Changes in Gut Microbiota by the Lactobacillus casei Anchoring the K88 Fimbrial Protein Prevented Newborn Piglets From Clinical Diarrhea.

In the last 20 years, accumulating evidence indicates that the gut microbiota contribute to the development, maturation, and regulation of the host immune system and mediate host anti-pathogen defenses. Lactobacillus casei (L.casei) is a normal flora of the gastrointestinal tract in mammals and, as a great mucosal delivery vehicle, has wide use in bioengineering. However, the diarrhea prevention role of commensal intestinal microbiota interfered by the recombinant L.casei (rL.casei) in newborn piglets is not well understood. In our study, newborn piglets orally fed with the rL.casei surface displayed the fimbrial protein K88 of enterotoxigenic Escherichia coli (ETEC) and their feces were collected for a period of time after feeding. The next-generation sequencing of these fecal samples showed that the relative abundance of L.casei was significantly increased. The oral administration of rL.casei altered the intestinal microbial community as evidenced by altered microbial diversity and microbial taxonomic composition. Remarkably, the functional enhancing of the intestinal bacterial community by rL.casei was positively correlated with membrane transport, replication, and repair (p < 0.05). The specific antibody detection indicates that high levels of anti-K88 secretory immunoglobulin A (sIgA) were induced in fecal samples and systemic immunoglobulin G was produced in serum. The diarrhea rate in piglets caused by ETEC K88 was decreased by about 24%. Thus, the oral administration of rL.casei not only activated the mucosal and humoral immune responses in vivo but also contributed to shape the intestinal probiotics in newborn piglets and to significantly reduce the diarrhea rates of newborn piglets.

The compound 2-benzylthio-5,8-dimethoxynaphthalene-1,4-dione leads to apoptotic cell death by increasing the cellular reactive oxygen species levels in Ras-mutated liver cancer cells.

The aim of the present study was to verify the pro-apoptotic anticancer potential of several 5,8-dimethoxy-1,4-phthoquinone (DMNQ) derivatives in Ras-mediated tumorigenesis. MTT assays were used to detect cellular viability and flow cytometry was performed to assess intracellular reactive oxygen species (ROS) levels and apoptosis. The expression levels of proteins were detected via western blotting. Among the 12 newly synthesized DMNQ derivatives, 2-benzylthio-5,8-dimethoxynaphthalene-1,4-dione (BZNQ; component #1) significantly reduced cell viability both in mouse NIH3T3 embryonic fibroblasts cells (NC) and H-RasG12V transfected mouse NIH3T3 embryonic fibroblasts cells (NR). Moreover, BZNQ resulted in increased cytotoxic sensitivity in Ras-mutant transfected cells. Furthermore, the reactive oxygen species (ROS) levels in H-RasG12V transfected HepG2 liver cancer cells (HR) were significantly higher compared with the levels in HepG2 liver cancer cells (HC) following BZNQ treatment, which further resulted in increased cellular apoptosis. Eliminating cellular ROS using an ROS scavenger N-acetyl-L-cysteine markedly reversed BZNQ-induced cellular ROS accumulation and cell apoptosis in HC and HR cells. Western blotting results revealed that BZNQ significantly downregulated H-Ras protein expression and inhibited the Ras-mediated downstream signaling pathways such as protein kinase B, extracellular signal-related kinase and glycogen synthase kinase phosphorylation and ß-catenin protein expression. These results indicated that the novel DMNQ derivative BZNQ may be a therapeutic drug for Ras-mediated liver tumorigenesis. The results of the current study suggest that BZNQ exerts its effect by downregulating H-Ras protein expression and Ras-mediated signaling pathways.

Peroxiredoxin I deficiency increases keratinocyte apoptosis in a skin tumor model via the ROS-p38 MAPK pathway.

