Heterogeneity and Lobularity of Pancreatic Pathology in Type 1 Diabetes during the Prediabetic Phase

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells are destroyed in the islets of Langerhans. One of its main pathological manifestations is the hyper-expression of Major Histocompatibility Complex I (MHC-I) by beta cells, which was first described over 3 decades ago yet its cause remains unknown. It might not only be a sign of beta cell dysfunction but could also render the cells susceptible to autoimmune destruction; for example, by islet-infiltrating CD8 T cells. In this report, we studied pancreas tissue from a 22-year-old non-diabetic male cadaveric organ donor who had been at high risk of developing T1D, in which autoantibodies against GAD and IA-2 were detected. Pancreas sections were analyzed for signs of inflammation. Multiple insulin-containing islets were identified, which hyper-expressed MHC-I. However, islet density and MHC-I expression exhibited a highly lobular and heterogeneous pattern even within the same section. In addition, many islets with high expression of MHC-I presented higher levels of CD8 T cell infiltration than normal islets. These results demonstrate the heterogeneity of human pathology that occurs early during the pre-diabetic, autoantibody positive phase, and should contribute to the understanding of human T1D.     

Fusogenic potential of sperm membrane lipids: nature's wisdom to accomplish targeted gene delivery

The membrane-membrane fusion during fertilization of oocyte by spermatozoa is believed to be mainly mediated by so called "fusion proteins". In the present study we have tried to demonstrate that beside the proteins, lipid components of membrane may play an important role in fusion of oocyte with spermatozoa. Conventional membrane-membrane fusion assays were used as means to demonstrate fusogenic potential of human sperm membrane lipids. The liposomes (spermatosomes) made of the lipids isolated from sperm membrane were found to undergo strong membrane-membrane fusion as evident from fluorescence dequenching and resonance energy transfer assays. Furthermore, the fusion of these liposomes with living cells (J774 A.1 macrophage cell line) was demonstrated to result in an effective transfer of a water-soluble fluorescent probe (calcein) to cytosol of the target cell. Lastly, the liposomes were demonstrated to behave like efficient vehicles for the in vivo cytosolic delivery of the antigens to target cells resulting in elicitation of antigen specific CD8(+) T cell responses.     

The Peptide Repertoire of HLA‐B27 may include Ligands with Lysine at P2 Anchor Position

The HLA‐B*27 peptidome has drawn significant attention due to the genetic association between some of the HLA‐B*27 alleles and the inflammatory rheumatic disease ankylosing spondylitis (AS), for which a comprehensive biological explanation is still lacking. This study aims to expand the known limits of the HLA‐B*27 peptidome to facilitate selection and testing of new peptides, possibly involved in the disease. The HLA peptidomes of HeLa and C1R cell lines stably transfected with the AS‐associated HLA‐B*27:05 allele, the nonassociated HLA‐B*27:09 allele, or their cysteine 67 to serine mutants (C67S), are analyzed on a very large scale. In addition, the peptidomes of HLA‐B*27:05 and HLA‐B*27:05‐C67S are analyzed from the spleens of rats transgenic for these alleles. The results indicate that C67S mutation increases the percentage of peptides with glutamine or lysine at their P2 position (P2‐Lys), in both HLA‐B*27:05 and HLA‐B*27:09. Furthermore, a small fraction of HLA‐B*27 peptides contains lysine at their second position (P2), in addition to the more commonly found peptides with arginine (P2‐Arg) or the less common glutamine (P2‐Gln) located at this anchor position. Overall these data indicate that peptides with P2‐Lys should be considered as real ligands of HLA‐B*27 molecules and taken into account while looking for putative peptides implicated in the AS.     

The proteomic landscape of renal tumors

Introduction: Renal cell carcinoma (RCC) is the most fatal of the common urologic cancers, with approximately 35% of patients dying within 5 years following diagnosis. Therefore, there is a need for non-invasive markers that are capable of detecting and determining the severity of small renal masses at an early stage in order to tailor treatment and follow-up. Proteomic studies have proved to be very useful in the study of tumors.

