Compostabilidad de envases de bolsas de PBAT+PLA y envases rígidos de PLA mediante tecnologías de pila y túnel

Los polímeros compostables suponen en torno al 30% de los bioplásticos destinados a envasado, siendo a su vez esta aplicación el principal destino de la producción de este tipo de materiales que, en el año 2013, superó 1,6 millones de toneladas. La presente tesis aborda la biodegradación de los residuos de envases domésticos compostables en medio aerobio para dos tipos de formato y materiales, envase rígido de PLA (Clase I) y dos tipos de bolsas de PBAT+PLA (Clases II y III). Sobre esta materia se han realizado diversos estudios en escala de laboratorio pero para otro tipo de envases y biopolímeros y bajo condiciones controladas del compost con alguna proyección particularizada en plantas. La presente tesis da un paso más e investiga el comportamiento real de los envases plásticos compostables en la práctica del compostaje en tecnologías de pila y túnel, tanto a escala piloto como industrial, dentro del procedimiento y con las condiciones ambientales de instalaciones concretas. Para ello, con el método seguido, se han analizado los requisitos básicos que debe cumplir un envase compostable, según la norma UNE – EN 13432, evaluando el porcentaje de biodegradación de los envases objeto de estudio, en función de la pérdida de peso seco tras el proceso de compostaje, y la calidad del compost obtenido, mediante análisis físico-químico y de fitotoxicidad para comprobar que los materiales de estudio no aportan toxicidad. En cuanto a los niveles de biodegrabilidad, los resultados permiten concluir que los envases de Clase I se compostan adecuadamente en ambas tecnologías y que no requieren de unas condiciones de proceso muy exigentes para alcanzar niveles de biodegradación del 100%. En relación a los envases de Clase II, se puede asumir que se trata de un material que se composta adecuadamente en pila y túnel industrial pero que requiere de condiciones exigentes para alcanzar niveles de biodegradación del 100% al afectarle de forma clara la ubicación de las muestras en la masa a compostar, especialmente en el caso de la tecnología de túnel. Mientras el 90% de las muestras alcanza el 100% de biodegradación en pila industrial, tan sólo el 50% lo consigue en la tecnología de túnel a la misma escala. En cuanto a los envases de Clase III, se puede afirmar que es un material que se composta adecuadamente en túnel industrial pero que requiere de condiciones de cierta exigencia para alcanzar niveles de biodegradación del 100% al poderle afectar la ubicación de las muestras en la masa a compostar. El 75% de las muestras ensayadas en túnel a escala industrial alcanzan el 100% de biodegradación y, aunque no se ha ensayado este tipo de envase en la tecnología de pila al no disponer de muestras, cabe pensar que los resultados de biodegrabilidad que hubiera podido alcanzar habrían sido, como mínimo, los obtenidos para los envases de Clase II, al tratarse de materiales muy similares en composición. Por último, se concluye que la tecnología de pila es más adecuada para conseguir niveles de biodegradación superiores en los envases tipo bolsa de PBAT+PLA. Los resultados obtenidos permiten también sacar en conclusión que, en el diseño de instalaciones de compostaje para el tratamiento de la fracción orgánica recogida selectivamente, sería conveniente realizar una recirculación del rechazo del afino del material compostado para aumentar la probabilidad de someter este tipo de materiales a las condiciones ambientales adecuadas. Si además se realiza un triturado del residuo a la entrada del proceso, también se aumentaría la superficie específica a entrar en contacto con la masa de materia orgánica y por tanto se favorecerían las condiciones de biodegradación. En cuanto a la calidad del compost obtenido en los ensayos, los resultados de los análisis físico – químicos y de fitotoxicidad revelan que los niveles de concentración de microorganismo patógenos y de metales pesados superan, en la práctica totalidad de las muestras, los niveles máximos permitidos en la legislación vigente aplicable a productos fertilizantes elaborados con residuos. Mediante el análisis de la composición de los envases ensayados se constata que la causa de esta contaminación reside en la materia orgánica utilizada para compostar en los ensayos, procedente