Keratinocyte hyperproliferation is an essential link in skin cancer pathogenesis. Peroxiredoxin I (Prx I) is known to regulate cancer cell proliferation, differentiation, and apoptosis, but its role in skin cancer remains unclear. This study aimed to elucidate the role and mechanism of Prx I in skin cancer pathogenesis. Dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were used to create a skin tumor model of the initiation/promotion stage of cancer. The role of Prx I in H2O2-induced keratinocyte apoptosis was also investigated. After DMBA/TPA treatment, Prx I deficiency was significantly associated with less skin tumors, lower Bcl-2 expression, and higher p-p38 and cleaved caspase-3 expressions in Prx I knockout tumors than in wild-type controls. H2O2 stimulation caused more cellular apoptosis in Prx I knockdown HaCaT cells than in normal HaCaT cells. The signaling study revealed that Bcl-2, p-p38, and cleaved caspase-3 expressions were consistent with the results in the tumors. In conclusion, the deletion of Prx I triggered the DMBA/TPA-induced skin tumor formation in vivo and in vitro by regulating the reactive oxygen species (ROS)-p38 mitogen-activated protein kinase (MAPK) pathway. These findings provide a theoretical basis for treating skin cancer.

Peroxiredoxin II Inhibits Alcohol-induced Apoptosis in L02 Hepatocytes Through AKT/ß-Catenin Signaling Pathway.

BACKGROUND: Peroxiredoxin II (PRDX2) performs unique roles in cells. It can reduce peroxides through cysteine residues, and helps prevent the effects of oxidative stress on cells. It is closely related to the occurrence and development of various diseases, especially alcoholic liver injury and even liver cancer. The metabolism of alcohol in hepatocytes leads to the increase in the levels of reactive oxygen species (ROS), oxidative stress, injury, and apoptosis. Therefore, this study focused on the investigating the protection conferred by PRDX2 against alcohol-induced apoptosis of hepatocytes. MATERIALS AND METHODS: PRDX2 inhibition of alcohol-induced apoptosis in L02 hepatocytes was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, fluorescence microscopy, flow cytometry, western blotting and hematoxylin and eosin staining. RESULTS: The results showed that the levels of reactive oxygen species, protein kinase B, ß-catenin, B-cell lymphoma-2 (BCL2), BCL-XL, BCL2-associated X, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase in PRDX2-silenced cells were increased significantly after the treatment of cells with ethanol. Similar results were obtained in an in vivo Prdx2-knockout mouse model of alcoholic liver injury. Therefore, PRDX2 may regulate the phosphorylation of the AKT signal protein by eliminating reactive oxygen species from cells, and it inhibits the downstream mitochondria-dependent apoptosis pathway, and, thereby, the apoptosis of cells. CONCLUSION: Thus, PRDX2 may be a potential molecular target for the prevention and treatment of alcoholic liver injury.

Peroxiredoxin I deficiency increases pancreatic ß­cell apoptosis after streptozotocin stimulation via the AKT/GSK3ß signaling pathway.

Apoptosis of pancreatic ß­cells is involved in the pathogenesis of type I and II diabetes. Peroxiredoxin I (Prx I) serves an important role in regulating cellular apoptosis; however, the role of Prx I in pancreatic ß­cell apoptosis is not completely understood. In the present study, the role of peroxiredoxin 1 (Prx I) during streptozotocin (STZ)­induced apoptosis of pancreatic ß­cells was investigated. The expression level of Prx I was decreased by STZ treatment in a time­dependent manner, and apoptosis of Prx I knockdown MIN6 cells was increased by STZ stimulation, compared with untransduced MIN6 cells. Furthermore, an intraperitoneal injection of STZ increased pancreatic islet damage in Prx I knockout mice, compared with wild­type and Prx II knockout mice. AKT and glycogen synthase kinase (GSK)­3ß phosphorylation significantly decreased following Prx I knockdown in MIN6 cells. However, phosphorylated ß­catenin and p65 levels significantly increased after STZ stimulation, compared with untransduced cells. The results of the present study indicate that deletion of Prx I mediated STZ­induced pancreatic ß­cell death in vivo and in vitro by regulating the AKT/GSK­3ß/ß­catenin signaling pathway, as well as NF­κB signaling. These findings provide a theoretical basis for treatment of pancreatic damage.

