Low-Temperature Multiple Micro-Dispensing on Microneedles for Accurate Transcutaneous Smallpox Vaccination.

Smallpox is an acute contagious disease caused by the variola virus. According to WHO guidelines, the smallpox vaccine is administrated by scarification into the epidermis using a bifurcated needle moistened with a vaccine solution. However, this invasive vaccination method involving multiple skin punctures requires a special technique to inoculate, as well as a cold chain for storage and distribution of vaccine solutions containing a live virus. Here, we report a transcutaneous smallpox vaccination using a live vaccinia-coated microneedle (MN) patch prepared by a low-temperature multiple nanoliter-level dispensing system, enabling accurate transdermal delivery of live vaccines and maintenance of bioactivity. The live vaccinia in hyaluronic acid (HA) solutions was selectively coated on the solid MN tips, and the coating amount of the vaccine was precisely controlled through a programmed multiple dispensing process with high accuracy under low temperature conditions (2-8 °C) for smallpox vaccination. Inoculation of mice (BALB/C mouse) with the MN patch coated with the second-generation smallpox vaccine increased the neutralizing antibody titer and T cell immune response. Interestingly, the live vaccine-coated MN patch maintained viral titers at -20 °C for 4 weeks and elevated temperature (37 °C) for 1 week, highlighting improved storage stability of the live virus formulated into coated MN patches. This coated MN platform using contact dispensing technique provides a simple and effective method for smallpox vaccination.

Antiviral activity of Bifidobacterium adolescentis SPM 0214 against herpes simplex virus type 1.

Herpes Simplex Virus type 1 (HSV-1) antibodies are found in up to 90 percent of the general population. About 30% of patients who have been exposed to HSV-1 develop recurrent infections, and this degree is continually increasing. In addition, resistance to all major anti-herpetic drugs such as acyclovir (ACV) has been increasingly reported. These observations underscore the importance of discovering new therapeutic tools for the treatment of HSV-1 infections. Bifidobacterium spp. has been studied in various fields including antibacterial and anticancer effect, but the antiviral activity was studied very little. The aim of this study was to test the antiviral activity of Bifidobacterium spp. against HSV-1. The Bifidobacterium adolescentis SPM 0214 used in this study through the screening of 23 Bifidobacterium spp. by plaque assay was assessed the cell viability assay in Vero cells. We also measured the plaque reduction assay and yield reduction assay after B. adolescentis SPM 0214 treatment at concentrations ranging between 10 and 104 µg/mL. The B. adolescentis SPM 0214 was not toxic to Vero cells, and the inhibition of plaque and yield formation was obviously increased compared to those of the control (no additive). Therefore, these results indicate that antiviral activity of B. adolescentis SPM 0214 against HSV-1.

In vitro evaluation of antibacterial activities and anti-inflammatory effects of Bifidobacterium spp. addressing acne vulgaris.

The objective of this study was to evaluate the antibacterial and anti-inflammatory effect of Bifidobacterium spp. In the first part of the study, the antibacterial activities of live and sonicated cells, from a total of 23 Bifidobacterium species, on the growth of 5 different strain of Staphylococcus aureus. Six strains, of sonicated Bifidobacterium, exhibited antibacterial activity against staphylococci samples, and seven Bifidobacterium strains exhibited antibacterial activity on the growth of S. aureus S.P.-N2. In the second part of the study, we tested the antimicrobial activity, of Bifidobacterium against Propionibacterium acne KCTC3320, using the co-culture method. The loss of P. acnes viability, caused by B. adolescentis SPM0308 and B. longum SPM1207, was 84% and 75%, respectively (*p < 0.05). In the third part of the study, the anti-inflammatory activity of B. adolescentis SPM0308 and B. longum SPM1207 were assessed; nitric oxide (NO), and tumor necrosis factor-α (TNF-α), production were tested using the murine macrophage RAW 264.7 cell line. Treatment of RAW 264.7 cells, with Bifidobacterium, decreased production of NO and TNF-α rather than LPS (100 ng/mL) treatment. The results suggest that B. adolescentis SPM0308 could be used as an effective control for P. acnes KCTC3320, and S. aureus, and reduce the risk of acne vulgaris development. We suggest that B. adolescentis SPM0308 may be a useful probiotic microorganism, for prevention of acne vulgaris, without adverse effects.

