Defining the expression hierarchy of latent T-cell epitopes in Epstein-Barr virus infection with TCR-like antibodies

Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. The latent gene products LMP1, LMP2A and EBNA1 are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines. Whilst CTL-based methodologies can be utilized to infer the presence of specific latent epitopes, they do not allow a direct visualization or quantitation of these epitopes. Here, we describe the characterization of three TCR-like monoclonal antibodies (mAbs) targeting the latent epitopes LMP1[subscript 125–133], LMP2A[subscript 426–434] or EBNA1[subscript 562–570] in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1[subscript 562–570] in association with HLA-A0201 was consistently observed in cell lines and EBV-associated tumor biopsies. These data highlight the discordance between MHC-epitope density and frequencies of associated CTL with implications for cell-based immunotherapies and/or vaccines for EBV-associated disease

Caloric restriction counteracts age-dependent changes in prolyl-4-hydroxylase domain (PHD) 3 expression

Caloric restriction remains the most reproducible measure known to extend life span or diminish age-associated changes. Previously, we have described an elevated expression of the prolyl-4-hydroxylase domain (PHD) 3 with increasing age in mouse and human heart. PHDs modulate the cellular response towards hypoxia by regulating the stability of the α-subunit of the transcriptional activator hypoxia inducible factor (HIF). In the present study we demonstrate that elevated PHD3, but not PHD1 or PHD2, expression is not restricted to the heart but does also occur in rat skeletal muscle and liver. Elevated expression of PHD3 is counteracted by a decrease in caloric intake (40% caloric restriction applied for 6 months) in all three tissues. Age-associated changes in PHD3 expression inversely correlated with the expression of the HIF-target gene macrophage migration inhibitory factor (MIF), which has been previously described to be involved in cellular HIF-mediated anti-ageing effects. These data give insight into the molecular consequences of caloric restriction, which influences hypoxia-mediated gene expression via PHD3

Expression hierarchy of T cell epitopes from melanoma differentiation antigens: unexpected high level presentation of tyrosinase-HLA-A2 Complexes revealed by peptide-specific, MHC-restricted, TCR-like antibodies.

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8(+) T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags

Direct visualization of distinct T cell epitopes derived from a melanoma tumor-associated antigen by using human recombinant antibodies with MHC- restricted T cell receptor-like specificity

Specificity in the cellular immune system is controlled and regulated by the T cell antigen receptor (TCR), which specifically recognizes peptide/major histocompatibility complex (MHC) molecules. In recent years many cancer-associated MHC-restricted peptides have been isolated and because of their highly restricted fine specificity, they are desirable targets for novel approaches in immunotherapy. Antibodies that would recognize tumor-associated MHC–peptide complexes with the same specificity as the TCR would be valuable reagents for studying antigen presentation by tumor cells, for visualizing MHC–peptide complexes on cells, and eventually for monitoring the expression of specific complexes during immunotherapy. To generate molecules with such a unique fine specificity, we selected a large nonimmune repertoire of phage Fab antibodies on recombinant HLA-A2 complexed with three common antigenic T cell, HLA-A2-restricted epitopes derived from the melanoma differentiation antigen gp100. We were able to isolate a surprisingly large panel of human recombinant Fab antibodies that exhibit a characteristic TCR-like binding specificity to each of the three gp100-derived epitopes, yet unlike TCRs, they did so with an affinity in the nanomolar range. These TCR-like antibodies recognize the native MHC–peptide complex expressed on the surface of antigen-presenting cells. Moreover, they can detect the specific MHC–peptide complexes on the surface of melanoma tumor cells. These results demonstrate the ability to isolate high-affinity human recombinant antibodies with the antigen-specific, MHC-restricted specificity of T cells, and this ability was demonstrated for three different epitopes of the same melanoma-derived antigen

Impactos ambientales de la huella hídrica y la generación de residuos de alimentos utilizados en las comidas de los trabajadores de un hospital público brasileño