Picrasma quassioides Extract Elevates the Cervical Cancer Cell Apoptosis Through ROS-Mitochondrial Axis Activated p38 MAPK Signaling Pathway.

BACKGROUND/AIM: Picrasma quassioides (P. quassioides) is used in traditional Asian medicine widely for the treatment of anemopyretic cold, eczema, nausea, loss of appetite, diabetes mellitus, hypertension etc. In this study we aimed to understand the effect of P. quassioides ethanol extract on SiHa cervical cancer cell apoptosis. MATERIALS AND METHODS: The P. quassioides extract-induced apoptosis was analyzed using the MTT assay, fluorescence microscopy, flow cytometry and western blotting. RESULTS: P. quassioides extract induced cellular apoptosis by increasing the accumulation of cellular and mitochondrial reactive oxygen species (ROS) levels and inhibiting ATP synthesis. Pretreatment with N-Acetylcysteine (NAC), a classic antioxidant, decreased the intracellular ROS production and inhibited apoptosis. In addition, the P38 MAPK signaling pathway is a key in the apoptosis of SiHa cells induced by the P. quassioides extract. CONCLUSION: The P. quassioides extract exerts its anti-cancer properties on SiHa cells through ROS-mitochondria axis and P38 MAPK signaling. Our data provide a new insight for P. quassioides as a therapeutic strategy for cervical cancer treatment.

Anti-tumor Properties of Picrasma quassioides Extracts in H-RasG12V Liver Cancer Are Mediated Through ROS-dependent Mitochondrial Dysfunction.

BACKGROUND: Picrasma quassioides (PQ) is a traditional Asian herbal medicine with anti-tumor properties that can inhibit the viability of HepG2 liver cancer cells. H-Ras is often mutated in liver cancer, however, the effect of PQ treatment on H-Ras mutated liver cancer is unclear. This study aimed to investigate the role of PQ on ROS accumulation and mitochondrial dysfunction in H-ras mutated HepG2 (HepG2G12V) cells. MATERIALS AND METHODS: PQ ethanol extract-induced HepG2G12V apoptosis was analyzed by the MTT assay, fluorescence microscopy, flow cytometry and western blotting. RESULTS: PQ treatment affected cell migration and colony formation in HepG2G12V cells. Cleaved-caspase-3, cleaved-caspase-9 and BCL2 associated agonist of cell death (BAD) expression levels were increased, while the levels of B-cell lymphoma-extra large (Bcl-xL) were decreased with PQ treatment. PQ treatment led to a reduction of H-Ras expression levels in liver cancer cells, thus reducing their abnormal proliferation. Furthermore, it led to increased expression levels of Peroxiredoxin VI, which regulates the redox signal in cells. CONCLUSION: Taken together these results provide a new functional significance for the role of PQ in treating HepG2G12V liver cancer.

Structure and Properties of PSf Hollow Fiber Membranes with Different Molecular Weight Hyperbranched Polyester Using Pentaerythritol as Core.

A homologous series of hyperbranched polyesters (HBPEs) was successfully synthesized via an esterification reaction of 2,2-bis(methylol)propionic acid (bis-MPA) with pentaerythritol. The molecular weights of the HBPEs were 2160, 2660, 4150 and 5840 g/mol, respectively. These HBPEs were used as additives to prepare polysulfone (PSf) hollow fiber membranes via non-solvent induced phase separation. The characteristic behaviors of the casting solution were investigated, as well as the morphologies, hydrophilicity and mechanical properties of the PSf membranes. The results showed that the initial viscosities of the casting solutions were increased, and the shear-thinning phenomenon became increasingly obvious. The demixing rate first increased and then decreased when increasing the HBPE molecular weight, and the turning point was 2660 g/mol. The PSf hollow fiber membranes with different molecular weights of HBPEs had a co-existing morphology of double finger-like and sponge-like structures. The starting pure water contact angle decreased obviously, and the mechanical properties improved.

