Induction of Partial Protection in Mice after Oral Administration of Lactococcus lactis Producing Brucella abortus L7/L12 Antigen

The Brucella abortus ribosomal protein L7/L12 is an immunodominant antigen and an interesting candidate for the development of oral live vaccines against brucellosis. Here, a recombinant Lactococcus lactis strain producing L7/L12 under the control of nisin inducible promoter was orally administered to BALB/c mice. Significant levels of anti-L7/L12 specific IgA detected in feces revealed an induced local humoral immune response. However, serum analysis did not reveal any anti-L7/L12 antibodies suggesting the absence of a systemic response. Nevertheless, the vaccinated mice showed a partial protective immunity against B. abortus virulent strain (S2308) challenged by intraperitoneal inoculation.     

Successful chemoimmunotherapy of murine L1210 lymphatic leukemia with cyclophosphamide and mafosfamide-treated leukemia cells

Summary Balb/c × DBA/2 F₁ mice (CD2F₁ mice) bearing L1210 lymphatic leukemia (10 L1210 cells i.p. injected on day 0) were subjected to chemoimmunotherapy. They received 100 mg/kg of cyclophosphamide i.p. on day + 8 and 10⁶ or 10⁷ immunogenic L1210 cells treated in vitro with mafosfamide — ASTA Z7654 (L1210-Maf cells) i.p. or i.p. + s.c. on days 0, + 3, + 6, + 9, + 12 after the leukemia implantation.About 30% of leukemia-bearing mice receiving cyclophosphamide and L1210-Maf cells after L1210 inoculation were able to reject the leukemia (as compared with 0% after injection of L1210-Maf cells only or 5% after cyclophosphamide administration). Better results (54% of cured mice) were obtained if 10⁷ L1210-Maf cells were injected i.p. + s.c. beside cyclophosphamide. Biological response modifiers (BRM's): levamisole, BCG, bestatin did not improve these results in the doses used in the experiment.Augmentation of anti-L1210 therapeutic response is dependent on the administration of cyclophosphamide and L1210-Maf cels. Cyclophosphamide not only reduces the tumor burden but probably can potentiate the L1210-Maf dependent antitumor immunity as well.     

重组人乳头瘤病毒16型L1 VLP的初步纯化、鉴定及免疫效果的初步研究

目的 对昆虫细胞-杆状病毒系统表达的人乳头瘤病毒(human papillomavirus,HPV)16型L1蛋白病毒样颗粒(virus-like particle,VLP)进行初步纯化研究,并在小鼠中检测纯化VLP的免疫原性.方法 采用蔗糖-氯化铯密度梯度超速离心纯化方法纯化HPV16 LI VLP.对纯化得到的VLP分别进行蛋白印迹法、十二烷基硫酸钠一聚丙烯酰胺凝胶电泳(SDS-PAGE)及电镜鉴定.然后用VLP免疫6周龄BALB/c小鼠.将小鼠随机分成6组(3个剂量组、2个佐剂组和1个对照组),间隔2周免疫3次,3次剂量相同.取血分离血清,采用间接ELISA法检测抗HPV16 L1 VLP抗体滴度.结果 经蔗糖-氯化铯密度梯度超速离心纯化后,用SDS-PAGE鉴定纯化的HPV16 LI VLP,纯度达到90%.电镜观察可见完整的VLP颗粒.间接EUSA检测结果显示,剂量组及佐剂组小鼠血清中均可检测到特异性抗HPV16 L1 VLP抗体,且抗体滴度随针次和剂量的增加而增高.结论 采用蔗糖-氯化铯密度梯度超速离心纯化法得到的HPV16 L1 VLP可用于免疫学初步研究.通过昆虫细胞表达产生的HPV16 LI VLP 具有较强的免疫原性,能诱导小鼠产生强的体液免疫应答.     