Antimicrobial activity of commonly used antibiotics and DNA fingerprint analysis of Pseudomonas aeruginosa obtained from clinical isolates and unchlorinated drinking water in Korea, 2010.

Pseudomonas aeruginosa exists in various environments, and can cause mild or serious infections resulting in a wide range of symptoms. In this study, we collected bacterial isolates from hospitalized patients and unchlorinated drinking water, in Korea, 2010. The water-borne and clinical isolates were compared using colony morphology, antimicrobial susceptibility testing, and random amplification of polymorphic DNA analysis. We first compared morphological features of the water-borne and clinical isolates. The clearest difference in colony morphology was colony shape; five water-borne isolate colonies (83%) had a smooth, circular morphology, while nine (75%) clinical isolate colonies had a rough, irregular morphology. Minimum inhibitory concentrations analyses were performed to determine antimicrobial resistant patterns; using ceftazidime, gentamicin, tigecycline, chloramphenicol, meropenem, and tobramycin according to Clinical and Laboratory Standard Institute (CLSI, 2009) methodology. All waterborne isolates were not resistant to gentamicin, tobramycin, and meropenem. The clinical isolates were resistant to every antibiotic except chloramphenicol. Genotyping was performed using the repetitive extragenic palindromic polymerase-chain-reaction. The DNA fingerprinting patterns did not reveal genetic similarity between the water-borne and clinical P. aeruginosa isolates. On the contrary, they showed that genetically distinct populations have been established in each of these environments. We have revealed significant morphological, clinical and genetic differences between water-borne and clinical isolates of the same bacterial species.

The combination of mixed lactic acid bacteria and dietary fiber lowers serum cholesterol levels and fecal harmful enzyme activities in rats.

Probiotics such as lactic acid bacteria (LAB) and prebiotics such as fiber are generally considered beneficial for health. These affect the microflora composition and fermentation metabolites and consequently contribute to local and systemic effects in humans. The beneficial effects of probiotics can be improved when combined with prebiotics. Here we investigated the effects of a mixed LAB supplement combined with dietary fiber on the population of LAB in the gut, as well as on serum cholesterol levels, fecal water content and microbial harmful enzyme activities. For animal studies, 0.2 mL of mixed LAB (Bifidobacterium longum SPM1205, Lactobacillus acidophilus, and SAFELAC isolated from Pediococcus pentosaceus) supplement (10(7) ∼ 10(8) colony forming units per day) was orally administered to male Sprague-Dawley rats every day for 2 weeks along with a diet containing 5% or 10% cellulose. The mixed LAB supplement combined with dietary cellulose significantly (p < 0.05) reduced serum total cholesterol and LDL levels. This combination also significantly (p < 0.05) increased the population of LAB and the fecal water content and significantly (p < 0.05) reduced microbial harmful enzyme (ß-glucosidase, ß-glucuronidase and tryptophanase) activities. These effects of LAB were particularly improved by its combination with 5% cellulose compared to the control (a diet without cellulose), and the 5% cellulose combination was more effective than the 10% cellulose combination. In conclusion, the incorporation of a fibrous diet such as cellulose with lactic acid bacteria improved the population of LAB, and daily consumption of this combination could reduce the serum cholesterol levels and activities of harmful enzymes such as ß-glucosidase, ß-glucuronidase, tryptophanase, urease in rats.

Antimicrobial activity of Bifidobacterium spp. isolated from healthy adult Koreans against cariogenic microflora.

