Active immunization with a structurally aggregated PD-L1 antigen breaks T and B immune tolerance in non-human primates and exhibits in vivo anti-tumoral effects in immunocompetent mouse tumor models.

Despite the clinical success of the programmed death ligand 1 (PD-L1) blocking therapy in cancer treatment, only a subset of patients exhibits durable responses, therefore further exploration of other immunotherapeutic alternatives are needed. This paper reported the development of the PKPD-L1Vac vaccine, a new protein vaccine candidate that uses aluminum phosphate as an adjuvant and as an antigen the extracellular domain of human PD-L1 fused to a 47 amino-terminal portion of the LpdA protein from N. meningitides (PKPD-L1). The PKPD-L1 antigen has different physical and biological characteristics than those found in the natural molecule and in others PD-L1 vaccine candidates. The quimeric protein has a reduced binding capacity to the PD-1 and CD80 receptors to decrease their pro-tumoral activity. Besides, the distinctive feature of the PKPD-L1 polypeptide to be structurally aggregated could be desirable for its immunogenic properties. PKPD-L1Vac elicited anti-PD-L1-specific IgG antibodies and T lymphocyte-mediated immunity in mice and non-human primates. The vaccine administration demonstrated antitumor activity on CT-26 and B16-F10 primary tumor models in mice. Moreover, the immunization with PKPD-L1Vac increased the tumor-infiltrating lymphocytes and decreased the proportion of CD3+CD8+PD1+high anergic T cells in CT-26 tumor tissues, suggesting that the vaccine may remodel the tumor microenvironment. In summary, the PKPD-L1Vac vaccine exhibits very promising preclinical results and deserves to move forward to a phase I clinical trial.

Dengue virus identification by transmission electron microscopy and molecular methods in fatal dengue hemorrhagic fever.

Dengue virus is the most significant virus transmitted by arthropods worldwide and may cause a potentially fatal systemic disease named dengue hemorrhagic fever. In this work, dengue virus serotype 4 was detected in the tissues of one fatal dengue hemorrhagic fever case using electron immunomicroscopy and molecular methods. This is the first report of dengue virus polypeptides findings by electron immunomicroscopy in human samples. In addition, not-previously-documented virus-like particles visualized in spleen, hepatic, brain, and pulmonary tissues from a dengue case are discussed.

Efecto adverso en la calidad proteica de los alimentos de dietas con alto contenido de fibra dietaria / Adverse effect on the food protein quality of diets high in dietary fiber

Modern diet tends to change eating habits and there is a tendency to consume more processed foods. These changes in eating habits towards more consumption of processed food, and the recognized benefic effects of dietary fiber by consumers, tend to increase the number of "high fiber" foods in the market. Although the beneficial effects of dietary fiber on human health is widely recognized, this increased consumption of dietary fiber may also have adverse effects on digestion, absorption and utilization offood proteins. Research in the past has shown that the consumption of high dietary fiber diets have an adverse effect on certain indicators of protein quality. Therefore it becomes very important to study the physicochemical properties of the various sources of dietary fiber, as well as the presence of other factors, associated to the fibrous fraction, in their possible negative influence on the protein quality of rich dietary fiber diets.

La dieta moderna cambia los hábitos alimenticios y existe una tendencia al consumo de alimentos más procesados. El cambio de hábito alimentario hacia alimentos más procesados tiende a incrementar, algunas veces por propósitos publicitarios, aquellos alimentos procesados "altos en fibra". Si bien los efectos benéficos de la fibra dietética a la salud humana son ampliamente reconocidos, este aumento en el consumo de fibra dietética puede también tener efectos adversos en la digestión, absorción y utilización de la proteína de los alimentos. En las investigaciones revisadas se obtuvo que el consumo de dietas altas en fibra dietética tiene efecto adverso en ciertos indicadores de calidad proteica, por lo que la inclusión de fuentes proteicas con altos contenidos de fibra impone la necesidad de estudiar las características físico-químicas de la fibra dietética, así como la presencia de factores que pudieran unirse a la fracción fibrosa e influir negativamente en la calidad proteica.

Effect of cryopreservation on fusion efficiency and in vitro development into blastocysts of bovine cell lines used in somatic cell cloning.