Enhanced immune response to DNA-based HPV16L1 vaccination by costimulatory molecule B7-2

We have investigated whether co-injection of DNA encoding the costimulatory molecule B7-2 augments immune response to the major capsid protein L1 of the high-risk human papillomavirus type 16 (HPV16L1). While immunoglobulin G (IgG) specific to HPV16L1 was detected in sera from mice injected intramuscularly with pcDNA-L1 that encodes HPV16L1, a significantly increased level of IgG was found in sera from mice immunised with pcDNA-L1 in conjunction with pLXHDmB7-2 DNA. Levels of IgG in the anti-sera were correlated with the inhibitory activity of the murine erythrocyte hemagglutination caused by the virus-like particles (VLP) and the binding of VLP to HeLa cells. Moreover, splenic cells isolated from mice co-injected with pLXHDmB7-2 had stronger proliferation and more IFN-gamma producing T cells (CD4(+) and CD8(+)) when stimulated with HPV16 VLP compared with cells from mice that had received pcDNA-L1 alone and mice of the control groups. Furthermore, in footpad swelling test, mice co-immunised with pLXHDmB7-2 had greater skin thickness over those immunised with pcDNA-L1 alone or control mice. We conclude that co-injection of DNA encoding B7-2 can enhance both humoral and cellular immune responses elicited by DNA-based vaccination against HPV16 infection in mice.     

Lentinan has a stimulatory effect on innate and adaptive immunity against murine Listeria monocytogenes infection

Lentinan, a (1-3)-beta glucan from Lentinus edodes, is licensed as an immunostimulatory drug. We tested the effect of lentinan in the well-established model system of the murine Listeria monocytogenes infection. Pre-treatment of bone marrow macrophages and dendritic cells with lentinan resulted in increased production of TNF-alpha and IL-12 after L. monocytogenes infection in vitro. After lentinan treatment bone marrow macrophages showed increased NO-production and enhanced cytotoxic activity against L. monocytogenes. Pre-treatment of mice with lentinan resulted in increased concentrations of TNF-alpha, IL-12 and IFN-gamma and also an increased number of L. monocytogenes specific CD8 T cells in the spleen. The bacterial burden in spleen and liver of mice was significantly reduced during primary and secondary Listeria infection after lentinan pre-treatment of mice. In summary these results show that lentinan enhances the protective CD8 T-cell response against L. monocytogenes probably by a mechanism that involves the IL-12-mediated augmentation of the specific antilisterial CD8 T-cell response.     

Protective immunity induced by DNA-library immunization against an intracellular bacterial infection

DNA-based immunization has shown to be a viable alternative approach to induce protective immunity against Brucella abortus infection. However, the use of a unique gene may not be sufficient to induce full protection. Therefore, a new strategy based on library immunization has been described to improve the level of protection against different pathogens and to identify new protective genes. In the present study, a B. abortus library was subcloned into the mammalian expression vector pCMV-Ubi. This plasmid was designed to create a fusion between the gene of interest with ubiquitin. The analysis of this Brucella-library showed approximately 72% of clones containing inserts with an average size of 500-2000 bp. Further, homology searches were performed using the BLASTn program, and all sequenced clones showed homology with Brucella genes, as expected. BALB/c mice immunised intramuscularly with the Brucella genomic expression library showed a strong specific total IgG antibody response to a Brucella protein extract, with production of IgG1 and IgG2a isotypes. Regarding cellular immunity, high levels of IFN-gamma and no IL-4 were detected in primed mouse splenocytes and partial protection against infection was reached in animals vaccinated with the Brucella library compared to the control group.     

Nasal priming with immunobiotic lactobacilli improves the adaptive immune response against influenza virus

The nasal priming with Lactobacillus rhamnosus CRL1505 modulates the respiratory antiviral innate immune response and improves protection against influenza virus (IFV) challenge in mice. However, the potential beneficial effect of the CRL1505 strain on the adaptive immune response triggered by IFV infection or vaccination was not evaluated before. In this work, we demonstrated that nasally administered L. rhamnosus CRL1505 is able to improve both the humoral and cellular adaptive immune responses induced by IFV infection or vaccination. Higher levels of IFV-specific IgA and IgG as well as IFN-γ were found in the serum and the respiratory tract of CRL1505-treated mice after IFV challenge. Lactobacilli treated mice also showed reduced concentrations of IL-17 and improved levels of IL-10 during IFV infection. The differential balance of inflammatory and regulatory cytokines induced by L. rhamnosus CRL1505 contributed to the protection against IFV by favoring an effective effector immune response without inducing inflammatory-mediated lung damage. The optimal immunomodulatory effect of the CRL1505 strain was achieved with viable bacteria. However, non-viable L. rhamnosus CRL1505 was also efficient in improving the adaptive immune responses generated by IFV challenges and therefore, emerged as an interesting alternative for vaccination of immunocompromised hosts. Similar to other immunomodulatory properties of lactobacilli, it was shown here that the adjuvant effect in the context of IFV vaccination was a strain dependent ability, since differences were found when L. rhamnosus CRL1505 and the immunomodulatory strain L. rhamnosus IBL027 were compared. This investigation represents a thorough exploration of the role of immunobiotic lactobacilli in improving humoral and cellular adaptive immune responses against IFV in the context of both infection and vaccination.     