OBJECTIVE: Dental caries is the main common infectious disease in the human oral cavity. Streptococcus mutans and Streptococcus sobrinus were reported to be the most important etiological factors in human dental caries. Thus, we examined the inhibitory effects of Bifidobacterium spp. cells and culture supernatants against S. mutans and S. sobrinus, including Streptococcus gordonii, and Aggregatibacter actinomycetemcomitans, which is associated with periodontal disease. METHODS: Mutans streptococci or A. actinomycetemcomitans and lactic acid bacteria (LAB) were mixed in 1:1 ratio and then incubated for 90 min at 37°C. After the incubation, the viability of mutans streptococci or A. actinomycetemcomitans was determined by plate count technique. We also investigated the morphological changes of S. mutans treated with LAB using scanning electron microscopy (SEM). RESULTS: In vitro viability of S. mutans, S. sobrinus, S. gordonii, and A. actinomycetemcomitans was affected by human intestinal LAB identified as Bifidobacterium adolescentis SPM1005 and Bifidobacterium longum SPM1207. Especially, B. adolescentis SPM1005 cells at 1.0 × 10(8) CFU had a strong growth-inhibiting effect against S. mutans and induced a 64% loss of its viability (p<0.05). In addition, swollen and disrupted S. mutans were observed after incubation with B. adolescentis SPM1005. However, the culture supernatant of this strain did not show such inhibitory activity. CONCLUSION: B. adolescentis SPM1005 cells decreased the growth of S. mutans, which is a risk factor for dental caries. Therefore, we suggest that this Bifidobacterium strain may be a useful probiotic microorganism for prevention of dental caries that does not have adverse effects.

Hypocholesterolemic effect of sonication-killed Bifidobacterium longum isolated from healthy adult Koreans in high cholesterol fed rats.

We have previously reported that live Bifidobacterium longum SPM1207, a strain isolated from healthy adult Koreans, significantly reduced serum cholesterol in broth and rat. We here examined the effect of oral administration of sonication-killed B. longum SPM1207 on serum cholesterol in rats in order to investigate whether this killed strain could be utilized as a potent probiotics for human and animals. Dietary treatments consisted of 3 treatment groups of 24 rats each randomly assigned to either normal diet, high cholesterol diet and saline (HCS), or high cholesterol diet and sonication-killed B. longum SPM1207 (HCKB) for 3 weeks. Although HDL-cholesterol levels in the serum were not significantly (p > 0.05) different between HCKB rats and HCS rats, total and LDL-cholesterol levels in the serum were significantly (p < 0.05) less increased in HCKB (total: 177.71 mg/dL, LDL-: 60.50 mg/dL) rats when compared to HCS (total: 237.17 mg/dL, LDL-: 71.50 mg/dL) rats. AI was significantly (p < 0.05) lower in HCKB (4.95 mg/dL) rats when compared to HCS (9.22 mg/dL) rats. Body weight increase and relative liver weight were significantly (p < 0.05) lower in HCKB rats when compared to HCS rats. Over the time, high cholesterol diet caused dry feces accompanied by decreased fecal water content (66.00 to 61.94%) but sonication-killed B. longum SPM1207 administration increased fecal water content (71.58 to 74.25%). The results in the current study provide evidence that the sonication-killed cells of B. logum SPM1207 isolated from healthy adult Koreans have a greater potential to be used as a cholesterol-lowering agent. Furthermore, the current study suggest that this killed specific strain may play role in part in blocking the body weight increase and relieving or eliminating constipation.

Efficacy of Lactic Acid Bacteria (LAB) supplement in management of constipation among nursing home residents.

BACKGROUND: Constipation is a significant problem in the elderly, specifically nursing home and/or extended-care facility residents are reported to suffer from constipation. Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as diarrhea and constipation effect. The objective of this study was to investigate the efficacy of this LAB supplement in the management of nursing home residents. METHODS: Nineteen subjects (8M, 11F; mean age 77.1 +/- 10.1) suffering with chronic constipation were assigned to receive LAB (3.0 x 10(11) CFU/g) twice (to be taken 30 minutes after breakfast and dinner) a day for 2 weeks in November 2008. Subjects draw up a questionnaire on defecation habits (frequency of defecation, amount and state of stool), and we collected fecal samples from the subjects both before entering and after ending the trial, to investigate LAB levels and inhibition of harmful enzyme activities. Results were tested with SAS and Student's t-test. RESULTS: Analysis of questionnaire showed that there was an increase in the frequency of defecation and amount of stool excreted in defecation habit after LAB treatment, but there were no significant changes. And it also affects the intestinal environment, through significantly increase (p < 0.05) fecal LAB levels. In addition, tryptophanase and urease among harmful enzyme activities of intestinal microflora were significantly decreased (p < 0.05) after LAB treatment. CONCLUSION: LAB, when added to the standard treatment regimen for nursing home residents with chronic constipation, increased defecation habit such as frequency of defecation, amount and state of stool. So, it may be used as functional probiotics to improve human health by helping to prevent constipation.

