 cardiomyocytes against oxidative stress-induced cell death. Three of the compounds significantly reduced the release of lactate dehydrogenase from H9c2 cardiomyocytes treated with buthionine-[S,R]-sulfoximine and reduced the uptake of propidium iodide by these cells. These effects were concentration-dependent. The three inhibitory compounds were identified as (-)-epicatechin, avicularin and quercitrin by spectroscopic techniques including one- and two-dimensional NMR and mass spectroscopy.

Cholera toxin B subunit-domain III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants.

Envelope glycoprotein E of the dengue virus, which plays a crucial role in its entry into host cells, has an immunogenic domain III (EIII, amino acids 297-394), which is capable of inducing neutralizing antibodies. However, mice immunized with EIII protein without adjuvant elicited low immune responses. To improve low immune responses, a DNA fragment, consisting of cholera toxin B subunit and EIII gene (CTB-EIII), was constructed and introduced into tobacco plant cells (Nicotiana tabacum L. cv. MD609) by Agrobacterium tumefaciens-mediated transformation methods. The integration and transcription of CTB-EIII fusion gene were confirmed in transgenic plants by genomic DNA PCR amplification and Northern blot analysis, respectively. The results of immunoblot analysis with anti-CTB and anti-dengue virus antibodies showed the expression of the CTB-EIII fusion protein in transgenic plant extracts. Based on the G(M1)-ELISA results, the CTB-EIII protein expressed in plants showed the biological activity for intestinal epithelial cell membrane glycolipid receptor, G(M1)-ganglioside, and its expression level was up to about 0.019% of total soluble protein in transgenic plant leaf tissues. The feasibility of using a plant-produced CTB-EIII fusion protein to generate immunogenicity against domain III will be tested in future animal experiments.

Expression and immunogenicity of enterotoxigenic Escherichia coli heat-labile toxin B subunit in transgenic rice callus.

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G(M1)-ganglioside, a receptor for biologically active LTB, was confirmed by G(M1)-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G(M1)-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.