A mWAP–hLF hybrid gene locus gave extremely high level expression of human lactoferrin in the milk of transgenic mice

The production of pharmaceuticals by current mammary gland bioreactor techniques is limited by the low expression level of foreign proteins. We describe here a novel method that solves this problem. A successive three-step gap-repair strategy was developed to replace the genomic coding sequence in mouse whey acidic protein (mWAP) gene locus with that of human lactoferrin (hLF) precisely from the start code to the end code. A 50-kb mWAP–hLF hybrid gene locus was constructed, and corresponding transgenic mice were generated. An extremely high-level expression of rhLF in the milk was demonstrated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot. The expression level ranged from 16.7 to 29.8 g/l among five transgenic lines, as indicated by the ELISA assay. Importantly, the expressed rhLF maintained the same antibacterial activity as the native hLF. Our strategy can very likely also be used for the efficient expression of other valuable pharmaceutical proteins. G. Shi and H. Chen contributed equally to this work.     

The Effects of Soy Milk Enriched with Lactobacillus casei and Omega-3 on the Tibia and L5 Vertebra in Diabetic Rats: a Stereological Study

Bone fragility, despite relatively high BMD values, is an important complication related to insulin resistance and oxidative stress in diabetes mellitus type 1. The present study aimed to compare the effects of soy milk (SM), soy milk containing Lactobacillus casei (PSM), and soy milk enriched with Lactobacillus casei and omega-3 (OPSM) on the stereology of the tibia and vertebra, and antioxidant activity in type 1 diabetic rats. Sixty-five male Sprague Dawley rats were randomly assigned into 5 groups of 13 animals each. Diabetes was induced by a single injection of STZ (60 mg/kg); two control groups (non-diabetic: CN and diabetic: CD) were selected and then fed with 1 mL of distilled water. Three treatment groups were fed 1 ml of SM, PSM, and OPSM via intragastric gavage for 60 days. Treatment with SM, PSM, and OPSM significantly decreased (P < 0.05) the number of the osteoclasts in both tibia and L5 vertebra, and plasma alkaline phosphatase level. Also, the osteoblast number, calcium level, catalase activity, and total antioxidant capacity were increased in the SM, PSM, and OPSM groups compared to the STZ group. OPSM had the greatest effects on the stereological and biochemical parameters compared to the SM and PSM groups. Soy milk combination with Lactobacillus casei and omega-3 can ameliorate the stereological changes in the tibia and vertebra. In addition, this combination increased the antioxidant activity and improved the redox homeostasis in diabetic rats. These results suggest the potential role of soy milk containing Lactobacillus casei enriched with omega-3 in preventing and delaying osteoporosis in diabetic patients.

     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

Lactobacillus casei DN-114 001 Inhibits the Ability of Adherent-Invasive Escherichia coli Isolated from Crohn's Disease Patients To Adhere to and To Invade Intestinal Epithelial Cells

Ileal lesions in 36.4% of patients with Crohn's disease are colonized by pathogenic adherent-invasive Escherichia coli. The aim of this study was to determine the in vitro inhibitory effects of the probiotic strain, Lactobacillus casei DN-114 001, on adhesion to and invasion of human intestinal epithelial cells by adherent-invasive E. coli isolated from Crohn's disease patients. The experiments were performed with undifferentiated Intestine-407 cells and with undifferentiated or differentiated Caco-2 intestinal epithelial cells. Bacterial adhesion to and invasion of intestinal epithelial cells were assessed by counting CFU. The inhibitory effects of L. casei were determined after coincubation with adherent-invasive E. coli or after preincubation of intestinal cells with L. casei prior to infection with adherent-invasive E. coli. Inhibitory effects of L. casei on adherent-invasive E. coli adhesion to differentiated and undifferentiated intestinal epithelial cells reached 75% to 84% in coincubation and 43% to 62% in preincubation experiments, according to the cell lines used. Addition of L. casei culture supernatant to the incubation medium increased L. casei adhesion to intestinal epithelial cells and enhanced the inhibitory effects of L. casei. The inhibitory effects on E. coli invasion paralleled those on adhesion. This effect was not due to a bactericidal effect on adherent-invasive E. coli or to a cytotoxic effect on epithelial intestinal cells. As Lactobacillus casei DN-114 001 strongly inhibits interaction of adherent-invasive E. coli with intestinal epithelial cells, this finding suggests that the probiotic strain could be of therapeutic value in Crohn's disease.     

