Fed-Batch Production of Tetanus Toxin by Clostridium Tetani

This study deals with the effects of the initial nitrogen source (NZ Case TT) level and the protocol of glucose addition during the fed-batch production of tetanus toxin by Clostridium tetani. An increase in the initial concentration of NZ Case TT (NZ(0)) accelerated cell growth, increased the consumption of the nitrogen source as well as the final yield of tetanus toxin, which achieved the highest values (50-60 L(f)/mL) for NZ(0) > 50 g/L. The addition of glucose at fixed times (16, 56, and 88 h) ensured a toxin yield (similar to 60 L(f)/mL) about 33% higher than those of fed-batch runs with addition at fixed concentration (similar to 45 L(f)/mL) and about 300% higher than those obtained in reference batch runs nowadays used at industrial,scale. The results of this work promise to substantially improve the present production of tetanus toxin and may be adopted for human vaccine production after detoxification and purification. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 88-92, 2010

Effect of different prebiotics on the fermentation kinetics, probiotic survival and fatty acids profiles in nonfat symbiotic fermented milk

The simultaneous effects of different binary co-cultures of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus rhamnosus and Bifidobacterium lactis with Streptococcus thermophilus and of different prebiotics on the production of fermented milk were investigated in this paper. In particular, we determined and compared the kinetics of acidification of milk either as such or supplemented with 4% (w/w) maltodextrin, oligofructose and polydextrose, as well as the probiotic survival, chemical composition (pH, lactose, lactic acid and protein contents), fatty acids profile and conjugate linoleic acid (CIA) content of fermented milk after storage at 4 degrees C for 24 h. Fermented milk quality was strongly influenced both by the co-culture composition and the selected prebiotic. Depending on the co-culture, prebiotic addition to milk influenced to different extent kinetic acidification parameters. All probiotic counts were stimulated by oligofructose and polydextrose, and among these B. lactis always exhibited the highest counts in all supplemented milk samples. Polydextrose addition led to the highest post-acidification. Although the contents of the main fatty acids were only barely influenced. the highest amounts of conjugated linoleic acid (38% higher than in the control) were found in milk fermented by S. thermophilus-L. acidophilus co-culture and supplemented with maltodextrin. (C) 2008 Elsevier B.V. All rights reserved.

Liquid-liquid extraction of proteases from fermented broth by PEG/citrate aqueous two-phase system

This work deals with the use of an aqueous two-phase system (ATPS) of PEG/citrate to remove proteases from a Clostridium perfringens fermentation broth. To plan the experimental tests and evaluate the corresponding results, three successive experimental designs were employed, for which the PEG molar mass (M-PEG) and concentration (C-PEG), the citrate concentration (C-C) and the pH were selected as independent variables, while the purification factor (PF), the partition coefficient (K), the activity yield (Y) and the selectivity (S) were selected as responses. PF of proteases in the top phase was shown to increase with increasing MPEG and decreasing Cc, whereas a completely opposite trend was observed for K. On the other hand, Y was favored by simultaneous decreases in both these variables, while S decreased with increasing Cc. Therefore, selecting a simultaneous increase in PF and Y as the most desirable result, the best performance of the system was obtained using M-PEG = 10-000 g/mol C-PEG = 22% (w/w) and C-c = 8.0% (w/w) at pH 8.5. Under these conditions, the activity yield was very high (131 %) but the purification factor (4.2) and the selectivity (4.3) were lower than those ensured by more selective purification methods. According to these results, the ATPS seems to be an interesting alternative primary concentration/decontamination step for vaccine preparation from C. perfringens fermented broth. (C) 2007 Elsevier B.V. All rights reserved.

Human Airway Epithelial Cell Responses to Neisseria lactamica and Purified Porin via Toll-Like Receptor 2-Dependent Signaling

The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin`s surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.

One-step biomimetic conversion of a furanoheliangolide into an eremantholide using Stryker`s reagent

The conversion of a furanoheliangolide structure (15-deoxygoyazensolide) into an eremantholide one (eremantholide C) was achieved by tandem hydride conjugate addition-intramolecular carbanion addition using Stryker`s reagent. (c) 2008 Elsevier Ltd. All rights reserved.