Synthesis and assembly of Porphyromonas gingivalis fimbrial protein in potato tissues.

Periodontal disease caused by the gram-negative oral anaerobic bacterium Porphyromonas gingivalis is thought to be initiated by the binding of P. gingivalis fimbrial protein to saliva-coated oral surfaces. To assess whether biologically active fimbrial antigen can be synthesized in edible plants, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein, fimA (amino acids 266-337), was cloned behind the mannopine synthase promoter in plant expression vector pPCV701. The plasmid was transferred into potato (Solanum tuberosum) leaf cells by Agrobacterium tumefaciens in vivo transformation methods. The fimA cDNA fragment was detected in transformed potato leaf genomic DNA by PCR amplification methods. Further, a novel immunoreactive protein band of ~6.5 kDa was detected in boiled transformed potato tuber extracts by acrylamide gel electrophoresis and immunoblot analysis methods using primary antibodies to fimbrillin, a monomeric P. gingivalis fimbrial subunit. Antibodies generated against native P. gingivalis fimbriae detected a dimeric form of bacterial-synthesized recombinant FimA(266-337) protein. Further, a protein band of ~160 kDa was recognized by anti-FimA antibodies in undenatured transformed tuber extracts, suggesting that oligomeric assembly of plant-synthesized FimA may occur in transformed plant cells. Based on immunoblot analysis, the maximum amount of FimA protein synthesized in transformed potato tuber tissues was approximately 0.03% of total soluble tuber protein. Biosynthesis of immunologically detectable FimA protein and assembly of fimbrial antigen subunits into oligomers in transformed potato tuber tissues demonstrate the feasibility of producing native FimA protein in edible plant cells for construction of plant-based oral subunit vaccines against periodontal disease caused by P. gingivalis.

Lactic acid bacteria affect serum cholesterol levels, harmful fecal enzyme activity, and fecal water content.

BACKGROUND: Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as lower cholesterol. Although present in many foods, most trials have been in spreads or dairy products. Here we tested whether Bifidobacteria isolates could lower cholesterol, inhibit harmful enzyme activities, and control fecal water content. METHODS: In vitro culture experiments were performed to evaluate the ability of Bifidobacterium spp. isolated from healthy Koreans (20 approximately 30 years old) to reduce cholesterol-levels in MRS broth containing polyoxyethanylcholesterol sebacate. Animal experiments were performed to investigate the effects on lowering cholesterol, inhibiting harmful enzyme activities, and controlling fecal water content. For animal studies, 0.2 ml of the selected strain cultures (108 approximately 109 CFU/ml) were orally administered to SD rats (fed a high-cholesterol diet) every day for 2 weeks. RESULTS: B. longum SPM1207 reduced serum total cholesterol and LDL levels significantly (p < 0.05), and slightly increased serum HDL. B. longum SPM1207 also increased fecal LAB levels and fecal water content, and reduced body weight and harmful intestinal enzyme activities. CONCLUSION: Daily consumption of B. longum SPM1207 can help in managing mild to moderate hypercholesterolemia, with potential to improve human health by helping to prevent colon cancer and constipation.

New antimicrobial drug resistance and epidemiological typing patterns of Staphylococci from clinical isolates and raw meats.