Comparative studies of fructose 1,6-diphosphate aldolase fromEscherichia coli 518 andLactobacillus casei var.rhamnosus ATCC 7469

A comparative study has been carried out with FDP aldolases fromEscherichia coli 518 andLactobacillus casei ATCC 7469, which had been purified 17.6- and 65-fold, respectively. The aldolase ofL.casei was stable only in the presence of mercaptoethanol, whereas that ofE.coli was strongly inhibited at low (1.0×10^–4 m) and activated at high concentrations (2.0×10^–1 m) of the same compound.p-Chloromercuric benzoic acid inhibited both aldolases, with 40% inhibition at 2×10^–5 m withE.coli aldolase against at 2×10^–4 m withL.casei aldolase. Significant differences were also observed in pH optima and Km values.E.coli aldolase exhibited a maximal activity at pH 9.0 and gave a Km value of 1.76×10^–3 m FDP with strong substrate inhibition above 7×10^–3 m, against pH 6.8–7.0 and a Km of 7.04×10^–3 m FDP forL.casei aldolase. Strong resistance ofL.casei aldolase against inhibition by EDTA, Ca²⁺ and Mn²⁺ was observed compared with complete inhibition at concentrations of 20mm, 40mm and 20mm, respectively, withE. coli aldolase. Polyacrylamide gel electrophoresis did not reveal any differences between the two enzyme preparations.The differences of the properties of FDP aldolases from different bacterial genera are discussed in relation to other Class II aldolases.     

Antibacterial effect of honey on the in vitro and in vivo growth of Escherichia coli

The study investigated the antibacterial effect of honey against pathogenic Escherichia coli. Honey showed inhibitory activity against the growth of E. coli (ATCC 25922) in agar plate assay. In liquid culture (48 h, 37 °C) the growth rate of bacterial cells decreased in the presence of honey (9.6 × 10⁵ c.f.u./ml) compared with sucrose (2.87 × 10⁸ c.f.u./ml). Rats fed with honey and orally inoculated with E. coli excreted significantly (P < 0.05) less bacterial cells in faeces compared to controls. Animals acclimatized to feeding of honey prior to E. coli inoculation showed a significant decrease in excreted bacterial load compared with the group provided with honey after bacterial inoculation. Consumption of honey also enhanced the concentration of short chain fatty acids in the intestine of rats (83 mM) compared with the control group (44.5 mM). The results show that honey possessed significant antibacterial activity against E. coli under in vitro and in vivo conditions, and indicate the potential benefit of consumption of honey regularly on the microbiological constitution of animals feeding on it.     

Protective action of reactivating factor of <Emphasis Type="Italic">Luteococcus japonicus</Emphasis> subsp. <Emphasis Type="Italic">casei</Emphasis> toward cells of <Emphasis Type="Italic">Escherichia coli</Emphasis> reparation mutants inactivated with UV-light

Reactivating factor (RF) from Luteococcus japonicus subsp. casei had a protective action on UV-irradiated cells of Escherichia coli AB1157 with a native reparation system and on cells of isogenic reparation mutants of E. coli UvrA⁻, RecA⁻, and PolA⁻: the effect resulted in multifold increase of survivability. Defense action of L. casei exometabolite is not connected with stimulating reparation systems in E. coli, and, probably, it is mediated by involvement of the exometabolite in the mechanism of cell division. RF did not provoke the reactivation of E. coli cells inactivated by UV-light.     