This study aimed to assess the environmental impacts of waste generation and the WF of raw materials used to provide meals to workers in a public hospital in southern Brazil over the course of the four seasons. This is a descriptive case study with a quant itative approach. The food raw materials that composed meals during 2019 were grouped by type of input. The items included from each food group were those which represented at least 85% (Multiple Criteria ABC Analysis) of the total amount used in kilograms within the respective group, in each month. The generation of residues from fruits, vegetables, and meat was estimated, as well as the WF of the items. For the statistical analysis, the Kruskal - Wallis test was used with a significance of 5%. Out of the 96 food inputs used, 49 items represented 86% of the total in kg, being the ones from which the environmental impacts were calculated. During the year, 435,411 meals were served. As for the number of diners, the highest frequency was observed in the winter a nd lowest in the summer. The annual waste percentage of the fruits acquired was 33.8%, being higher in the summer than in other seasons. Animal products were responsible for 64.2% of the WF, being higher in the winter. Assessing user frequencies, climatic conditions, and raw - material selection are important measures for the appropriate management of foodservices, as well as for assessing their environmental impacts.Este estudo teve como objetivo avaliar os impactos ambientais da geração de resíduos e da pegada hídrica (PH) das matérias - primas utilizadas na alimentação dos trabalhadores de um hospital público do sul do Brasil ao longo das quatro estações. Trata - se de um estudo de caso descritivo com abordagem quantitativa. As matérias - primas alimentares que compunham as refeições durante 2019 foram agrupadas por tipo de insumo. Os itens incluídos de cada grupo de alimentos foram aqueles que representaram pelo menos 85% (Análise Curva ABC) da quantidade total utilizada em quilogramas (kg) dentro do respectivo grupo, em cada mês. Foi estimada a geração de resíduos de frutas, hortaliças e carnes, bem como a PH dos itens. Para a análise estatística, foi utilizado o teste de Kruskal - Wallis com significância de 5%. Dos 96 insumos alimentares utilizados, 49 itens representaram 86% do total em kg, sendo aqueles a partir dos quais foram calculados os impactos ambientais. No ano, foram servidas 435.411 refeições. Quanto ao número de comensais, a maior frequência foi observada no inverno e a menor no verão. As frutas tiveram um total de 33,8% de desperdício, sendo que no verão foi observado a maior quantidade. Os produtos de origem animal foram responsáveis por 64,2% da PH, e no i nverno foi identificado os maiores valores. A avaliação das frequências dos usuários, as condições climáticas e a seleção de matérias - primas são medidas importantes para o gerenciamento adequado dos serviços de alimentação, bem como para avaliar seus impactos ambientais.Este estudio tuvo como objetivo evaluar los impactos ambientales de la generación de residuos y el huela hídrica (HH) de las materias primas utilizadas par a proporcionar alimentos a los trabajadores de un hospital público en el sur de Brasil durante las cuatro estaciones Del año. Este es un estudio de caso descriptivo con un enfoque cuantitativo. Las materias primas alimentarias que componían las comidas dur ante 2019 se agruparon por tipo de insumo. Los ítems incluidos de cada grupo de alimentos fueron aquellos que representaron al menos el 85% (Análisis ABC) de la cantidad total utilizada en kilogramos (kg) dentro del grupo respectivo, en cada mes. Se estimó la generación de residuos de frutas, verduras y carnes, así como la HH de los artículos. Para el análisis estadístico se utilizó la prueba de Kruskal - Wallis con una significancia del 5%. De los 96 insumos alimentarios utilizados, 49 ítems representaron el 86% del total en kg, siendo a partir de los cuales se calcularon los impactos ambientales. Durante el año se sirvieron 435,411 comidas. En cuanto al número de comensales, la mayor frecuencia se observó en invierno y la menor en verano. El porcentaje de de sperdicio anual de los frutos adquiridos fue del 33,8%, siendo mayor en verano que en otras temporadas. Los productos animales fueron responsables del 64,2% de la HH, siendo mayor en invierno. La evaluación de la frecuencia de los usuarios, las condiciones climáticas y la selección de materias primas son medidas importantes para la gestión adecuada de los servicios alimentarios, así como para evaluar sus impactos ambientale

The length of lipids bound to human CD1d molecules modulates the affinity of NKT cell TCR and the threshold of NKT cell activation.

CD1d-restricted lymphocytes recognize a broad lipid range. However, how CD1d-restricted lymphocytes translate T cell receptor (TCR) recognition of lipids with similar group heads into distinct biological responses remains unclear. Using a soluble invariant NKT (iNKT) TCR and a newly engineered antibody specific for alpha-galactosylceramide (alpha-GalCer)-human CD1d (hCD1d) complexes, we measured the affinity of binding of iNKT TCR to hCD1d molecules loaded with a panel of alpha-GalCer analogues and assessed the rate of dissociation of alpha-GalCer and alpha-GalCer analogues from hCD1d molecules. We extended this analysis by studying iNKT cell synapse formation and iNKT cell activation by the same panel of alpha-GalCer analogues. Our results indicate the unique role of the lipid chain occupying the hCD1d F' channel in modulating TCR binding affinity to hCD1d-lipid complexes, the formation of stable immunological synapse, and cell activation. These data are consistent with previously described conformational changes between empty and loaded hCD1d molecules (Koch, M., V.S. Stronge, D. Shepherd, S.D. Gadola, B. Mathew, G. Ritter, A.R. Fersht, G.S. Besra, R.R. Schmidt, E.Y. Jones, and V. Cerundolo. 2005. Nat. Immunol 6:819-826), suggesting that incomplete occupation of the hCD1d F' channel results in conformational differences at the TCR recognition surface. This indirect effect provides a general mechanism by which lipid-specific lymphocytes are capable of recognizing both the group head and the length of lipid antigens, ensuring greater specificity of antigen recognition

Defective carotid body function and impaired ventilatory responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1α

To investigate whether the transcriptional activator hypoxia-inducible factor 1 (HIF-1) is required for ventilatory responses to hypoxia, we analyzed mice that were either wild type or heterozygous for a loss-of-function (knockout) allele at the Hif1a locus, which encodes the O(2)-regulated HIF-1α subunit. Although the ventilatory response to acute hypoxia was not impaired in Hif1a(+/−) mice, the response was primarily mediated via vagal afferents, whereas in wild-type mice, carotid body chemoreceptors played a predominant role. When carotid bodies isolated from wild-type mice were exposed to either cyanide or hypoxia, a marked increase in sinus nerve activity was recorded, whereas carotid bodies from Hif1a(+/−) mice responded to cyanide but not to hypoxia. Histologic analysis revealed no abnormalities of carotid body morphology in Hif1a(+/−) mice. Wild-type mice exposed to hypoxia for 3 days manifested an augmented ventilatory response to a subsequent acute hypoxic challenge. In contrast, prior chronic hypoxia resulted in a diminished ventilatory response to acute hypoxia in Hif1a(+/−) mice. Thus partial HIF-1α deficiency has a dramatic effect on carotid body neural activity and ventilatory adaptation to chronic hypoxia