The antimicrobial susceptibilities of Staphylococcus isolated from clinical isolates and raw meats were tested for six different antimicrobial agents that are in widespread clinical use in Korea and four new antimicrobials, linezolid, quinupristin/dalfopristin, daptomycin, and tigecycline. And this study analyzed the mecA genes and genetic patterns of MRSA by performing epidemiological studies using the PCR method. 46%, 51%, and 79% of clinical isolates were identified as MRSA in 1998, 1999, and 2005, respectively, and the mecA gene was detected in 82% of these isolates. Of the 133 staphylococci isolated from raw meats, 18% of the isolates were found to be resistant to methicillin, but none of these isolates showed the presence of the mecA gene. New antimicrobials, which have rarely or not yet been used in Korean hospitals, showed high activity against all staphylococcal isolates including methicillin-resistant isolates. The randomly amplified polymorphic DNA (RAPD) patterns of MRSA isolates differed significantly between clinical isolates and raw meat isolates.

Association between an AluI polymorphism in the calcitonin receptor gene and quantitative ultrasound parameters in Korean men.

OBJECTIVE: The purpose of this study was to investigate the association between an AluI RFLP of the calcitonin receptor (CTR) gene and quantitative ultrasound (QUS) parameters in Korean men, and the interaction with nutrition as a lifestyle factor. MATERIALS AND METHODS: Broadband ultrasound attenuation, speed of sound and stiffness index of the calcaneus were measured using an ultrasound bone densitometer in 201 Korean men (mean age +/- SD: 51.6 +/- 11.7 years). The PCR-RFLP method was used to analyze an AluI polymorphism in the CTR gene. RESULTS: In all subjects, the distribution of CC, CT and TT genotypes occurred with frequencies of 87.1, 12.4 and 0.5%, respectively. When stratified by omnivore and vegetarian groups, there was a significant association between an AluI polymorphism in the CTR gene and QUS parameters such as speed of sound and stiffness index in only vegetarian subjects. CONCLUSION: Our data suggest that the AluI polymorphism of the CTR gene can be useful as a genetic marker in the interindividual susceptibility of QUS parameters by the interaction with nutritional status as a lifestyle factor.

Genetic variation in the renin-angiotensin system and response to endurance training.

OBJECTIVE: To investigate the relationship between genetic variation in the renin-angiotensin system and the effect of 12-week endurance training in Korean women. MATERIALS AND METHODS: Seventeen women who participated in an endurance training program for 12 weeks were genotyped for the angiotensinogen M235T polymorphism, angiotensin II type 1 receptor A1166C polymorphism, angiotensin-converting enzyme (ACE) T-3892C polymorphism, and angiotensin II type 2 receptor C3123A polymorphism. The following clinical parameters were measured before and after the endurance training program: blood pressure, body composition, ventilatory response, total cholesterol, triglyceride, and glucose. RESULTS: Of the genetic markers investigated, the frequency of the T allele for the ACE T-3892C polymorphism was significantly associated with the response in body mass index and VO(2max) after 12 weeks of endurance training (p< 0.05). None of the other polymorphisms were significantly associated with the effect of training. CONCLUSION: The significant association between ACE T-3892C and the change in body mass index and VO(2max) in Korean women are attributed to training, suggesting that this genetic variation is a useful genetic marker for clarifying the interindividual response to endurance training.

The protein polymorphism of haptoglobin in Korean elite athletes.

OBJECTIVE: To investigate protein polymorphism of the haptoglobin (Hp) and the relationship between Hp phenotypes and anthropometric or biochemical parameters in elite Korean male athletes. MATERIALS AND METHODS: Serum samples were collected from 120 Korean male elite athletes. The Hp phenotypes were determined by polyacrylamide gel electrophoresis, followed by peroxidase staining. Then anthropometric or biochemical measurements were made: body composition, blood pressures, ventilatory responses, cholesterol (total, LDL cholesterol and HDL cholesterol), triglyceride, apolipoprotein A1, lipoprotein (a), creatine phosphokinase and lactate dehydrogenase. RESULTS: The gene frequencies of the Hp1-1, Hp2-1 and Hp2-2 phenotypes in Korean male athletes were 12, 37 and 51%, respectively; this polymorphism was significantly associated with the VO(2max) index in the athletes. An excess of the Hp1 allele was also observed in marathon runners compared with the other sporting activities, although it did not have any statistical significance. CONCLUSION: Hp polymorphism exists in elite Korean male athletes and Hp phenotype may be a useful marker for endurance performance in these male athletes.