Leishmania (Viannia) braziliensis transfectants overexpressing the miniexon gene lose virulence in vivo

The miniexon gene has a central role in the processing of polycistronic pre-mRNA of kinetoplastids. It is added to the 5` extremity of each mRNA, supplying the 5`-capped structure to the molecule. Previous studies in Leishmania (Leishmania) major showed that the overexpression of the miniexon array attenuates the Virulence of the parasite in in vivo assays. The results presented here extend those findings to Vionnia subgenus. Leishmania (Vionnia) braziliensis was transfected with a cosmid harboring a tandem array of one hundred miniexon gene copies and then characterized by Northern blot analysis. The overexpression of the exogenous gene was confirmed and its effect on the virulence of L (V.) braziliensis was investigated in hamsters. In BALB/c mice we could not detect parasites during the course of 15 weeks of infection. In addition, hamsters infected with transfectants overexpressing the miniexon gene exhibited only a minor footpad swelling of late onset and failed to develop progressive lesion, these attenuated parasites could be recovered from the inoculation site 1 year after infection. The persistence of parasites in the host indicates that a stable line overexpressing the miniexon may be tested as live vaccine against leishmaniasis. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

DNAhsp65 vaccination induces protection in mice against Paracoccidioides brasiliensis infection

Heat-shock proteins are Molecules with extensive data showing their potential as immunomodulators of different types of diseases, The gene of HSP65 from Mycobacterium leprae has shown prophylactic and immunotherapeutic effects against a broad arrays of experimental models including tuberculosis, leishmaniasis, arthritis and diabetes. With this in mind, we tested the DNAhsp65 vaccine using an experimental model of Paraccocidiodomycosis, an important endemic mycosis in Latin America. The intramuscular immunization with DNAhsp65 induced, in BALB/c mice, an increase of Th1-levels cytokines and a reduction of fungal burdens resulted in a marked reduction of collagen and lung remodeling. DNAhsp65 may be an attractive candidate for prevention, therapy and as an adjuvant for mycosis treatment. (C) 2008 Elsevier Ltd. All rights reserved.

Strongyloides venezuelensis: The antigenic identity of eight strains for the immunodiagnosis of human strongyloidiasis

The antigens of eight strains of Strongyloides venezuelensis were identified by means of the indirect immunofluorescence antibody (IFAT), enzyme-linked immunosorbent assay (ELISA) and immunoblot (IB) tests. Infective larvae (L3) from these strains were obtained from Rattus norvegicus feces cultures. For IFAT, sections of U were used while the ELISA and IB, tests were conducted with alkaline extract. Ninety serum samples were tested: 30 from patients with S. stercoralis, 30 from patients with other parasitic diseases, and 30 from healthy subjects (free of parasites). Average sensitivity and specificity among all eight strains, both for IFAT and ELISA, were, respectively, 93% and 100%. In the IB, anti-S. stercoralis IgG recognized a single antigenic fraction with 45 kDa. Serum samples from patients with S. stercoralis revealed antigens from different strains of S. venezuelensis, indicating antigenic identity for possible use in the synthesis of recombinant antigen that could be useful in immunodiagnosis and vaccine against this parasite. (C) 2008 Elsevier Inc. All rights reserved.

Evaluation of Environmental Mycobacteria Contamination in a Specific Pathogen Free Animal Facility from a Tropical Country

P>With the evidence showing the protection variability of bacille Calmette-Guerin, new potential vaccines for tuberculosis have been tested around the world. One of the general concerns in tuberculosis vaccine development is the possibility of priming the host immune system with prior exposure to environmental mycobacteria antigens, which can change the efficacy of subsequent vaccination. As there is a great homology between the species from Mycobacterium genera, the previous contact of experimental animals with environmental mycobacteria could sensitize the mice and, in this way, could influence subsequent vaccine research. The aim of our study was to investigate critical points in an animal facility to search for environmental mycobacteria that eventually could be in direct or indirect contact with the experimental animals. Samples were collected from surfaces of walls, floor, animal cages and shelves and analysed using the Ogawa-Kudoh decontamination method. Samples of drinking water, food and sawdust were collected for analysis by the NALC/NaOH decontamination method. Also, the samples were cultivated directly in broth medium, without any method for decontamination. After decontamination methods, we observed bacterial colony growth in 4.31% of the total of samples analysed. These samples were stained with Ziehl-Neelsen and we did not detect any acid-fast bacilli, suggesting that the animal facility analysed is free from contamination by environmental mycobacteria and is not a source of mycobacterial antigens. Furthermore, our study showed a new paradigm in tuberculosis vaccine development: concern about the animal facility environment in terms of immune system priming of experimental animals by nascent bacterial contaminants.