A Facile Way to Prepare Hydrophilic Homogeneous PES Hollow Fiber Membrane via Non-Solvent Assisted Reverse Thermally Induced Phase Separation (RTIPS) Method.

Sulfonated polyethersulfone (SPES) was used as an additive to prepare hydrophilic poly(ethersulfone) (PES) hollow fiber membranes via non-solvent assisted reverse thermally induced phase separation (RTIPS) process. The PES/SPES/N,N-dimethylacetamide (DMAc)/ polyethylene glycol 200 (PEG200) casting solutions are lower critical solution temperature (LCST) membrane forming systems. The LCST and phase separation rate increased with the increase of SPES concentrations, while the casting solutions showed shear thinning. When the membrane forming temperature was higher than the LCST, membrane formation mechanism was controlled by non-solvent assisted RTIPS process and the also membranes presented a more porous structure on the surface and a bi-continuous structure on the cross section. The membranes prepared by applying SPES present higher pure water flux than that of the pure PES membrane. The advantages of the SPES additive are reflected by the relatively high flux, good hydrophilicity and excellent mechanical properties at 0.5 wt.% SPES content.

Insulin neuroprotection and the mechanisms.

OBJECTIVE: To analyze the mechanism of neuroprotection of insulin and which blood glucose range was benefit for insulin exerting neuroprotective action. DATA SOURCES: The study is based on the data from PubMed. STUDY SELECTION: Articles were selected with the search terms "insulin", "blood glucose", "neuroprotection", "brain", "glycogen", "cerebral ischemia", "neuronal necrosis", "glutamate", "γ-aminobutyric acid". RESULTS: Insulin has neuroprotection. The mechanisms include the regulation of neurotransmitter, promoting glycogen synthesis, and inhibition of neuronal necrosis and apoptosis. Insulin could play its role in neuroprotection by avoiding hypoglycemia and hyperglycemia. CONCLUSIONS: Intermittent and long-term infusion insulin may be a benefit for patients with ischemic brain damage at blood glucose 6-9 mmol/L.

Passive protection of mice pups through oral or intranasal immunization of dams with recombinant Lactobacillus casei vaccine against ETEC F41.

Mucosal immunization is advantageous over other routes of antigen delivery because it can induce both mucosal and systemic immune responses. In this study, we have developed fimbriae protein of enterotoxigenic Escherichia coli (ETEC) F41 was stably expressed on the surface Lactobacillus casei 525. The method of expressing vaccine antigens in L. casei induces both systemic and mucosal immunity after oral or intranasal administration. We demonstrate that an oral or intranasal vaccine based on live recombinant L. casei 525 protects infant mice from ETEC F41 infection. This platform technology can be applied to design oral or intranasal vaccine delivery vehicles against several microbial pathogens.

[Molecular mechanism of ORFV intervention strategies based on the UPS of host cell: a review].

In order to compete the antiviral effects of the host cell in the process of infection, ORFV(known as Orf virus) relies on a series of functional genes developed through long-term population evolution, such as interferon resistance genes, Bcl-2 protein genes and cell cycle inhibitor gene and so on, with these weapons this virus is able to effectively counteract immune clearance and immune regulation from a host cell. Concurrently, ORFV also focuses on exploiting signal transduction pathways of the ubiquitin-proteasome system(UPS), circumvents the intracellular signal transduction and CD8+ T activation, for shielding virus particles towards maturation and releasing outside. This review introduced inner link between the UPS of host cell and intervention mechanism by virus, and analyzed the key roles of certains components in UPS, these all together showed the evolution tendency of ORFV that was involved in the designing of inhibition to immune response and for intracellular immune escape upon the selection pressure in host cell infected.

