Development of a Listeria monocytogenes based vaccine against prostate cancer

Prostate specific antigen (PSA) is a likely immunotherapeutic target antigen for prostate cancer, the second leading cause of cancer-related death in American men. Previously, we demonstrated that attenuated strains of Listeria monocytogenes (Lm) can be used as effective vaccine vectors for delivery of tumor antigens causing regression of established tumors accompanied by strong immune responses toward these antigens in murine models of cancer. In the present study, we have developed and characterized a recombinant live attenuated L. monocytogenes/PSA (Lm–LLO–PSA) vaccine with potential use for the treatment of pCa. Human PSA gene was cloned into and expressed by an attenuated Lm strain. This recombinant bacterial vaccine, Lm–LLO–PSA was tested for stability, virulence, immunogenicity and anti-tumor effects in a murine model for pCa. Immunization with Lm–LLO–PSA was shown to lower the number of tumor infiltrating T regulatory cells and cause complete regression of over 80% of tumors formed by an implanted genetically modified mouse prostate adenocarcinoma cell line, which expressed human PSA. Lm–LLO–PSA was immunogenic in C57BL/6 mice and splenocytes from mice immunized with Lm–LLO–PSA showed significantly higher number of IFN-γ secreting cells over that of the naïve animals in response to a PSA H2Db-specific peptide, as measured by both, ELISpot and intracellular cytokine staining. In addition, using a CTL assay we show that the T cells specific for PSA were able to recognize and lyse PSA-peptide pulsed target cells in vitro. In a comparison study with two other PSA-based vaccines (a pDNA and a vaccinia vaccine), Lm–LLO–PSA was shown to be more efficacious in regressing established tumors when used in a homologues prime/boost regimen. Together, these results indicate that Lm–LLO–PSA is a potential candidate for pCa immunotherapy and should be further developed.     

Purification and Characterization of Hieronymain III. Comparison with Other Proteases Previously Isolated from Bromelia hieronymi Mez

A new proteolytic enzyme, named hieronymain III, has been purified by ion-exchange chromatography from unripe fruits of Bromelia hieronymi Mez. The new peptidase belongs to the cysteine catalytic type, as well as hieronymain I and II, the other two peptidases previously isolated from this species. Hieronymain III showed optimum alkaline pH range (8.6–9.3) and the molecular mass (MALDI-TOF) was 23713 Da. The N-terminal sequence (AVPQSIDWRRYGAVTTSRNQG) exhibited a higher percentage identity with hieronymain II (93%) than with hieronymain I (71%). The three peptidases showed notable differences on synthetic substrates degradation: whereas hieronymain III was the only one able to hidrolyze Z-Arg-Arg-p-nitroanilide, hieronymain I and II could degrade Z-Phe-Arg-p-nitroanilide; on the other hand, PFLNA was only split by hieronymain I. Finally, the three proteases showed different preferences on N-α-CBZ-p-nitrophenyl aminoacid ester substrates. From a biotechnological point of view, cleavage specificity differences are significant enough to use these enzymes as potential tools in that area.     

Hypothalamic angiotensinergic-noradrenergic systems interaction in fructose induced hypertension