In Vitro Antibacterial Activity of 4-Phenyl-1-(2-phenyl-allyl)pyridinium bromide: A Novel Class of Pyridinium Based Antibacterial Compounds

The continuing increase in the incidence of multi drug resistant pathogenic bacteria and shortage of new antimicrobial agents are the prime driver in efforts to identify the novel antimicrobial classes. In vitro antibacterial activity of 4-phenyl-1-(2-phenylallyl) pyridinium bromide was tested against Gram positive Staphylococcus aureus, Streptococcus species, Bacillus subtilis, and Gram negative Klebsiella aerogenes and Escherichia coli using disk diffusion method. Among them S. aureus showed strong antibacterial activity (21.99 ± 0.03 mm) while E. coli showed very little activity (8.97 ± 0.06 mm) towards the compound. The MIC of 4-phenyl-1-(2-phenyl-allyl)-pyridinium bromide for 90% S. aureus was ≤20 μg/ml and was compared with phenoxymethylpenicillin, cloxacillin, erythromycin and vancomycin. When 4-phenyl-1-(2-phenyl-allyl)pyridinium bromide showed MIC at ≤20 μg/ml, all others showed MIC at ≤100 μg/ml. Strong antibacterial activity of 4-phenyl-1-(2-phenyl-allyl)pyridinium bromide against S. aureus indicates that there is a possibility to use it as an effective antibacterial agent.     

Evaluation of the probiotic and functional potential of Lactobacillus agilis 32 isolated from pig manure

Escherichia coli is a symbiotic bacterium in humans and animals and an important pathogen of humans and animals. Prevention and suppression of E. coli infection is of great concern. In this study, we isolated a strain of Lactobacillus agilis 32 from pig manure and evaluated its biological characteristics, and found that its bacterial survival rate was 25% after 4 h of treatment at pH 2, and under the condition of 0·5% bile concentration, its survival rate exceeds 30%. In addition, L. agilis 32 has a cell surface hydrophobicity of 77·8%, and exhibits 67·1% auto-aggregation and 63·2% aggregation with Enterotoxigenic E. coli 10 (ETEC 10). FITC fluorescence labelling showed that the fluorescence intensity of cecum was significantly higher than that of duodenum, jejunum or colon (P < 0·05), but no significant difference from ileum. Lactobacillus agilis 32 bacterial culture and CFS showed average inhibition zone diameters of 14·2 and 15·4 mm respectively. Lactobacillus agilis 32 CFS treatment can significantly reduce the pathogenicity of ETEC 10. These results show that L. agilis 32 is an active and potential probiotic, and it has a good antibacterial effect on ETEC10, which provides basic research for probiotics to prevent and treat intestinal diarrhoea pathogen infection.     

Prevention of Pathogenic Escherichia coli Infection in Mice and Stimulation of Macrophage Activation in Rats by an Oral Administration of Probiotic Lactobacillus casei I-5

Lactobacillus casei I-5 isolated from an alcohol fermentation broth enhanced immunity and prevented pathogenic infection as a probiotic. Mice fed with I-5 cells for 11 days prior to an intraperitoneal challenge with pathogenic Escherichia coli Juhl exhibited a high survival rate compared with the control group. Rats fed with I-5 cells for 10 days significantly increased the phagocytosis of peritoneal macrophages. In a cell culture system employing peritoneal macrophages from rats, the I-5 administration activated NF-κB stimulated by LPS. It also enhanced LPS-stimulated IL-12 and TNF-α production, but not IL-6 production. These results show that L. casei I-5 effectively prevented infection by pathogenic E. coli possibly through the activation of peritoneal macrophages. The strain would be useful to prevent pathogenic microbial infections in humans and farm animals.     

Enhanced Gene Transduction into Skeletal Muscle of Mice In Vivo with Pluronic Block Copolymers and Ultrasound Exposure

The pluronic block copolymers are able to enhance the ultrasound-induced gene delivery in vitro. In the present study, the effects of pluronics on the efficiency of gene transfer into skeletal muscle in vivo under sonoporation were investigated. Plasmid DNA encoding green fluorescent protein (GFP) in combination with three different pluronics, F127, L61, and P85, was injected into the tibialis anterior (TA) muscle of mice with and without adjunct ultrasound (1 MHz, 3 W/cm² 1 min, 20% duty cycle). Mice were killed 1 week after injection. The TA muscles were removed and snap frozen immediately in isopentane cooled by liquid nitrogen and sections of 7 μm thick were cut. Transfection efficiency was assessed by counting the number of GFP-positive fibers under fluorescence microscopy, and tissue damage by hematoxylin and eosin staining. The results suggested that all three pluronics significantly enhanced transgene expression in skeletal muscle (P < 0.01), especially the P85 showed significantly higher efficiency than the other two pluronics (P < 0.05). Ultrasound synergistically enhanced the gene delivery efficiency with P85 (P < 0.01), but was unable to do so with F127 and L61 groups. In short, P85 displays significantly synergistic effect with ultrasound for enhancing plasmid DNA transduction in skeletal muscle of mice in vivo.     