Development, characterization and anti-tumor effect of a sequential sustained-release preparation containing ricin and cobra venom cytotoxin.

Cobra venom cytotoxin (CVC) loaded in poly (lactide-co-glycolide) (PLGA) microspheres was mixed with ricin and encapsulated in a thermosensitive PLGA-PEG-PLGA hydrogel for this study. This sequential sustained-release preparation (SSRP) containing ricin and CVC could avoid burst release effect of CVC from microspheres. In addition, in SSRP, the two biotoxins have different drug release rates and antitumor mechanisms, which can be complementary to each other. Ricin has a faster release rate than CVC. It can combine with the tumor cell membrane and enter the cell, inhibiting protein synthesis within 2 weeks. Whereas CVC releases slowly in 5 weeks directly dissolving the tumor cell membrane and killing the cells which are less-sensitive to ricin. The in vivo experiments showed that intratumoral injection of SSRP could inhibit hepatocellular carcinoma growth significantly, and the tumor growth inhibition rate reached 73.5%. It appears that a new medicine preparation for cancer local treatment should be further studied for clinical applications.

Immunization with recombinant Lactobacillus casei strains producing K99, K88 fimbrial protein protects mice against enterotoxigenic Escherichia coli.

To exploit a safe and effective vaccine for the prevention against K99 or K88 infections of enterotoxigenic Escherichia coli (ETEC), we have developed a mucosal delivery vehicle based on Lactobacillus casei CICC 6105 using poly-γ-glutamate synthetase A (PgsA) as an anchoring matrix. To evaluate the immunization effect of the recombinant strains (harboring plasmids pLA-K99-K88-LTB, pLA-K99, and pLA-K88), anti-ETEC K99 or K88 antibody responses, T-cell proliferation, and cytokines by intracellular staining (ICS) were investigated after specific pathogen-free (SPF) C57BL/6 mice orally inoculated with these recombinant strains. After oral vaccination into C57BL/6 mice, all recombinant strains were proved to be immunogenic and able to elicit high levels of mucosal immunoglobulin A (IgA) titers in bronchoalveolar lavage fluids, intestinal fluids and prominent systemic immunoglobulin G and IgG subclasses (IgG1, IgG2b, and IgG2a) responses in sera. Using the T-cell proliferation assay, the stimulation index (SI) of groups immunized with pLA-K99/L. casei and pLA-K88/L. casei reached to 2.73 and 2.64, respectively, versus 2.56 in a group immunized with pLA-K99-K88-LTB/L. casei. A detailed analysis of the cell-mediated immune responses by ICS showed the number of specific CD8(+) T cells expressing cytokines (IFN-γ, TNF-α, and IL-2) and granule-associated proteins (CD107a) was higher than that of specific CD4(+) T cells secreted by immune spleen cells upon restimulation in vitro with peptides. Next, the results showed that DCs activated in vitro with recombinant L. casei enhance specific T-cell proliferation and promote T cells to produce both Th1 and Th2 cytokines. More than 80% of the vaccinated mice were protected after challenge with a lethal dose of standard strains C83912 and C83902. These results demonstrate that recombinant L. casei can induce specific humoral and mucosal antibodies and cellular immune response against protective antigens upon oral administration.

[Impact of unilateral acute testicular ischemia on the contralateral testis of rabbits in anesthetic and conscious states].