The outcome of the process of cloning by nuclear transfer depends on multiple factors that affect its efficiency. Donor cells should be carefully selected for their use in somatic nuclear transfer, and the protocols used for keeping frozen cell banks are of cardinal importance. Here we studied the effect of two protocols for freezing donor cells on fusion rate and development into blastocysts. Our hypothesis is that freezing affects cell membranes in a way that interferes with the fusion process upon cloning but without hampering normal cell development in vitro. We found that freezing cell lines without controlling the cooling rate gives lower yields in the fusion step and in the final development into blastocysts, compared with cells frozen with a controlled cooling rate of approximately 1 degrees C/min. Transmission electron microscopy of the cells subjected to different freezing procedures showed major damage to the cells frozen with a non-controlled protocol. We conclude that freezing of donor cells for cloning is a critical step in the procedure and should be monitored carefully using a method that allows for a step-wise, controlled cooling rate.

Plan de cuidados al paciente geriátrico hospitalizado con síndrome confusional agudo / Care's plan of geriatric patient with acuse confesional syndrome at hospital

¿Sabemos cuidar a un paciente geriátrico hospitalizado con síndrome confusional agudo? ¿En qué medida podemos mejorar? En este artículo proponemos un Plan de Cuidados, que tomando como referencia teórica el Modelo de Autocuidado de Dorothea E. Orem, puede servir de guía en la práctica profesional a la enfermera que cuida pacientes ancianos hospitalizados. Partiendo de una valoración geriátrica focalizada, se identifican una serie de diagnósticos enfermeros que se asocian con mayor frecuencia a esta situación, y se proponen las intervenciones encaminadas a resolverlos. Dado que el síndrome confusional agudo es una complicación que se presenta con bastante frecuencia en los pacientes geriátricos hospitalizados, consideramos que la enfermera tiene un papel primordial, tanto en la identificación del mismo, como en su tratamiento, instaurando un plan de intervención que impida su cronificación. Por último, destacamos la importancia de incluir en el Plan de Cuidados a la familia del paciente o al cuidador principal, ya que una participación activa de estos puede contribuir de forma significativa a la recuperación del paciente (AU)

Characterization of the HCV core virus-like particles produced in the methylotrophic yeast Pichia pastoris.

Little is known about the mechanism of hepatitis C virion assembly. So the capacity of the entire Hepatitis C virus core protein (HCcAg) produced in Pichia pastoris to form particles either in its native soluble state or after detergent treatment of HCcAg associated to cell debris were studied. Size exclusion chromatography suggested that HCcAg assembled into high molecular weight structures. HCcAg was also specifically recognized by a serum from a chronic HCV carrier patient. This antigen migrated with buoyant density values similar to those obtained for native nucleocapsid particles from infected patients when analyzed using sucrose density gradient centrifugation. The analysis by electron microscopy of purified HCcAg showed aggregates resembling virus-like particles (VLPs) with an average diameter of 30 nm. These results indicated that the HCcAg obtained from P. pastoris assembled into VLPs resembling HCV nucleocapsid particles in a mature stage. Such HCcAg aggregates characterized here could be a valuable tool to elucidate the mechanisms of HCV nucleocapsid assembly.

Recombinant Opc protein from Neisseria meningitidis reconstituted into liposomes elicits opsonic antibodies following immunization.

The reconstitution of recombinant bacterial outer membrane proteins (OMPs) into their native conformations after purification has been the major problem in their use as effective vaccines. Liposomes have been shown to be an attractive approach, providing a native-like environment for these antigens. The meningococcal recombinant Opc (rOpc) protein, produced as inclusion bodies in Escherichia coli, was incorporated into phospholipid vesicles consisting of dipalmitoyl phosphatidylcholine and cholesterol. The incorporation of rOpc into the lipid bilayer was demonstrated, and the reconstitution of some native epitopes was tested using a set of monoclonal antibodies. Subcutaneous immunization of Balb/c mice with rOpc-containing vesicles resulted in the generation of a high level of specific antibodies. The elicited antibodies reacted with the native meningococcal protein and showed opsonic activity.

Plasmid DNA-recombinant Opc protein complexes for nasal DNA immunization.