del residuo de origen doméstico de la denominada “fracción resto”. Esta conclusión confirma la necesidad de realizar una recogida selectiva de la fracción orgánica en origen, existiendo estudios que evidencian la mejora de la calidad del residuo recogido en la denominada “fracción orgánica recogida selectivamente” (FORM). Compostable polymers are approximately 30% of bioplastics used for packaging, being this application, at same time, the main destination for the production of such materials exceeded 1.6 million tonnes in 2013. This thesis deals with the biodegradation of household packaging waste compostable in aerobic medium for two format types and materials, rigid container made of PLA (Class I) and two types of bags made of PBAT + PLA (Classes II and III). There are several studies developed about this issue at laboratory scale but for other kinds of packaging and biopolymers and under composting controlled conditions with some specifically plants projection. This thesis goes one step further and researches the real behaviour of compostable plastic packaging in the composting practice in pile and tunnel technologies, both at pilot and industrial scale, within the procedure and environmental conditions of concrete devices. Therefore, with a followed method, basic requirements fulfilment for compostable packaging have been analysed according to UNE-EN 13432 standard. It has been assessed the biodegradability percentage of the packaging studied, based on loss dry weight after the composting process, and the quality of the compost obtained, based on physical-chemical analysis to check no toxicity provided by the studied materials. Regarding biodegradability levels, results allow to conclude that Class I packaging are composted properly in both technologies and do not require high exigent process conditions for achieving 100% biodegradability levels. Related to Class II packaging, it can be assumed that it is a material that composts properly in pile and tunnel at industrial scale but requires exigent conditions for achieving 100% biodegradability levels for being clearly affected by sample location in the composting mass, especially in tunnel technology case. While 90% of the samples reach 100% of biodegradation in pile at industrial scale, only 50% achieve it in tunnel technology at the same scale. Regarding Class III packaging, it can be said that it is a material properly composted in tunnel at industrial scale but requires certain exigent conditions for reaching 100% biodegradation levels for being possibly affected by sample location in the composting mass. The 75% of the samples tested in tunnel at industrial scale reaches 100% biodegradation. Although this kind of packaging has not been tested on pile technology due to unavailability of samples, it is judged that biodegradability results that could be reached would have been, at least, the same obtained for Class II packaging, as they are very similar materials in composition. Finally, it is concluded that pile technology is more suitable for achieving highest biodegradation levels in bag packaging type of PBAT+PLA. Additionally, the obtained results conclude that, in the designing of composting devices for treatment of organic fraction selectively collected, it would be recommended a recirculation of the refining refuse of composted material in order to increase the probability of such materials to expose to proper environmental conditions. If the waste is grinded before entering the process, the specific surface in contact with organic material would also be increased and therefore biodegradation conditions would be more favourable. Regarding quality of the compost obtained in the tests, physical-chemical and phytotoxicity analysis results reveal that pathogen microorganism and heavy metals concentrations exceed, in most of the samples, the maximum allowed levels by current legislation for fertilizers obtained from wastes. Composition analysis of tested packaging verifies that the reason for this contamination is the organic material used for composting tests, comes from the household waste called “rest fraction”. This conclusion confirms the need of a selective collection of organic fraction in the origin, as existing studies show the quality improvement of the waste collected in the so-called “organic fraction selectively collected” (FORM).