Areas covered: In this review, we will detail the current knowledge obtained by the different proteomic approaches, focusing on MS-based strategies, used to investigate RCC biology in order to identify diagnostic, prognostic and predictive biomarkers on tissue, cultured cells and biological fluids.

Expert commentary: Currently, no reliable biomarkers or targets for RCC have been translated into the clinical setting. Moreover, despite the efforts of proteomics and other -omics disciplines, only a small number of them have been observed as shared targets between the different analytical platforms and biological specimens. The difficulty to define a specific molecular pattern for RCC and its subtypes highlights a peculiar profile and a heterogeneity that must be taken into account in future studies.     

The controlling roles of Trp60 and Trp95 in beta2-microglobulin function, folding and amyloid aggregation properties

Amyloidosis associated to hemodialysis is caused by persistently high β₂-microglobulin (β₂m) serum levels. β₂m is an intrinsically amyloidogenic protein whose capacity to assemble into amyloid fibrils in vitro and in vivo is concentration dependent; no β₂m genetic variant is known in the human population. We investigated the roles of two evolutionary conserved Trp residues in relation to β₂m structure, function and folding/misfolding by means of a combined biophysical and functional approach. We show that Trp60 plays a functional role in promoting the association of β₂m in class I major histocompatibility complex; it is exposed to the solvent at the apex of a protein loop in order to accomplish such function. The Trp60 → Gly mutation has a threefold effect: it stabilizes β₂m, inhibits β₂m amyloidogenic propensity and weakens the interaction with the class I major histocompatibility complex heavy chain. On the contrary, Trp95 is buried in the β₂m core; the Trp95 → Gly mutation destabilizes the protein, which is unfolded in solution, yielding nonfibrillar β₂m aggregates. Trp60 and Trp95 therefore play differential and complementary roles in β₂m, being relevant for function (Trp60) and for maintenance of a properly folded structure (Trp95) while affecting in distinct ways the intrinsic propensity of wild-type β₂m towards self-aggregation into amyloid fibrils.     

Perpetuation of immunological memory through common MHC-I binding modes of peptidomimic and antigenic peptides

Understanding the molecular mechanisms of immunological memory assumes importance in vaccine design. We had earlier hypothesized a mechanism for the maintenance of immunological memory through the operation of a network of idiotypic and anti-idiotypic antibodies (Ab2). Peptides derived from an internal image carrying anti-idiotypic antibody are hypothesized to facilitate the perpetuation of antigen specific T cell memory through similarity in peptide-MHC binding as that of the antigenic peptide. In the present work, the existence of such peptidomimics of the antigen in the Ab2 variable region and their similarity of MHC-I binding was examined by bioinformatics approaches. The analysis employing three known viral antigens and one tumor-associated antigen shows that peptidomimics from Ab2 variable regions have structurally similar MHC-I binding patterns as compared to antigenic peptides, indicating a structural basis for memory perpetuation.     

Identification of SIV Nef CD8 T cell epitopes restricted by a MHC class I haplotype associated with lower viral loads in a macaque AIDS model

Virus-specific CD8⁺ T-cell responses are crucial for the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. Multiple studies on HIV-infected individuals and SIV-infected macaques have indicated association of several major histocompatibility complex class I (MHC-I) genotypes with lower viral loads and delayed AIDS progression. Understanding of the viral control mechanism associated with these MHC-I genotypes would contribute to the development of intervention strategy for HIV control. We have previously reported a rhesus MHC-I haplotype, 90-120-Ia, associated with lower viral loads after SIVmac239 infection. Gag_206–216 and Gag_241–249 epitope-specific CD8⁺ T-cell responses have been shown to play a central role in the reduction of viral loads, whereas the effect of Nef-specific CD8⁺ T-cell responses induced in all the 90-120-Ia⁺ macaques on SIV replication remains unknown. Here, we identified three CD8⁺ T-cell epitopes, Nef_9–19, Nef_89–97, and Nef_193–203, associated with 90-120-Ia. Nef_9–19 and Nef_193–203 epitope-specific CD8⁺ T-cell responses frequently selected for mutations resulting in viral escape from recognition by these CD8⁺ T cells, indicating that these CD8⁺ T cells exert strong suppressive pressure on SIV replication. Results would be useful for elucidation of the viral control mechanism associated with 90-120-Ia.     