Attempts to use thiabendazole to improve the immune response in dexamethasone-treated or stressed cattle

Thiabendazole was evaluated in two separate experiments for its ability to enhance the immune response in dexamethasone-treated or stressed cattle. In the first experiment the cattle received either no drug treatment (controls), dexamethasone intramuscularly (IM), or dexamethasone IM plus thiabendazole orally. All animals were inoculated with heat-killed Brucella abortus strain 19, equine ferritin, tetanus toxoid, and live Corynebacterium equi at the time dexamethasone therapy was initiated. Dexamethasone (0.04 mg/kg/day IM for 3 days) significantly (p less than 0.05) inhibited the lymphocyte blastogenic response to mitogens and the antibody response to ferritin and tetanus toxoid. Thiabendazole given orally (16 mg/kg/day) beginning 24 h prior to antigen and dexamethasone administration and continued for 6 days failed to prevent the dexamethasone-induced suppression of the lymphocyte blastogenic or antibody responses. In the second experiment 51 cattle were divided into a control group and a thiabendazole-treated group. The animals were stressed by weaning, injection of antigen (equine ferritin, tetanus toxoid, B. abortus strain 19 and killed bovine viral diarrhea virus) and castration of the bulls on the day that thiabendazole therapy was started. Thiabendazole administered orally for 5 days at a dosage of 20 mg/kg did not enhance the antibody response to any of the antigens, and was associated with a significantly lower antibody response to B. abortus.     

Synthesis, antitumor activity, and mechanism of action of benzo[a]pyrano[3,2-h]acridin-7-one analogues of acronycine

Twenty-two derivatives belonging to the cis-1,2-diacyloxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[a]pyrano[3,2-h]acridin-7-one series were synthesized in nine steps starting from 3,5-dimethoxyacetanilide (5) and 2-methoxy-1-naphthalenecarboxylic acid (7). Most of them exhibited submicromolar cytotoxicity when tested against murine leukemia (L1210) and human epidermoid carcinoma (KB-3-1) cell lines. The cytotoxic activity correlated strongly with the ability of the compounds to form covalent adducts with purified DNA. Among the most active compounds, 25, with IC50 values of 0.7 and 0.15 microM against L1210 and KB-3-1, respectively, was selected for evaluation in vivo against Colon 38 adenocarcinoma implanted in mice. This compound was active at 3 mg/kg i.v. (day 12 and 24) with 3/7 tumor free mice by day 80.     

Kedarcidin, a new chromoprotein antitumor antibiotic. I. Taxonomy of producing organism, fermentation and biological activity

Strain L585-6 (ATCC 53650) is an actinomycete isolated from a soil sample collected in Maharastra State, India. It produces a new chromoprotein antitumor antibiotic, designated kedarcidin. Taxonomic studies demonstrated that strain L585-6 is an unidentified and unknown actinomycete. Kedarcidin shows potent antitumor activity against implanted P388 leukemia (3.3 micrograms/ml/kg) and B16 melanoma (2 micrograms/kg) in mice. Kedarcidin also shows potent antimicrobial activity against Gram-positive bacteria but no activity against Gram-negative bacteria.     