OBJECTIVE: To compare the impact of unilateral acute testicular ischemia on the hemodynamics and histology of the contralateral testis of the rabbits under consistent anesthesia with that of the rabbits in the conscious state. METHODS: Forty-two healthy male white rabbits were randomly divided into an anesthetic group (Group A) to receive injection of sodium pentobarbital (PS) and a non-anesthetic group (Group B), each including a control group of 5 animals (A0 and B0), an incomplete testicular ischemia group of 8 (A1 and B1), and a complete testicular ischemia group of 8 (A2 and B2). Testicular ischemia models were constructed by color Doppler ultrasonography. Contrast-enhanced ultrasound (CEUS) was used to observe the perfusion of the contralateral testes before and after ligating and loosening the unilateral spermatic cord in each experimental group. The control animals also underwent CEUS and measurement of the heart rate (HR) and blood pressure (BP) at the corresponding time. Histological structure changes in the contralateral testes of the rabbits were observed in both anesthetic and conscious states. RESULTS: PS anesthesia markedly suppressed the HR and BP of the rabbits. The parameters in Groups of A0, A1 and A2 showed no statistically significant changes after unilateral ligation of the spermatic cord, while Groups B1 and B2 displayed significantly decreased peak-base difference (PBD) and prolonged arrival time (AT) and half time of descending peak intensity (HT). Groups A1, B1 and B2 showed significantly increased PBD and prolonged HT shortly after loosening the spermatic cord. Focal pathological and ultrastructural changes were observed in the contralateral testes of the ischemic rabbits, but no significant difference was found in Johnson's score in comparison with the controls. The apoptotic cells were remarkably increased in Groups A1, B1 and B2. CONCLUSION: Acute testicular ischemia may induce injury to the contralateral testis to some degree, and a reflexive sympathetic response may cause hemodynamic changes in the non-anesthetic state. And the neural and vascular inhibitory effects of anesthesia could make insignificant changes of blood perfusion in the contralateral testis.

[Ischemic postconditioning reduces testis ischemia-reperfusion injury in rabbits].

OBJECTIVE: To investigate the protective effect of ischemic postconditioning (IP) against different degrees of testicular ischemia-reperfusion (IR) injury in rabbits. METHODS: Forty-two white male rabbits were equally randomized into 7 groups: a control, 3 IR (R1, R2 and R3), and 3 IP (P1, P2 and P3) groups. Testicular models of different degrees of ischemia were established in the IR and IP groups. Before reperfusion, ultrasonography showed homogeneous echoes with slightly decreased blood flow in R1 and P1, heterogeneous echoes with obviously decreased blood flow in R2 and P2, lamellar or fragmental low echo areas absent of blood flow signals in R3 and P3. Then the IR groups were directly subjected to perfusion, and the IP groups to 3 episodes of 30-second reperfusion followed by 30-second ischemia. All the groups underwent contrast-enhanced ultrasonography (CEUS) before reperfusion and, after 3 days, examined for the contents of malonaldehyde (MDA), superoxide dismutase (SOD) and histology, and observed for the pathological changes of the testicular tissue. RESULTS: Before reperfusion, no significant differences were found in the CEUS parameters beta, time-to- peak (TTP), peak-base intensity (PBD) and half of declining time (DT/2) between R1 and P1, R2 and P2, and R3 and P3 (P>0.05). There were remarkable differences in MDA and SOD between R1 and P1, and R2 and P2 (P<0.05), but not between R3 and P3 (P >0.05). Johnson's score, apoptosis index and ultrastructure showed marked differences between R1 and P1 (P<0.05) but not between R2 and P2, and R3 and P3 (P >0.05). CONCLUSION: IP can attenuate IR-induced testis injury, but the effect varies with the degree of ischemia, and its pathological manifestation differs from the biochemical one.

[Ultrasonographic appearance of experimental testicular ischemia and protective effect of allopurinol].