Novel strategies of essential oils,chitosan, and nano- chitosan for inhibition of multi-drug resistant: E. coli O157:H7 and Listeria monocytogenes

Despite the wide range of available antibiotics, food borne bacteria demonstrate a huge spectrum of resistance. The current study aims to use natural components such as essential oils (EOs), chitosan, and nano-chitosan that have very influential antibacterial properties with novel technologies like chitosan solution/film loaded with EOs against multi-drug resistant bacteria. Two strains of Escherichia coli O157:H7 and three strains of Listeria monocytogenes were used to estimate antibiotics resistance. Ten EOs and their mixture, chitosan, nano-chitosan, chitosan plus EO solutions, and biodegradable chitosan film enriched with EOs were tested as antibacterial agents against pathogenic bacterial strains. Results showed that E. coli O157:H7 51,659 and L. monocytogenes 19,116 relatively exhibited considerable resistance to more than one single antibiotic. Turmeric, cumin, pepper black, and marjoram did not show any inhibition zone against L. monocytogenes; Whereas, clove, thyme, cinnamon, and garlic EOs exhibited high antibacterial activity against L. monocytogenes with minimum inhibitory concentration (MIC) of 250–400 μl 100^?1 ml and against E. coli O157:H7 with an MIC of 350–500 μl 100^?1 ml, respectively. Among combinations, clove, and thyme EOs showed the highest antibacterial activity against E. coli O157:H7 with MIC of 170 μl 100^?1 ml, and the combination of cinnamon and clove EOs showed the strongest antibacterial activity against L. monocytogenes with an MIC of 120 μl 100^?1 ml. Both chitosan and nano-chitosan showed a promising potential as an antibacterial agent against pathogenic bacteria as their MICs were relatively lower against L. monocytogenes than for E. coli O157:H7. Chitosan combined with each of cinnamon, clove, and thyme oil have a more effective antibacterial activity against L. monocytogenes and E. coli O157:H7 than the mixture of oils alone. Furthermore, the use of either chitosan solution or biodegradable chitosan film loaded with a combination of clove and thyme EOs had the strongest antibacterial activity against L. monocytogenes and E. coli O157:H7. However, chitosan film without EOs did not exhibit an inhibition zone against the tested bacterial strains.     

Reduction of Sulfide, Ammonia Compounds, and Adhesion Properties of Lactobacillus casei Strain KE99 In Vitro

The ability of Lactobacillus casei strain KE99 to reduce sulfide, ammonia, and to adhere to bio-surfaces was characterized and compared with three lactobacillus reference strains. Sulfide reduction by strain KE99 in MRS broth increased exponentially after 10-h growth and reached a maximum (>300 ppm reduction) within 48 h. KE99 demonstrated a maximum reduction of sulfide under anaerobic (341 ppm) growth conditions at pH 6.0-8.0 range. Maximum anaerobic reduction of sulfide was demonstrated by L. casei 393 at pH 7.0 (272 ppm); L. rhamnosus at pH 8.0 (277 ppm); and L. reuteri at pH 7.0 (244 ppm). KE99 reduced sulfide more (p < 0.0001) in MRS broth spiked with Na₂S (374 ppm) than (NH₄)₂S (340 ppm) salts. Ammonia reduction by strain KE99 and the three lactobacillus reference strains in MRS broth was low. Ammonia reduction reached a maximum within 36 h and remained unchanged over extended incubations of 48 h to 72 h or further. KE99 reduced ammonium sulfate (37 ppm) more readily than the nitrate (31 ppm), hypophosphate (29 ppm), or chloride (20 ppm) salts of ammonia. KE99 and the three reference strains of lactobacilli demonstrated avid binding to Bio-coat™ (Cn type-I, Cn type-IV, laminin, fibronectin), Matrigel™, and Caco-2 cell monolayers in vitro. The number of lactobacilli binding to Caco-2 was estimated at 74/cell with strain KE99, which was significantly higher compared with 40/cell (p < 0.0001), 26/cell (0.0001), and 64/cell (p < 0.002) with L. casei 393, L. reuteri, and L. rhamnosus, respectively. The interaction of KE99 to immobilized Cn type-I was saturable and reached an equilibrium within 1 h at room temperature. KE99 binding to Cn type-I occurred at a wide pH range and was biphasic with maximum binding at pH 5.5 and 7.5. Inhibition and binding-displacement experiments with different salts and sugars suggested that the KE99 binding to immobilized Cn type-I may involve a combination of electrostatic and lectin-type interactions. KE99 effectively detached the Cn-adherent E. coli O157:H7 in the range of 55% (ATCC43895) to 76% (ATCC43894). The binding-displacement values for L. casei 393, L. reuteri and L. rhamnosus to detach Cn-adherent E. coli O157:H7 (ATCC43894) were 66 ± 4%, 59 ± 2%, and 64 ± 2%, respectively. Also, a reconstituted solution of the freeze-dried KE99 preparation effectively detached the Cn-adherent E. coli O157:H7 in a dose-dependent manner that reached a binding-displacement equilibrium of 85% at a 1% wt/vol KE99 concentration. Received: 25 May 2001 / Accepted: 2 July 2001     