Deconstructing Tick Saliva NON-PROTEIN MOLECULES WITH POTENT IMMUNOMODULATORY PROPERTIES

Dendritic cells (DCs) are powerful initiators of innate and adaptive immune responses. Ticks are blood-sucking ectoparasite arthropods that suppress host immunity by secreting immunomodulatory molecules in their saliva. Here, compounds present in Rhipicephalus sanguineus tick saliva with immunomodulatory effects on DC differentiation, cytokine production, and costimulatory molecule expression were identified. R. sanguineus tick saliva inhibited IL-12p40 and TNF-alpha while potentiating IL-10 cytokine production by bone marrow-derived DCs stimulated by Toll-like receptor-2, -4, and -9 agonists. To identify the molecules responsible for these effects, we fractionated the saliva through microcon filtration and reversed-phase HPLC and tested each fraction for DC maturation. Fractions with proven effects were analyzed by micro-HPLC tandem mass spectrometry or competition ELISA. Thus, we identified for the first time in tick saliva the purine nucleoside adenosine (concentration of similar to 110pmol/mu l) as a potent anti-inflammatory salivary inhibitor of DC cytokine production. We also found prostaglandin E(2) (PGE(2) similar to 100 nM) with comparable effects in modulating cytokine production by DCs. Both Ado and PGE(2) inhibited cytokine production by inducing cAMP-PKA signaling in DCs. Additionally, both Ado and PGE(2) were able to inhibit expression of CD40 in mature DCs. Finally, flow cytometry analysis revealed that PGE(2), but not Ado, is the differentiation inhibitor of bone marrow-derived DCs. The presence of non-protein molecules adenosine and PGE(2) in tick saliva indicates an important evolutionary mechanism used by ticks to subvert host immune cells and allow them to successfully complete their blood meal and life cycle.

The prospects for immunotherapy in smoking cessation

Context Smoking is a major preventable cause of death and disability that is maintained by dependence on nicotine. Smoking cessation reduces mortality and morbidity. Although existing pharmacological aids to smoking cessation and relapse prevention (nicotine replacement therapy and bupropion) improve on unassisted quitting and behavioural methods, they are only modestly effective. More effective pharmacological methods are required that improve compliance, reduce side-effects, and can be used in combination with existing cessation methods. Starting point A nicotine vaccine is a promising immunotherapeutic approach to smoking cessation and relapse prevention. Such a vaccine would induce the immune system to form specific antibodies to nicotine to prevent it from crossing the blood-brain barrier to act on receptor sites in the central nervous system. Recent studies in rats provide proof of principle by showing that nicotine-specific antibodies can prevent the reinstatement of nicotine self-administration (N Lindblom et al, Respiration 2002; 69: 254–60) and block dopamine release in the shell of the nucleus accumbens (Sde Villiers et al, Respiration 2002; 69: 247–53). A phase 1 trial of a human cocaine vaccine has also recently been successfully completed (T Kosten et al, Vaccine 2002; 20: 1196–204). A safe and effective human nicotine vaccine would potentially have fewer side-effects and better compliance than existing smoking-cessation pharmacotherapies. It could also be used in combination with some of them (eg, bupropion). Where next? The most promising clinical application of a human nicotine vaccine is likely to be in relapse prevention in abstinent smokers. A vaccine may also have a role in preparing smokers to quit. Clinical trials of safety and efficacy in human smokers and ex-smokers are warranted. If a nicotine vaccine proves to be safe and effective, the health-care system will need to ensure that it is registered for clinical use and that the poorer members of the community (among whom smoking prevalence is now highest in developed countries) have access to the vaccine. The community will need to be appropriately informed about the role of a nicotine vaccine to ensure that it is not prematurely used for preventive purposes in children and adolescents.