The nasal mucosa may provide a simple, non-invasive route to deliver DNA encoding genes that stimulate a specific immune response. Based on this, a new approach using pCMVbeta-gal plasmid DNA complexed to the Opc meningococcal outer membrane protein was assayed for. Optimal conditions of interaction were established between recombinant Opc protein and pCMVbeta-gal plasmid DNA. Complexes were fully characterized by electrophoresis analysis, DNAse resistance assay and transmission electron microscopy. DNA-protein complexes were also evaluated in in vitro transfection experiments. After the characterisation of complexes, Balb/c mice were intranasal (i.n.) and intramuscularly (i.m.) immunized. The humoral immune response against beta-galactosidase was measured by ELISA. The proliferative response in the spleen lymph nodes was also measured. Complexes administered by i.n. route induced both systemic and mucosal antibody responses. This behavior was not observed with the naked DNA. Finally, a lymphoproliferative response specific to beta-galactosidase induced by DNA-protein complexes was also detected.

Immunogenicity of recombinant class 1 protein from Neisseria meningitidis refolded into phospholipid vesicles and detergent.

The possibility of eliciting bactericidal antibodies against a recombinant class 1 protein (P1) from Neisseria meningitidis, joined to the first 45 amino acids of the neisserial LpdA protein (PM82), was examined. P1 was produced in Escherichia coli as intracellular inclusion bodies, from which it was purified and reconstituted by (a) inclusion into phospholipid vesicles and detergent and (b) refolding in 0.1% SDS. When Balb/c mice were immunised, high titres of subtype-specific bactericidal antibodies against P1 were obtained in both cases. These results suggest that in spite of being a denaturing agent, it is possible to use SDS to reconstitute the P1 protein in a conformation that exposes the immunodominat regions.

Assembly of truncated HCV core antigen into virus-like particles in Escherichia coli.

Core protein is one of the most conserved and immunogenic of the hepatitis C virus proteins. Several pieces of experimental evidence suggest its ability for formation of virus like particles alone or in association with other viral proteins in mammalian or yeast cells with great similarity to those detected in patient sera and liver extract. In this work we report an Escherichia coli-derived truncated hepatitis C core protein that is able to aggregate. SDS-PAGE and size exclusion chromatography patterns bring to mind the aggregation of monomers of recombinant protein Co.120. The Co.120 protein migrated with buoyant density of 1.28 g/cm(3) when analyzed using CsCl density gradient centrifugation. Spherical structures with an average diameter of 30 nm were observed using electron microscopy. We report here that VLPs are generated when the first 120 aa of HCV core protein are expressed in E. coli.

Characterization of the oligosaccharides assembled on the Pichia pastoris-expressed recombinant aspartic protease.

Aspartic protease, widely used as a milk-coagulating agent in industrial cheese production, contains three potential N-glycosylation sites. In this study, we report the characterization of N-linked oligosaccharides on recombinant aspartic protease secreted from the methylotrophic yeast Pichia pastoris using a combination of mass spectrometric, 2D chromatographic, chemical and enzymatic methods. The carbohydrates from site I (Asn79) were found to range from Man6-17GlcNAc2 with 50% bearing a phospho-diester-motif, site II (Asn113) was not occupied and site III (Asn188) contained mostly uncharged species ranging from Man-13GlcNAc2. These charged groups are not affecting the transport through the secretion pathway of the recombinant glycoprotein. Changes from a molasses-based medium to a minimal salts-based medium led to a clear reduction of the degree of phosphorylation of the N-glycan population.

Ultrastructural and immunocytochemical evidences of core-particle formation in the methylotrophic Pichia pastoris yeast when expressing HCV structural proteins (core-E1).

Particulate antigens of the Hepatitis C virus (HCV) are reported for the first time by transmission electron microscopy in Pichia pastoris. The yeast was cloned to express the first 339 NH2-terminal amino acids of the HCV polyprotein (C-E1.339 polypeptide). The C-E1.339 polypeptide covers the putative 191 aa of the core protein (aa 1-191) and 148 aa of the E1 envelope antigen (aa 192-339). Virus-like particles (VLP) with diameters ranging from 20 nm to 30 nm were specifically observed in those cells expressing the HCV polyprotein. The VLP appeared along the membrane of the endoplasmic reticulum, but were fundamentally localized in vacuoles, either free or inside autophagic bodies. Clustered particles, chains of particles, high-density reticular structures, and crystalloid bodies were also detected, the last one being an orderly arrangement of particles with 20 nm diameters. The crystal-associated particles are well differentiated from the intracellular VLP because of their uniform size and shape. We argue that membrane components are retained in the architecture of the VLP, conferring to this particle certain heterogeneity. Both kinds of particles, the VLP formed after treatment with NP-40 and the crystal-associated particles, were core protein-positives. Whether they reflect mature HCV nucleocapsid or intermediary states in the viral nucleocapsid morphogenesis remains unknown. We conclude that, like mammalian cell lines, the P. pastoris yeast could be an appropriate host for the analysis of HCV polyprotein processing and, eventually, virus assembly.