OBJECTIVE: Several studies suggest the importance of the interaction between the renin angiotensin and sympathetic nervous systems in blood pressure control, especially in clinical situations such as the metabolic syndrome. Previously, we have demonstrated changes in noradrenergic hypothalamic control of blood pressure in an animal model of insulin resistance and hypertension. The aim of the present study was to evaluate the effects of the interaction between the noradrenergic and angiotensinergic systems on hypothalamic blood pressure regulation in fructose hypertensive rats. METHODS: In control (C) and fructose-fed hypertensive (F) rats, we studied: 1) the effects of hypothalamic perfusion of irbesartan (AT(1) angiotensin receptor antagonist, 50 and 500 microg ml(-1)) and metoprolol (beta(1) adrenergic receptor antagonist, 10 and 100 microg ml(-1)) on blood pressure, heart rate and noradrenaline intrahypothalamic levels, by means of the microdialysis technique; and 2) the effects of intrahypothalamic microinjection of angiotensin II alone or after metoprolol pre-administration, on blood pressure and heart rate. RESULTS: Meanwhile irbesartan perfusion did not modify neither mean arterial pressure (MAP) nor heart rate or noradrenaline hypothalamic levels in the C group, its highest dose diminished MAP (DeltaMAP: F: - 16.3+/-1 mm Hg, p<0.05) and noradrenaline levels (% of basal levels: 58+/-7%, p<0.05) in the F group, without affecting heart rate. Intrahypothalamic perfusion of metoprolol diminished MAP only in the F group (DeltaMAP: F: -12.1+/-1.1 mm Hg, p<0.05), but did not modify heart rate in both groups. On the other hand, it diminished noradrenaline hypothalamic levels in C (% of basal levels: 53+/-6%, p<0.05) but not in the F group. The pressor response to angiotensin II microinjection was increased in F rats (DeltaMAP: F: 13.3+/-1.5 mm Hg vs. C: 6.9+/-1.8 mm Hg; p<0.05). Previous administration of metoprolol markedly abolished this increment. CONCLUSIONS: Our results suggest the existence of an increase in AT(1) and beta(1) adrenergic receptors tone in the hypothalamus of F rats, which could be related to the increase in blood pressure present in this experimental model. On the other hand, considering that the enhanced pressor response to angiotensin II intrahypothalamic injection in F rats was abolished by previous administration of a beta(1) adrenergic receptor antagonist, these results would indicate that beta(1) adrenergic receptors activation participates in the pressor response to angiotensin II in this experimental model of insulin resistance and hypertension.     

Cry1A(b)16 toxin from Bacillus thuringiensis: Theoretical refinement of three‐dimensional structure and prediction of peptides as molecular markers for detection of genetically modified organisms

Transgenic maize produced by the insertion of the Cry transgene into its genome became the second most cultivated crop worldwide. Cry gene from Bacillus thuringiensis kurstaki expresses protein derivatives of crystalline endotoxins which confer insect resistance onto the maize crop. Mandatory labeling of processed food containing or made by genetically modified organisms is in force in many countries, so, it is very urgent to develop fast and practical methods for GMO identification, for example, biosensors. In the absence of an available empirical structure of Cry1A(b)16 protein, a theoretical model was effectively generated, in this work, by homology modeling and molecular dynamics simulations based on two available homologous protein structures. Molecular dynamics simulations were carried out to refine the selected model, and an analysis of its global structure was performed. The refined models of Cry1A(b)16 showed a standard fold and structural characteristics similar to those seen in Bacillus thuringiensis Cry1A(a) insecticidal toxin and Bacillus thuringiensis serovar kurstaki Cry1A(c) toxin. After in silico analysis of Cry1A(b)16, two immunoreactive candidate peptides were selected and specific polyclonal antibodies were produced resulting in antibody–peptide interaction. Biosensing devices are expected to be developed for detection of the Cry1A(b) protein as a marker of transgenic maize in food. Proteins 2017; 85:1248–1257. © 2017 Wiley Periodicals, Inc.     

Effect of salinity on the quantity and quality of carotenoids accumulated by Dunaliella salina (strain CONC-007) and Dunaliella bardawil (strain ATCC 30861) Chlorophyta

Dunaliella salina and D. bardawil are well-known microalgae accumulating high levels of beta-carotene under growth-limiting conditions. In both taxa, this pigment is primarily composed of the isomers 9-cis and all-trans. The 9-cis beta-carotene occurs only in natural sources and is the most attractive from a commercial point of view. The conditions that enhance the preferred accumulation of 9-cis beta-carotene in D. salina are controversial and they have not been well established yet. This study examined the effect of salinity on the quantity and quality of total carotenoids and beta-carotene isomers accumulated by D. salina (strain CONC-007) and D. bardawil (strain ATCC 30861) grown in two media with different nutritional compositions (PES and ART) and at salt concentrations of 1M, 2M and 3M NaCl. Total carotenoids were determined by spectrophotometry and beta-carotene isomers, by HPLC. The highest carotenoid contents per cell were obtained at 2M NaCl in both taxa. In both media, an increase of the 9-cis/all-trans beta-carotene ratio was observed in D. bardawil when the salt concentration increased, with a maximum value of 2.6 (in ART medium at 3M NaCl). In D. salina this ratio did not exhibit the same pattern, and the salt concentrations for maximal ratios were different in both media. The highest ratio obtained for this strain was 4.3 (in ART medium at 2M NaCl).     