Role of reactive oxygen species in the antibacterial mechanism of silver nanoparticles on Escherichia coli O157:H7

In this study, the conditions and mechanism of antibacterial activity of hydrophilic polymer coated silver nanoparticles (AgNPs) against E. coli O157:H7 (CMCC44828) as model pathogen was studied. The AgNPs were coated with amphiphilic polymer that introduced carboxyl groups on the surface to make it water-soluble. The AgNPs were exposed to various treatment conditions of pH and temperature before these were combined with the E. coli. The mechanism of the antibacterial activity was studied through the formation of reactive oxygen species (ROS) that was later suppressed with antioxidant to establish correlation with the AgNPs antimicrobial activity. Studies were carried out at both anaerobic and aerobic conditions. The results indicated that 5 mg/L AgNPs inhibited ~50% of the growth of 10⁶ colony forming units per milliliter (cfu/mL) E. coli cells in liquid Luria–Bertani (LB) medium. This dose-dependent antimicrobial activity was higher at increased temperature (37°C) but was lower when the AgNPs were treated with acid at pH 2 before exposure to the bacteria. It was also established that the conditions of higher antimicrobial effect generated more ROS that was dependent on the presence of oxygen. The antibacterial activity was suppressed in the presence of an antioxidant.     

Effects of Lactobacillus rhamnosus ATCC 7469 on Different Parameters Related to Health Status of Rainbow Trout (Oncorhynchus mykiss) and the Protection Against Yersinia ruckeri

In the current study, we investigated the effect of a probiotic bacterium (Lactobacillus rhamnosus ATCC 7469) microencapsulated with alginate and hi-maize starch and coated with chitosan on improving growth factors, body composition, blood chemistry, and the immune response of rainbow trout (initial weight: 18.41 ± 0.32 g). Four experimental diets were formulated to feed fish for 60 days. They were control diet without any additive (C), diet added with beads without probiotic (E), a probiotic sprayed to the diet (L.r), and encapsulated probiotic supplemented diet (E-L.r). The results indicated that feeding with E-Lr significantly improved weight gain (84.98 g) and feed conversion ratio (0.95) compared to the other groups (P < 0.05). Also, fish fed E-Lr diet had a significantly higher value of whole-body protein (17.51%), total protein in the blood (4.98 g/dL), lysozyme (30.66 U/mL), alternative complement pathway hemolytic activity (134 U/mL), superoxide dismutase (203 U/mg protein), and catalase (528.33 U/mg protein) (P < 0.05) as compared to those fed the control diet. Similarly, a higher relative expression of immune-related genes such as interleukin-1 (Il-1) and tumor necrosis factor-alpha (TNF-1α) were reported in those fed E-L.r and L.r diets respectively. Interestingly, the fish fed dietary E-L.r had a significantly lower value of lipid in the whole body (4.82%) and cholesterol in the blood (160.67%) in comparison with those fed the control diet (P < 0.05). At the end of the experiment, all groups were challenged by Yersinia ruckeri where the survival rate of rainbow trout fed dietary E-L.r (70.36%) was statistically higher than that of the others (P < 0.05). Overall, the results suggested that encapsulated probiotic Lact. rhamnosus ATCC 7469 acted better than unencapsulated probiotic and has a potential to improve growth performance, flesh quality, and the immune response of rainbow trout.