Levansucrase from Acetobacter diazotrophicus SRT4 is secreted via periplasm by a signal-peptide-dependent pathway.

Acetobacter diazotrophicus SRT4 secretes a constitutive levansucrase (LsdA) (EC 2.4.1.10) that is responsible for sucrose utilization. Immunogold electron microscopical studies revealed that LsdA accumulates in the periplasm before secretion. The periplasmic and extracellular forms of the enzyme were purified to homogeneity. Both proteins exhibited similar physical and biochemical characteristics indicating that LsdA adopts its final conformation in the periplasm. The N-terminal sequence of mature LsdA was pGlu-Gly-Asn-Phe-Ser-Arg as determined by PSD-MALDI-TOFMS (post-source decay-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry). Comparison of this sequence with the predicted precursor protein revealed the cleavage of a 30-residue typical signal peptide followed by the formation of the pyroglutamic acid (pGlu) residue. Thus, in contrast with other Gram-negative bacteria, A. diazotrophicus secretes levansucrase by a signal-peptide-dependent mechanism.

Physico-chemical characterization of recombinant hepatitis B surface antigen by a multidimensional approach.

Initially, our work was directed to respond to the question: why hepatitis B surface antigen (HBsAg) produces a very broad peak in preparative size-exclusion chromatography (SEC). For this purpose, we used a multidimensional approach based on SEC fractionation of purified HBsAg followed by the individual analysis of SEC fractions by a battery of assays, such as SEC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay and transmission electron microscopy. As a result, HBsAg particles were shown to be heterogeneous in terms of particle assembly. In order to elucidate the origin of HBsAg heterogeneity, we included here the denaturing SEC into a multidimensional approach. The data from denaturing SEC evidenced the fragmentation of protein monomers within the HBsAg particle that, probably, occurs during fermentation broth, rather than during in vitro HBsAg processing. The fractions isolated from widely separated regions of HBsAg peak differed in the extent of protein fragmentation, suggesting that the variable extent of protein degradation within HBsAg particles may be one of the factors responsible for broadening of the HBsAg peak in SEC.

High cytoplasmic expression in E. coli, purification, and in vitro refolding of a single chain Fv antibody fragment against the hepatitis B surface antigen.

A single-chain Fv (scFv) antibody fragment against the hepatitis B surface antigen (HBsAg) was expressed in Escherichia coli in the form of two independent fusion proteins, with either 60 ('long') or 27 ('short') amino acid N-terminal encoding sequences related to human interleukin-2. Both fusion proteins were expressed insolubly and at high levels in the bacterial cytoplasm (approximately 30% of total bacterial protein in MM294 cells at a laboratory scale). When recombinant cells were cultured in 5-1 fermentors, expression and optical density increased 2- and 4-fold, respectively, compared to a previous periplasmic insoluble version of the same anti HBsAg scFv. After extraction and solubilization in urea, the cytoplasmic scFvs were purified using immobilized metal ion affinity chromatography, followed by DTT treatment, and refolding by dialysis against a basic pH buffer containing EDTA. The refolded scFvs recognized the recombinant HBsAg in ELISA. Results of an ELISA where antigen affinity chromatography repurified scFvs were used as standards, indicated that refolding efficiencies were high: 56.2% for the 'short' fusion scFv, and 50.6% for the 'long' fusion scFv. Corrected final yields of active scFv were 30.3 and 27.3 mg l-1, respectively, for the aforementioned fusion proteins, 5-6 times better than those reported for the periplasmic scFv variant.

Aggregation of recombinant hepatitis B surface antigen induced in vitro by oxidative stress.