Cytotoxicity of cortisone-resistant lymphocytes from mice treated with a group A streptococcus or Freund's complete adjuvant against tumor cells

The cortisone-resistant lymphocytes (CR lymphocytes) of mice treated with a group A streptococcus, Su strain, or Freund's complete adjuvant (FCA) were examined for their cytotoxicity on Ehrlich carcinoma cells and sarcoma-180 cells. Female mice of the ddY strain, 7-8 weeks of age, were injected subcutaneously with streptococci or FCA in emulsion, and they were killed 14 days later. To obtain CR lymphocytes, mice treated with and without agents were injected intraperitoneally with hydrocortisone acetate (125 mg/kg) 2 days before killing. Tumor cells and CR lymphocytes from thymus, spleen or mesenteric lymph node were suspended in Hanks balanced salt solution supplemented with 2% bovine albumin. The cytotoxicity of CR lymphocytes on tumor cells was examined by the Winn test: Tumor growth was observed in mice inoculated s.c. with the mixture of tumor cells (T) and CR lymphocytes (L) at a T/L ratio of 1/10 (10(6) tumor cells/mouse). The mesenteric and thymic CR lymphocytes of mice treated with streptococci or FCA were more effective than the corresponding lymphocytes of untreated mice in suppressing the tumor growth in animals given the cell mixture. This suggests that the treatment of mice with streptococci or FCA results in an enhancement in the cytotoxicity of mesenteric and thymic CR lymphocytes against the tumor cells.     

In vitro activity of eugenol, an active component from Ocimum sanctum, against multiresistant and susceptible strains of Neisseria gonorrhoeae

In view of the widespread emergence of resistant isolates, an attempt was made to isolate and characterise the component(s) of Ocimum sanctum with activity against Neisseria gonorrhoeae. Bioassay-guided purification of the hexane extract of leaves of O. sanctum was carried out, which yielded H12c as the active compound. H12c was characterised and was determined to be eugenol, with a minimum inhibitory concentration of 85-256 mg/L. The antigonorrhoeal efficacy of H12c was better against multiresistant strains. The 50% lethal dose (LD50) of H12c was found to be 2g/kg body weight in rats. In view of its efficacy and lower toxicity, eugenol may be a potentially suitable molecule to be developed clinically in response to emerging resistant isolates of N. gonorrhoeae.     

款冬花多糖对荷瘤小鼠的抑瘤率及对白血病小鼠生存期的影响

目的研究款冬花多糖对荷瘤小鼠的抑瘤率和对白血病小鼠生存期的影响,并探讨其抗肿瘤机制。方法以小鼠肉瘤(S180)、小鼠肝癌(H22)瘤株及小鼠网状细胞白血病(L615)瘤株造模,设正常对照组、阳性药物组、款冬花高剂量组和款冬花低剂量组4组,考察款冬花多糖对各组小鼠抑瘤率和对白血病生存期的影响。结果款冬花多糖对S180、H22小鼠的抑瘤率分别为55.76%、45.61%;对L615白血病小鼠生命延长率>55.76%。结论款冬花多糖是抗肿瘤、抗白血病的有效药物,可干扰肿瘤细胞的有丝分裂过程和提高机体免疫力。     

Microcapsules formulated in the enteric coating copolymer Eudragit L100 as delivery systems for oral vaccination against infections by gastrointestinal nematode parasites

Microcapsules using the copolymer of methacrylic acid (Eudragit L100) were formulated for oral delivery of vaccines against the enteral/parenteral nematode parasite Trichinella spiralis. Antigenic preparations from first stage larvae (L1) of T. spiralis were microencapsulated in Eudragit L100. The microcapsules prepared by the spray drying method were resistant to acid pH, although the antigen was rapidly released under neutral and basic environmental conditions. The native protein conformation and biological activity was preserved in the microcapsules, as assessed by SDS-PAGE and ELISA. When administered to NIH mice, the antigen loaded microcapsules protected against infection by T. spiralis at both the intestinal and muscular levels, the worm burden diminishing by 45.58 and 53.33%, respectively. Furthermore, following administration of the microparticles an increase of the serum IgG1 response, a marker for the Th2 type response, was evident. These results indicate that microcapsules formulated with anionic biocompatible polymers such as Eudragit may be useful for oral vaccination against nematode infections.     

Production of monoclonal antibodies capable of neutralizing dermonecrotic activity of Loxosceles intermedia spider venom and their use in a specific immunometric assay.