A new rapid and comprehensive peptidome analysis by one-step direct transfer technology for 1-D electrophoresis/MALDI mass spectrometry

We have developed a new target plate for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). This target plate enables direct electric transfer of analytes from the 1-dimensional gel electrophoresis (1-DE) gel to the target plate in one step. Incorporated with a one-step direct transfer technique, this novel 1-DE/MALDI-MS (1-DE/MS) system eliminates staining, extracting, loading, and many other time-consuming intermediate processes, thereby greatly reducing analysis time while providing high throughput proteome analysis. Furthermore, in peptidome analysis, during the 1-DE step this system separates or removes the high molecular weight plasma proteins in blood and the various low molecular weight substances in tissue extracts, which interfere with mass spectrometry. This system can therefore be used for peptide profiling of any biological sample without special pretreatment. In view of these advantages, the 1-DE/MS system will greatly improve the usefulness of current peptidomic modalities in the discovery and validation of biomarker molecules in various body fluids and tissue extracts, permitting early detection, diagnosis, and treatment of diseases.     

Current advances in tumor proteomics and candidate biomarkers for hepatic cancer

Tumor proteomics apply proteomics techniques to tumor biological research, mainly by screening candidate biomarkers for early tumor diagnosis, prognosis and treatment. Hepatocellular carcinoma (HCC) is a type of malignant tumor with one of the highest death rates in the world. With the advent of the post-genomic age, tumor biological research developing the technology of proteomics has become a major focus of researchers. The discovery of novel candidate biomarkers is one of crucial problems for the early diagnosis of HCC. In general, there are three distinct types of candidate biomarkers for HCC based on different areas: biochemical biomarkers, antigenic biomarkers and epigenetic biomarkers. This review mainly discusses current advances in the problems and prospects of candidate biomarker for the early diagnosis of HCC, discovered by technologies of tumor proteomics.     

Selective autophagy of MHC-I promotes immune evasion of pancreatic cancer

ABSTRACT

Major histocompatibility complex class I (MHC-I) is a key molecule in anti-tumor adaptive immunity. MHC-I is essential for endogenous antigen presentation by cancer cells and subsequent recognition and clearance by CD8⁺ T cells. Defects in MHC-I expression occur frequently in several cancers, leading to impaired antigen presentation, immune evasion and/or resistance to immune checkpoint blockade (ICB) therapy. Pancreatic ductal adenocarcinoma (PDAC), a deadly malignancy with dismal patient prognosis, is resistant to ICB and shows frequent downregulation of MHC-I independent of genetic mutations abrogating MHC-I expression. Previously, we showed that PDAC cells exhibit elevated levels of autophagy and lysosomal biogenesis, which together support the survival and growth of PDAC tumors via both cell-autonomous and non-cell-autonomous mechanisms. In our recent study, we have identified NBR1-mediated selective macroautophagy/autophagy of MHC-I as a novel mechanism that facilitates immune evasion by PDAC cells. Importantly, autophagy or lysosome inhibition restores MHC-I expression, leading to enhanced anti-tumor T cell immunity and improved response to ICB in transplanted tumor models in syngeneic host mice. Our results highlight a previously unknown function of autophagy and the lysosome in regulation of immunogenicity in PDAC, and provide a novel therapeutic strategy for targeting this deadly disease.