OBJECTIVE: To investigate the correlation of the ultrasonographic appearance of different degrees of experimentally induced acute unilateral testicular ischemia with the protective effect of allopurinol. METHODS: Forty-two male white rabbits were equally randomized into 7 groups: sham-operation control, ischemic A, B and C, and treatment D, E and F. Models of different degrees of unilateral testicular ischemia were established in the ischemic and treatment groups under the dynamic observation by color Doppler ultrasound. The ischemic testes showed slightly decreased homogeneous echoes and flow signals in groups A and D, obviously decreased heterogeneous echoes and flow signals in groups B and E, and radial or fragmental low-echo areas and disappearance of flow signals in groups C and F. The ischemic groups received reperfusion after the appearance of the above ultrasonographic changes, while the treatment groups following the intraperitoneal injection of allopurinol at 200 mg/kg. Contrast-enhanced ultrasonography (CEUS) was performed on the bilateral testes before and 3 days after the reperfusion. After 3 days of breeding, the histological changes and malondialdehyde (MDA) contents of the ischemic testes were observed, and the correlation was analyzed between the protective effect of allopurinol and the ultrasonographic appearance of different degrees of acute unilateral testicular ischemia. RESULTS: CEUS showed fast wash-in and fast wash-out in the sham-operation control group, slow wash-in and slow wash-out in groups A and B and extensive central filling defect in group C before the reperfusion. Fast wash-in and slow wash-out were observed in all the ischemic groups 3 days after the reperfusion, most obviously in group C. Groups D, E and F exhibited the same CEUS appearance as A, B and C before and 3 days after the reperfusion. Johnsen's scores were significantly increased in groups D (9.10 +/- 0.23) and E (7.03 +/- 0.20) in comparison with A (8.53 +/- 0.22) and B (5.82 +/- 0.33) (P < 0.05), but with no significant differences between C (2.30 +/- 0.53) and F (2.45 +/- 0.33) (P > 0.05). The rates of apoptosis were significantly decreased in groups D ([1.68 +/- 0.43]%) and E ([12.53 +/- 0.59]%) compared with A ([7.12 +/- 0.84]%) and B ([20.87 +/- 1.59]%) (P < 0.05), but with no significant differences between C ([52.93 +/- 2.62 ]%) and F ([51.23 +/- 2.53 ]%) (P > 0.05). Significant decreases of MDA contents in the ischemic testes were observed in groups D ([0.64 +/- 0.05] nmol/mg prot), E ([1.59 +/- 0.06] nmol/mg prot) and F ([3.10 +/- 0.17] nmol/mg prot) in comparison with A ([1.38 +/- 0.07] nmol/mg prot), B ([2.11 +/- 0.08] nmol/mg prot) and C ([3.25 +/- 0.14] nmol/mg prot) (P < 0.05). CONCLUSION: Allopurinol contributes to the recovery of spermatogenesis when testicular ischemia is sonographically shown to be mild or moderate, but produces no significant effect when it is shown to be severe. Ultrasonography helps to choose the right therapy of testicular torsion and predict spermatogenesis of ischemic testes after reperfusion.

Immunogenicity and protective efficacy of orally or intranasally administered recombinant Lactobacillus casei expressing ETEC K99.

In an effort to develop a safe and effective vaccine for the prevention of enterotoxigenic Escherichia coli (ETEC) K99 infections, we have developed a surface antigen display system using pgsA (poly-gamma-glutamate synthetase A) as an anchoring matrix. The recombinant fusion proteins comprised of pgsA and fimbriae protein of ETEC K99 were stably expressed on Lactobacillus casei. Surface localization of the fusion protein was verified by immunoblotting, immunofluorescence microscopy and flow cytometry. Specific Pathogen Free (SPF) BALB/c mice orally or intranasally vaccinated with recombinant L. casei resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA against ETEC K99, as demonstrated by enzyme-linked immunosorbent assays using purified fimbriae peptides. The serum antibody isotypes elicited were predominantly IgG1 and IgG2a. Vaccinated SPF BALB/c mice were evaluated by oral challenge with standard-type ETEC C83912 after the last booster immunization. More than 80% of immunized mice survived regardless of the immune route. The antibody titers elicited following oral immunization were lower than those following intranasal immunization but the protective efficacy was in the same order of magnitude. These results indicate that mucosal immunization with recombinant L. casei expressing ETEC K99 fimbriae protein on its surface provides an effective means for eliciting a protective immune response against the ETEC K99.