We have produced 13 mAbs for Loxosceles intermedia crude venom. Twelve were reactive against proteins of 32-35 kDa and one of these Li mAb(7) showed high neutralizing potency for the dermonecrotic activity of L. intermedia venom. This Li mAb(7) showed no cross-reactivity, with Loxosceles laeta (Brazil), L. laeta (Perú) and Loxosceles gaucho venoms. The mAbs were produced by immunization with the crude venom and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia whole venom or dermonecrotic fraction (DNF) as antigens coated onto microtitre plates. A sensitive two-site immunometric assay was designed and shown to be useful for identifying and quantifying DNF from L. intermedia in biological samples. The Li mAb(7) coated onto microtitre plates and hyperimmune horse anti-L. intermedia IgGs prepared by immunoaffinity chromatography and conjugated to horseradish peroxidase were used to set up a sandwich-type ELISA. Measurable absorbance signals were obtained with 0.2 ng of L. intermedia crude venom per assay.     

Synthesis and in vivo antitumor activity of 2-amino-9H-purine-6-sulfenamide, -sulfinamide, and -sulfonamide and related purine ribonucleosides

A number of 6-sulfenamide, 6-sulfinamide, and 6-sulfonamide derivatives of 2-aminopurine and certain related purine ribonucleosides have been synthesized and evaluated for antileukemic activity in mice. Amination of 6-mercaptopurine ribonucleoside (7a) and 6-thioguanosine (7b) with chloramine solution gave 9-beta-D-ribofuranosylpurine-6-sulfenamide (8a) and 2-amino-9-beta-D-ribofuranosylpurine-6-sulfenamide (sulfenosine, 8b), respectively. Selective oxidation of 8a and 8b with 3-chloroperoxybenzoic acid (MCPBA) gave (R,S)-9-beta-D-ribofuranosylpurine-6-sulfinamide (9a) and (R,S)-2-amino-9-beta-D-ribofuranosylpurine-6-sulfinamide (sulfinosine, 9b), respectively. However, oxidation of 8a and 8b with excess of MCPBA gave 9-beta-D-ribofuranosylpurine-6-sulfonamide (10a) and 2-amino-9-beta-D-ribofuranosylpurine-6-sulfonamide (sulfonosine, 10b), respectively. Similarly, amination of 5'-deoxy-6-thioguanosine (7c) afforded the 6-sulfenamide derivative (8c), which on controlled oxidation gave (R,S)-2-amino-9-(5-deoxy-beta-D-ribofuranosyl)purine-6-sulfinamide (9c) and the corresponding 6-sulfonamide derivative (10c). Treatment of 6-thioguanine (12) with aqueous chloramine solution gave 2-amino-9H-purine-6-sulfenamide (13). Oxidation of 13 with 1 molar equiv of MCPBA afforded (R,S)-2-amino-9H-purine-6-sulfinamide (14), whereas the use of 4 molar equiv of MCPBA furnished 2-amino-9H-purine-6-sulfonamide (15). The resolution of R and S diastereomers of sulfinosine (9b) was accomplished by HPLC techniques. The structures of (R)-9b and 10b were assigned by single-crystal X-ray diffraction studies. (R)-9b exists in the crystal structure in four crystallographically independent conformations. Of the 18 compounds evaluated, 13 exhibited very significant anti-L1210 activity in mice. Sulfenosine (8b) at 22 mg/kg per day X 1 showed a T/C of 170, whereas sulfinosine (9b) at 173 mg/kg per day X 1 showed a T/C of 167 against L1210 leukemia. The 5'-deoxy analogue of sulfinosine (9c) at 104 mg/kg per day also showed a T/C of 172. A single treatment with 8b, 9b, and 9c reduced body burdens of viable L1210 cells by more than 99.8%.     

Microcapsules formulated in the enteric coating copolymer Eudragit L100 as delivery systems for oral vaccination against infections by gastrointestinal nematode parasites

Microcapsules using the copolymer of methacrylic acid (Eudragit L100) were formulated for oral delivery of vaccines against the enteral/parenteral nematode parasite Trichinella spiralis. Antigenic preparations from first stage larvae (L1) of T. spiralis were microencapsulated in Eudragit L100. The microcapsules prepared by the spray drying method were resistant to acid pH, although the antigen was rapidly released under neutral and basic environmental conditions. The native protein conformation and biological activity was preserved in the microcapsules, as assessed by SDS-PAGE and ELISA. When administered to NIH mice, the antigen loaded microcapsules protected against infection by T. spiralis at both the intestinal and muscular levels, the worm burden diminishing by 45.58 and 53.33%, respectively. Furthermore, following administration of the microparticles an increase of the serum IgG1 response, a marker for the Th2 type response, was evident. These results indicate that microcapsules formulated with anionic biocompatible polymers such as Eudragit may be useful for oral vaccination against nematode infections.     