Critical sets for families of Latin squares

In this paper I give details of new constructions for critical sets in latin squares. These latin squares, of order n, are such that they can be partitioned into four subsquares each of which is based on the addition table of the integers module n/2, an isotopism of this or a conjugate.

Mucosal immunisation with papillomavirus virus-like particles elicits systemic and mucosal immunity in mice

It has been shown previously that recombinant virus-like particles (VLPs) of papillomavirus can induce VLP-specific humoral and cellular immune responses following parenteral administration. To test whether mucosal administration of bovine papillomavirus type 1 (BPV1) VLPs could produce mucosal as well as systemic immune responses to VLPs, 50 mu g chimeric BPV1 VLPs containing an HPV16 E7 CTL epitope (BPVL1/E7 VLP) was administered intranasally to mice. After two immunisations, L1-specific serum IgG and IgA were observed. L1-specific IgG and IgA were also found in respiratory and vaginal secretions. Both serum and mucosal antibody inhibited papillomavirus VLP-induced agglutination of RBC, indicating that the antibody induced by mucosal immunisation may recognize conformational determinants associated with virus neutralisation. For comparison, VLPs were given intramuscularly, and systemic and mucosal immune responses were generally comparable following systemic or mucosal delivery. However, intranasal administration of VLP induced significantly higher local IgA response in lung, suggesting that mucosally delivered HPV VLP may be more effective for mediating local mucosal immune responses. Intranasal immunisation with HPV6b L1 VLP produced VLP-specific T proliferative responses in splenocytes, and immunisation with BPVL1 VLP containing an HPV16 E7 CTL epitope induced E7-specific CTL responses. We conclude that immunisation with papillomavirus VLPs via mucosal and intramuscular routes, without adjuvant, can elicit specific antibody at mucosal surfaces and also systemic VLP epitope specific T cell responses. These findings suggest that mucosally delivered VLPs may offer an alternative HPV VLP vaccine strategy for inducing protective humoral immunity to anogenital HPV infection, together with cell-mediated immune responses to eliminate any cells which become infected. (C) 1998 Academic Press.

Post translational modifications of recombinant human Papillomavirus type 6b major capsid protein

We have determined the post-translational modifications of the major capsid protein, L1 of human papillomavirus (HPV) type 6b. Since this virus cannot be cultured in the laboratory to obtain sufficient material for a study, a recombinant L1 protein produced in a vaccinia virus expression system was used in this investigation. Our results show that this protein is phosphorylated at serine residues and is also glycosylated. No myristoylation or palmitoylation was detected. The fraction of L1 protein incorporated into virus-like particles was not glycosylated. Since recombinant L1 protein is a potential human vaccine candidate, knowledge of the post-translation modifications of this protein may prove useful for the design of anti-HPV vaccines. (C) 1999 Elsevier Science B.V. All rights reserved.

Immune responses induced by BCG recombinant for human papillomavirus L1 and E7 proteins

Recombinant bacille Calmette-Guerin (BCG) based vaccine delivery systems could potentially share the safety and effectiveness of BCG. We therefore prepared recombinant BCG vaccines which expressed the L1 late protein of the human papillomavirus (HPV) 6b or the E7 early protein of the HPV 16. The two recombinants were evaluated as immunogens in C57BL/6J and BALB/c mice, and compared with a conventional protein/adjuvant system using E7 or L1 mixed with Quil-A adjuvant. rBCG6bL1 and rBCG16E7 primed specific immune responses, represented by DTH, T-proliferation and antibody, and rBCG16E7 induced cytotoxic immune response to E7 protein. The magnitude of the observed responses were less than those elicited by protein/adjuvant vaccine. As recombinant BCG vaccines expressing HPV6bL1 or HPV16E7 persist at low levels in the immunised host, they may be beneficial to prime or retain memory responses to antigens, but are unlikely to be useful as a single component vaccine strategy. (C) 2000 Elsevier science Ltd. All rights reserved.