T cell positive selection by a high density, low affinity ligand

Interaction of the αβ T cell receptor (TCR) with major histocompatibility (MHC) molecules occupied with any of a large collection of peptides derived from self proteins is a critical step in driving T cell “positive” selection in the thymus. Interaction with this same pool of self-peptide/MHC ligands deletes T cells with potential self-reactivity. To examine how T cells survive both of these processes to form a self-tolerant mature repertoire, mice were constructed whose entire class II MHC IEk specific repertoire was positively selected on a single peptide covalently attached to the IEk molecule. In these mice T cells were identified that could respond to a variant of the positively selecting peptide bound to IEk. The affinities of the TCRs from these T cells for the positively selecting ligand were extremely low and at least 10-fold less than those for the activating ligand. These results support the theory that positive selection is driven by TCR affinities lower than those involved in T cell deletion or activation and that, if present at high concentration, even very low affinity ligands can positively select.

Evidence for the recent horizontal transfer of long terminal repeat retrotransposon

The evolutionary dynamics existing between transposable elements (TEs) and their host genomes have been likened to an “arms race.” The selfish drive of TEs to replicate, in turn, elicits the evolution of host-mediated regulatory mechanisms aimed at repressing transpositional activity. It has been postulated that horizontal (cross-species) transfer may be one effective strategy by which TEs and other selfish genes can escape host-mediated silencing mechanisms over evolutionary time; however, to date, the most definitive evidence that TEs horizontally transfer between species has been limited to class II or DNA-type elements. Evidence that the more numerous and widely distributed retroelements may also be horizontally transferred between species has been more ambiguous. In this paper, we report definitive evidence for a recent horizontal transfer of the copia long terminal repeat retrotransposon between Drosophila melanogaster and Drosophila willistoni.

Pig but not human interferon-γ initiates human cell-mediated rejection of pig tissue in vivo

Split-thickness pig skin was transplanted on severe combined immunodeficient mice so that pig dermal microvessels spontaneously inosculated with mouse microvessels and functioned to perfuse the grafts. Pig endothelial cells in the healed grafts constitutively expressed class I and class II major histocompatibility complex molecules. Major histocompatibility complex molecule expression could be further increased by intradermal injection of pig interferon-γ (IFN-γ) but not human IFN-γ or tumor necrosis factor. Grafts injected with pig IFN-γ also developed a sparse infiltrate of mouse neutrophils and eosinophils without evidence of injury. Introduction of human peripheral blood mononuclear cells into the animals by intraperitoneal inoculation resulted in sparse perivascular mononuclear cell infiltrates in the grafts confined to the pig dermis. Injection of pig skin grafts on mice that received human peripheral blood mononuclear cells with pig IFN-γ (but not human IFN-γ or heat-inactivated pig IFN-γ) induced human CD4+ and CD8+ T cells and macrophages to more extensivley infiltrate the pig skin grafts and injure pig dermal microvessels. These findings suggest that human T cell-mediated rejection of xenotransplanted pig organs may be prevented if cellular sources of pig interferon (e.g., passenger lymphocytes) are eliminated from the graft.

CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates

Selective inhibition of T cell costimulation using the B7-specific fusion protein CTLA4-Ig has been shown to induce long-term allograft survival in rodents. Antibodies preventing the interaction between CD40 and its T cell-based ligand CD154 (CD40L) have been shown in rodents to act synergistically with CTLA4-Ig. It has thus been hypothesized that these agents might be capable of inducing long-term acceptance of allografted tissues in primates. To test this hypothesis in a relevant preclinical model, CTLA4-Ig and the CD40L-specific monoclonal antibody 5C8 were tested in rhesus monkeys. Both agents effectively inhibited rhesus mixed lymphocyte reactions, but the combination was 100 times more effective than either drug alone. Renal allografts were transplanted into nephectomized rhesus monkeys shown to be disparate at major histocompatibility complex class I and class II loci. Control animals rejected in 5–8 days. Brief induction doses of CTLA4-Ig or 5C8 alone significantly prolonged rejection-free survival (20–98 days). Two of four animals treated with both agents experienced extended (>150 days) rejection-free allograft survival. Two animals treated with 5C8 alone and one animal treated with both 5C8 and CTLA4-Ig experienced late, biopsy-proven rejection, but a repeat course of their induction regimen successfully restored normal graft function. Neither drug affected peripheral T cell or B cell counts. There were no clinically evident side effects or rejections during treatment. We conclude that CTLA4-Ig and 5C8 can both prevent and reverse acute allograft rejection, significantly prolonging the survival of major histocompatibility complex-mismatched renal allografts in primates without the need for chronic immunosuppression.