Expression of Babesia bovis rhoptry-associated protein 1 (RAP1) in Brucella abortus S19

Brucella abortus strain 19 (live vaccine) induces a strong humoral and cellular immune response and therefore, it is an attractive vector for the delivery of heterologous antigens. The objective of the present study was to express the rhoptry-associated protein (RAP1) of Babesia bovis in B. abortus S19, as a model for heterologous expression of immunostimulatory antigens from veterinary pathogens. A plasmid for the expression of recombinant proteins fused to the aminoterminal of the outer membrane lipoprotein OMP19 was created, pursuing the objective of increasing the immunogenicity of the recombinant antigen being expressed by its association to a lipid moiety. Recombinant strains of B. abortus S19 expressing RAP1 as a fusion protein either with the first amino acids of beta-galactosidase (S19pBB-RAP1) or B. abortus OMP19 (S19pBB19-RAP1) were generated. Plasmid stability and the immunogenicity of the heterologous proteins were analyzed. Mice immunized with S19pBB-RAP1 or S19pBB19-RAP1 developed specific humoral immune response to RAP1, IgG2a being the predominant antibody isotype. Furthermore, a specific cellular immune response to recombinant RAP1 was elicited in vitro by lymphocytes from mice immunized with both strains. Therefore, we concluded that B. abortus S19 expressing RAP1 is immunostimulatory and may provide the basis for combined heterologous vaccines for babesiosis and brucellosis.     

Postembryonic development of serotonin-immunoreactive neurons in the central nervous system of the blowfly

Summary Neurons immunoreactive with antibodies to serotonin (5-HT) were mapped in the thoracico-abdominal ganglia of the blowfly, Calliphora erythrocephala, during postembryonic development. Reconstructions from serial sections of tissue processed with a preincubation PAP-method permitted a detailed analysis of the morphological changes occurring in 5-HT-immunoreactive (5-HTi) neurons.All the 5-HTi cell bodies in the thoracico-abdominal ganglia of the 3rd instar larva, except two in the metathoracic ganglion, retain their immunochemical phenotype throughout pupal development. Hence, all the adult 5-HTi neurons in these ganglia differentiate during embryonic development. The finer processes of the larval 5-HTi neurons undergo a substantial regression during the first 24 h of pupal development, and thereafter new branches form on the primary processes of the same cell bodies. The slight change in relative position of 5-HTi cell bodies and the reorganization of the neuropil into an adult pattern occur during the first half of pupal development. The neuropil mass and extent of 5-HTi processes continue to increase during the following days and appear to be fully developed two days (80% of pupal development) before hatching.On the basis of the presented data, some of the basic processes are discussed that lead to the transformation of the larval nervous system into its adult form.     

Mapping of serotonin-immunoreactive neurons in the larval nervous system of the flies Calliphora erythrocephala and Sarcophaga bullata

Summary Serotonin-immunoreactive (5-HTi) neurons were mapped in the larval central nervous system (CNS) of the dipterous flies Calliphora erythrocephala and Sarcophaga bullata. Immunocytochemistry was performed on cryostat sections, paraffin sections, and on the entire CNS (whole mounts).The CNS of larvae displays 96–98 5-HTi cell bodies. The location of the cell bodies within the segmental cerebral and ventral ganglia is consistent among individuals. The pattern of immunoreactive fibers in tracts and within neuropil regions of the CNS was resolved in detail. Some 5-HTi neurons in the CNS possess axons that run through peripheral nerves (antenno-labro-frontal nerves).The suboesophagealand thoracico-abdominal ganglia of the adult blowflies were studied for a comparison with the larval ventral ganglia. In the thoracico-abdominal ganglia of adults the same number of 5-HTi cell bodies was found as in the larvae except in the metathoracic ganglion, which in the adult contains two cell bodies less than in the larva. The immunoreactive processes within the neuropil of the adult thoracico-abdominal ganglia form more elaborate patterns than those of the larvae, but the basic organization of major fiber tracts was similar in larval and adult ganglia. Some aspects of postembryonic development are discussed in relation to the transformation of the distribution of 5-HTi neurons and their processes into the adult pattern.