Vaccination with liposomal leishmanial antigens adjuvanted with monophosphoryl lipid-trehalose dicorynomycolate (MPL-TDM) confers long-term protection against visceral leishmaniasis through a human administrable route

The development of a long-term protective subunit vaccine against visceral leishmaniasis depends on antigens and adjuvants that can induce an appropriate immune response. The immunization of leishmanial antigens alone shows limited efficacy in the absence of an appropriate adjuvant. Earlier we demonstrated sustained protection against Leishmania donovani with leishmanial antigens entrapped in cationic liposomes through an intraperitoneal route. However, this route is not applicable for human administration. Herein, we therefore evaluated the immune response and protection induced by liposomal soluble leishmanial antigen (SLA) formulated with monophosphoryl lipid-trehalose dicorynomycolate (MPL-TDM) through a subcutaneous route. Subcutaneous immunization of BALB/c mice with SLA entrapped in liposomes or with MPL-TDM elicited partial protection against experimental visceral leishmaniasis. In contrast, liposomal SLA adjuvanted with MPL-TDM induced significantly higher levels of protection in liver and spleen in BALB/c mice challenged 10 days post-vaccination. Protection conferred by this formulation was sustained up to 12 weeks of immunization, and infection was controlled for at least 4 months of the challenge, similar to liposomal SLA immunization administered intraperitoneally. An analysis of cellular immune responses of liposomal SLA + MPL-TDM immunized mice demonstrated the induction of IFN-γ and IgG2a antibody production not only 10 days or 12 weeks post-vaccination but also 4 months after the challenge infection and a down regulation of IL-4 production after infection. Moreover, long-term immunity elicited by this formulation was associated with IFN-γ production also by CD8? T cells. Taken together, our results suggest that liposomal SLA + MPL-TDM represent a good vaccine formulation for the induction of durable protection against L. donovani through a human administrable route.     

Antimutagenic effect of essential oil of sage (Salvia officinalis L.) and its monoterpenes against UV-induced mutations in Escherichia coli and Saccharomyces cerevisiae.

Mutagenic and antimutagenic potential of essential oil (EO) of cultivated sage (S. officinalis L.) and its monoterpenes: thujone, 1,8-cineole, camphor and limonene against UVC-induced mutations was studied with Salmonella/microsome, E. coli WP2, E. coli K12 [Simi?, D., Vukovi?-Gaci?, B., Knezevi?-Vukcevi?, J., 1998. Detection of natural bioantimutagens and their mechanisms of action with bacterial assay-system. Mutat. Res. 402, 51-57] and S. cerevisiae D7 reversion assays. The toxicity of EO differed, depending on the strain used. The most sensitive were permeable strains TA100, TA102, E. coli K12 IB112 and non-permeable WP2. Mutagenic potential of EO and monoterpenes was not detected, with or without S9. EO reduced the number of UV-induced revertants in a concentration-dependent manner, reaching 50-70% of inhibition at the maximum non-toxic concentrations: 3 microl/plate (TA102), 5 microl/plate (WP2), 7.5 microl/plate (IB112), 30 microl/plate (E. coli K12 SY252) and 60 microl/plate (D7). The metabolic activation had no effect on antimutagenic potential of EO. Similar toxicity of monoterpenes was observed in TA100, E. coli SY252 and D7, with the exception of limonene (less toxic to D7). Reduction of UV-induced revertants by non-toxic concentrations of monoterpenes, tested with SY252 and D7, reached 40-50% at 15-20 microl/plate of thujone, 10 microl/plate of cineole and 1-10 microg/plate of camphor. Limonene showed antimutagenic effect only in D7. Our data recommend sage monoterpenes for further chemoprevention studies.