In order to examine whether oxygen radicals could be responsible for aggregation of recombinant hepatitis B surface antigen (HBsAg) during its assembly in yeast, purified HBsAg was oxidized with ammonium peroxodisulphate (AP) and analyzed by non-denaturing and denaturing size exclusion chromatography, immunoassay and immunoelectron microscopy. As a result, peroxodisulphate radicals induced a reproducible aggregation of HBsAg. At 44 mM AP, the aggregation process took a few hours and the resulting structures were large, branched and non-antigenic. During more gentle oxidation with 9 mM AP and 20-80 microM Cu2+, a continuous structural modification to HBsAg delaying for tens of hours preceded the aggregation event. During this pre-aggregation period, peroxidation of HBsAg lipids and covalent cross-linking of S protein chains occurred that led a complete loss of antigenicity of oxidized particles. In contrast, yeast-derived HBsAg aggregate is decomposed to S monomers under reducing conditions and recognized by anti-HBsAg polyclonal and monoclonal antibodies, suggesting that is has been assembled in vivo from antigenic and reversibly cross-linked particles. Based on these observations, we conclude that oxidation, at least with respect to the specific molecular sites oxidized by AP, is not a primary event in HBsAg aggregate formation in vivo. Since oxidized HBsAg was shown to be irreversibly cross-linked and non-antigenic, there are no suitable techniques for detection HBsAg oxidation in biological samples. Hence, at present, the magnitude of the in-vivo oxidative damage to HBsAg cannot be evaluated and thus, whether the plasma-derived HBsAg undergoes radical-induced oxidation in the course of viral hepatitis remains to be established. If this occurs, this process is expected to contribute to low HBsAg levels in chronic hepatitis B carriers, failure of the currently available immunoassays to identify HBsAg-positive blood donors and inconsistency in the results provided by HBsAg- and anti-HBsAg-based tests in several recent reports.

Aggregation of recombinant hepatitis B surface antigen in Pichia pastoris.

The combination of immunoaffinity and size-exclusion chromatography (SEC) is a powerful tool to analyze multiprotein particle assembly. This approach was used to investigate the source of aggregation of recombinant hepatitis B surface antigen (HBsAg) detected in purified material. As HBsAg aggregation does not originate in the stresses, such as the concentration of HBsAg solutions, temperature and chaotropic agents, it is less probable that the HBsAg aggregate is produced during the process. To test whether aggregation takes place in vivo, crude yeast extract containing the expressed HBsAg was fractioned on a Sephacryl S-400 column just after cell disruption, and each fraction immunopurified individually. As a result, the HBsAg aggregate was isolated from a fraction corresponding to the elution of large particle aggregates only, not native HBsAg particles. It was biologically active, which demonstrates aggregate formation by specific assembly of partially or wholly folded HBsAg intermediates.

Characterization of a virus isolated from the cerebrospinal fluid of patients with epidemic neuropathy.

A previously unknown disease, termed epidemic neuropathy (EN), occurred in Cuba between 1991 and 1993. When samples of cerebrospinal fluid (CSF) from 45 patients with EN and 11 controls were inoculated into cultures of VERO cells, almost all (93%) of the samples from the cases of EN but only one (9%) of the control samples produced a slowly progressing cytopathological effect (CPE). Although the results of other studies indicated the presence of a picornavirus-like virus in CSF samples from EN cases, the CPE and other physico-chemical characteristics observed were not those expected of picorn-viruses. Several aetiological factors may have contributed to EN but at least one virus could have played a major role.

Evidence for the denaturation of recombinant hepatitis B surface antigen on aluminium hydroxide gel.

Despite the complexity of the subject of protein-alum interactions, a valuable information can be obtained by analyzing the adsorbed and desorbed protein by common physico-chemical methods. In the present work, to approach the adsorption of hepatitis B surface antigen (HBsAg) on alum, the experimental data were supported by complementary analyses of the adsorbed protein by immunoelectron microscopy and the desorbed protein by denaturing size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. First, the depletion of HBsAg was investigated. The aspects assessed were the conditions, recovery and chromatographic performance of the desorbed protein. The results obtained strongly suggested the loss of particulate structure of HBsAg after adsorption on alum. This conclusion was further reinforced by direct immunoelectron microscopic visualization of HBsAg in the adsorbed state.

Large-scale production in Pichia pastoris of the recombinant vaccine Gavac against cattle tick.

A gene coding for the Bm86 tick protein was recently cloned, expressed in Pichia pastoris and shown to induce an inmunological response in cattle against ticks. Moreover, the Gavac vaccine (Heber Biotec S.A., Havana, Cuba), which contains this recombinant protein, has proved to control the Boophilus microplus populations under field conditions. This paper reviews the development and large-scale production of this vaccine, the efficacy of the resulting product and the strategy followed in designing its production plant. The production plant fulfills biosafety requirements and GMP.