[Effects of intratumoral injection of microspheres containing cobra venom cytotoxin on transplanted human hepatoma in nude mice].

OBJECTIVE: To evaluate the safety and efficacy of intratumoral injection of polylactic-co-glycolic acid (PLGA) microspheres containing cobra venom cytotoxin in nude mice with transplanted human hepatoma. METHODS: Cytotoxic activity of cytotoxin from cobra venom was determined by using methyl thiazolyl tetrazolium method in vitro. Microspheres containing cobra venom cytotoxin were prepared with a double emulsion-solvent evaporation method. Forty BALB/c nude mice were inoculated subcutaneously in right flank with hepatoma BEL-7404 cells. Thirty-two mice whose tumor size reached about 1.0 cm in diameter, were randomly assigned into normal saline group, blank microsphers group, cytotoxin group and cytotoxin-PLGA group. Nude mice were intratumorally injected with normal saline, blank microspheres, cytotoxin or cytotoxin-PLGA microspheres respectively. Internal echo characteristics and blood flow of tumors were observed by high-frequency ultrasound every week after treatment. Twenty-six days after treatment, the tumors were removed to calculate the inhibition rate of tumor growth. The tumor, heart, liver and kidney tissues were obtained for histopathological examination. RESULTS: The cytotoxin separated and purified from crude cobra venom caused intense cytotoxic effects to the BEL-7404 cells in vitro. The diameter of PLGA microspheres containing cobra venom cytotoxin was about (34.45+/-9.85)microm. Encapsulation rate was up to (78.13+/-8.92)%, and cumulative amount of cobra venom cytotoxin released from the PLGA microspheres in vitro during 30 days was up to 84.3%. After intratumoral injection, tumor volumes and weights in the cytotoxin-PLGA group were lower than those in the normal saline group, with a tumor growth inhibition rate of 52.36%. Observed under a light microscope, most tumor tissues were necrotic. No obvious morphological change could be seen on the liver, kidney and heart tissues. CONCLUSION: The above findings indicate that intratumoral injection of cytotoxin-PLGA microspheres has strong antitumor effect and can obviously lessen systemic toxicity, which may provide an effective and feasible method for hepatocellular carcinoma treatment.

Induction of immune responses in mice after oral immunization with recombinant Lactobacillus casei strains expressing enterotoxigenic Escherichia coli F41 fimbrial protein.

In an effort to develop a safe and effective vaccine for the prevention of enterotoxigenic Escherichia coli (ETEC) F41 infections, we have developed a surface antigen display system using poly-gamma-glutamate synthetase A (PgsA) as an anchoring matrix. The recombinant fusion proteins comprised of PgsA and fimbrial protein of F41 were stably expressed in Lactobacillus casei 525. Surface localization of the fusion protein was verified by immunoblotting, immunofluorescence microscopy, and flow cytometry. Oral inoculation of recombinant L. casei 525 into specific-pathogen-free BALB/c mice resulted in significant mucosal immunoglobulin A (IgA) titers that remained elevated for >16 weeks. High levels of IgG responses in sera specific for F41 fimbriae were also induced, with prominent IgG1 titers as well as IgG2a and IgG2b titers. The helper T-cell (Th) response was Th2-cell dominant, as evidenced by increased mucosal and systemic interleukin-4-producing T cells and a concomitant elevation of serum IgG1 antibody responses. More than 80% of the mice were protected against challenge with a 2 x 10(4)-fold 50% lethal dose of standard-type F41 (C83919). The induced antibodies were important for eliciting a protective immune response against F41 infection. These results indicated that the use of recombinant L. casei 525 could be a valuable strategy for future vaccine development for ETEC.