     

Antagonistic activity of two potential probiotic bacteria from fish intestines and investigation of their effects on growth performance and immune response in rainbow trout (Oncorhynchus mykiss)

The probiotic activity of two bacterial strains, Lactobacillus casei and Lactobacillus plantarum, isolated from common carp intestines was studied using antagonistic tests in vitro against Yersinia ruckeri. Randomly assigned to triplicate groups were 450 rainbow trout (mean weight, 20 ± 3 g) fed three different diets: a commercial feed, or the same feed incorporated into either 5 × 10⁷ CFU g^?1 of L. casei or L. plantarum. After a 30‐day feeding trial, 30 fish in each group were challenged with Yersinia ruckeri by intraperitoneal injection. Growth parameters were significantly increased in both treatment groups. Immune parameters such as lysozyme activity, alternative complement activity and total immunoglobulin level were significantly higher in the L. casei group than in fish fed the control diet, while no significant differences were revealed between the L. plantarum and control groups. Mortality rates of fish fed L. casei and L. plantarum were lower than in fish fed the control diet after challenging with Y. ruckeri.     

The Effect of Lactobacillus casei Consumption in Improvement of Obsessive–Compulsive Disorder: an Animal Study

Obsessive–compulsive disorder (OCD) is an important neuropsychiatric disorder worldwide. Common treatments of OCD include serotonergic antidepressants, which can cause potentially serious side effects. We assessed the effects of Lactobacillus casei (L. casei) Shirota consumption in an animal model of OCD. OCD-like symptoms were induced in rats by the chronic injection of the D2/D3 dopamine agonist quinpirole hydrochloride. Rats were classified into five groups of 6 rats. Four groups were injected chronically with quinpirole (0.5 mg/kg, twice weekly for 5 weeks). They were fed with L. casei Shirota (10⁹ CF/g, daily for 4 weeks) (group 1), fluoxetine (10 mg/kg, daily for 4 weeks) (group 2), combination of L. casei Shirota and fluoxetine (group 3), and normal saline (positive control group). The last group did not receive dopamine agonist and was only injected with saline (negative control group). Expression levels of brain-derived neurotrophic factor (Bdnf), solute carrier family 6 member 4 (Slc6a4), and 5-hydroxytryptamine receptor type 2A (Htr2a) were assessed in orbitofrontal cortex tissues of all rats. Behavioral tests showed improvement of OCD signs in rats treated with L. casei Shirota, fluoxetine, and a combination of drugs. Quantitative PCR analysis showed a remarkable decrease in the expression of Bdnf and an increase in the expression of Htr2a in quinpirole-treated rats. After treatment with L. casei Shirota and fluoxetine, the expression level of Bdnf was increased remarkably, whereas Htr2a expression was decreased. The current study showed the effectiveness of L. casei Shirota in the treatment of OCD in a rat model. The beneficial effects of this probiotic are possibly exerted through the modulation of serotonin-related genes expression.

     

<Emphasis Type="Italic">Trigonella foenum-graecum</Emphasis> (Fenugreek) Protects Against Selenite-Induced Oxidative Stress in Experimental Cataractogenesis