Recombinant human granzyme A binds to two putative HLA-associated proteins and cleaves one of them

The release of cytotoxic granule contents by cytotoxic T lymphocytes triggers apoptotic target cell death. Cytotoxic granules contain a pore-forming protein, perforin, and a group of serine proteases called granzymes. We expressed human granzyme A in bacteria as a proenzyme capable of in vitro activation by enterokinase. The recombinant activated enzyme has catalytic activity against substrates with Arg, preferably, or Lys at the P1 position, comparable to trypsin. An enzymatically inactive recombinant granzyme A, with the active site Ser mutated to Ala, was produced and used with affinity chromatography to identify potential substrates. Two granzyme A-binding cytoplasmic proteins of molecular mass 33 and 44 kDa were isolated and identified by tryptic fragment sequencing as PHAP I and II, ubiquitous putative HLA-associated proteins, previously coisolated by binding to an HLA class II peptide. PHAP II forms an SDS-stable complex with recombinant mutant granzyme A and coprecipitates with it from cytoplasmic extracts. PHAP II, either purified or in cell lysates, is cleaved by the recombinant enzyme at nanomolar concentrations to a 25-kDa fragment. PHAP II begins to be degraded within minutes of initiation of cytotoxic T lymphocyte attack. PHAP I and II are candidate participants in the granzyme A pathway of cell-mediated cytotoxicity.

The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4+ T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4+ T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the β cell-reactive CD4+ T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a Haemophilus influenzae TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4+ T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and/or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.

Enhanced immunogenicity of HIV-1 vaccine construct by modification of the native peptide sequence

Viral proteins are not naturally selected for high affinity major histocompatibility complex (MHC) binding sequences; indeed, if there is any selection, it is likely to be negative in nature. Thus, one should be able to increase viral peptide binding to MHC in the rational design of synthetic peptide vaccines. The T1 helper peptide from the HIV-1 envelope protein was made more immunogenic for inducing T cell proliferation to the native sequence by replacing a residue that exerts an adverse influence on peptide binding to an MHC class II molecule. Mice immunized with vaccine constructs combining the more potent Th helper (Th) epitope with a cytotoxic T lymphocyte (CTL) determinant developed greatly enhanced CTL responses. Use of class II MHC-congenic mice confirmed that the enhancement of CTL response was due to class II-restricted help. Thus, enhanced T cell help is key for optimal induction of CTL, and, by modification of the native immunogen to increase binding to MHC, it is possible to develop second generation vaccine constructs that enhance both Th cell activation and CTL induction.

Interaction of pigeon cytochrome c-(43–58) peptide analogs with either T cell antigen receptor or I-Ab molecule

We determined that a pigeon cytochrome c-derived peptide, p43–58, possesses two anchor residues, 46 and 54, for binding with the I-Ab molecule that are compatible to the position 1 (P1) and position 9 (P9) of the core region in the major histocompatibility complex (MHC) class II binding peptides, respectively. In the present study to analyze each binding site between P1 and P9 of p43–58 to either I-Ab or T cell antigen receptor (TCR), we investigated T cell responses to a series of peptides (P2K, P3K, P4K, P5K, P6K, P7K, and P8E) that sequentially substituted charged amino acid residues for the residues at P2 to P8 of p43–58. T cells from C57BL/10 (I-Ab) mice immunized with P4K or P6K did not mount appreciable proliferative responses to the immunogens, but those primed with other peptides (P2K, P3K, P5K, P7K, and P8E) showed substantial responses in an immunogen-specific manner. It was demonstrated by binding studies that P1 and P9 functioned as main anchors and P4 and P6 functioned as secondary anchors to I-Ab. Analyses of Vβ usage of T cell lines specific for these analogs suggested that P8 interacts with the complementarity-determining region 1 (CDR1)/CDR2 of the TCR β chain. Furthermore, sequencing of the TCR on T cell hybridomas specific for these analogs indicated that P5 interacts with the CDR3 of the TCR β chain. The present findings are consistent with the three-dimensional structure of the trimolecular complex that has been reported for TCR/peptide/MHC class I molecules.