Cataract is the opacification in eye lens and leads to 50% of blindness worldwide. The present study was undertaken to evaluate the anticataract potential of Trigonella foenum-graecum Linn seeds (fenugreek) in selenite-induced in vitro and in vivo cataract. In vitro enucleated rat lenses were maintained in organ culture containing Dulbecco’s modified Eagles medium (DMEM) alone or in addition with 100 μM selenite and served as the normal and control groups, respectively. For the test group, the medium was supplemented with selenite and T. foenum-graecum aqueous extract. The lenses were incubated for 24 h at 37°C. After incubation, the lenses were processed for the estimation of reduced glutathione (GSH), lipid peroxidation product (malondialdehyde), and the antioxidant enzymes. In vivo selenite cataract was induced in 9-day-old rats by subcutaneous injection of sodium selenite (25 μmol/kg body weight). Animals in the test group were injected with different doses of aqueous extract of T. foenum-graecum 4 h before the selenite challenge. A fall in GSH and a rise in malondialdehyde levels were observed in control as compared to normal lenses. T. foenum-graecum significantly (P < 0.01) restored glutathione and decreased malondialdehyde levels. A significant restoration in the activities of antioxidant enzymes such as superoxide dismutase (P < 0.01), catalase, (P < 0.01), glutathione peroxidase (P < 0.01), and glutathione-S-transferase (P < 0.01) was observed in the T. foenum-graecum supplemented group as compared to control. In vivo, none of the eyes was found with nuclear cataract in treated group as opposed to 72.5% in the control group. T. foenum-graecum protects against experimental cataract by virtue of its antioxidant properties. Further studies are warranted to explore its role in human cataract.     

Human papillomavirus type 16 E6-specific antitumor immunity is induced by oral administration of HPV16 E6-expressing Lactobacillus casei in C57BL/6 mice

Given that local cell-mediated immunity (CMI) against the human papillomavirus type 16 E6 (HPV16 E6) protein is important for eradication of HPV16 E6-expressing cancer cells in the cervical mucosa, the HPV16 E6 protein may be a target for the mucosal immunotherapy of cervical cancer. Here, we expressed the HPV16 E6 antigen on Lactobacillus casei (L. casei) and investigated E6-specific CMI following oral administration of the L. casei-PgsA-E6 to mice. Surface expression of HPV16 E6 antigens was confirmed and mice were orally inoculated with the L. casei-PgsA or the L. casei-PgsA-E6. Compared to the L. casei-PgsA-treated mice, significantly higher levels of serum IgG and mucosal IgA were observed in L. casei-PgsA-E6-immunized mice; these differences were significantly enhanced after boost. Consistent with this, systemic and local CMI were significantly increased after the boost, as shown by increased counts of IFN-γ-secreting cells in splenocytes, mesenteric lymph nodes (MLN), and vaginal samples. Furthermore, in the TC-1 tumor model, animals receiving the orally administered L. casei-PgsA-E6 showed reduced tumor size and increased survival rate versus mice receiving control (L. casei-PgsA) immunization. We also found that L. casei-PgsA-E6-induced antitumor effect was decreased by in vivo depletion of CD4⁺ or CD8⁺ T cells. Collectively, these results indicate that the oral administration of lactobacilli bearing the surface-displayed E6 protein induces T cell-mediated cellular immunity and antitumor effects in mice.     

Secretory Expression and Purification of Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> Heat-Labile Enterotoxin B Subunit and its Applications on Intranasal Vaccination of Hantavirus

In order to further study the B subunit of the Escherichia coli heat-labile enterotoxin (LTB), we obtained the LTB gene from pathogenic E. coli, cloned it into the pET22b (+) prokaryotic expression vector, and expressed it as a fusion protein with His tag in E. coli BL21 (DE3). The recombinant LTB was expressed and purified by Ni²⁺ affinity chromatography. The biological activity of the purified recombinant LTB was assayed in a series of monosialotetrahexosylganglioside (GM1)-ELISA experiments. The recombinant LTB (rLTB) was efficiently expressed under the induction of 10 g/l lactose at 37°C for 6 h and yielded up to 31% of the total bacterial protein. Fused with pelB signal peptide, rLTB was successfully localized to the periplasmic space. GM1-ELISA experiments showed that the rLTB obtained retains strong GM1 ganglioside-binding activity. The ELISA result of hantavirus nucleoprotein-specific secretory immunoglobulin A (sIgA) and IgG showed that intranasal administration of inactivated hantavirus with rLTB significantly increased the levels of hantavirus-specific sIgA (P < 0.01) and IgG (P < 0.01) in comparison with inactivated hatavirus alone. In summary, we have developed a method for the efficient secretory expression and purification of rLTB, and the inactivated hantavirus co-administered intranasally with rLTB could effectively induce both mucosal and humoral immune responses specific to hantavirus. Shouchun Cao and Ying Zhang contributed equally to this work.