Endocytosis and cancer

Eukaryotic cells use endocytosis to internalise plasma membrane, surface receptors and their ligands, viruses and various extracellular soluble molecules. Endocytosis has been regarded as a long-term mechanism of signal attenuation via receptor clearance from the cell surface. However, additional, and quite unexpected, functions for endocytosis have emerged, which, together with its attenuation function, project a central role for this process in cellular homeostasis and control of proliferation. Subversion of endocytic control is thus predicted to play a causative role in hyperproliferative conditions, first and foremost cancer.     

Association of β<Subscript>2</Subscript>-microglobulin with the α<Subscript>3</Subscript> domain of H-2D<Superscript>b</Superscript> heavy chain

MHC class I molecules are heterotrimeric complexes composed of heavy chain, ₂-microglobulin (₂m) and short peptide. This trimeric complex is generated in the endoplasmic reticulum (ER), where a peptide loading complex (PLC) facilitates transport from the cytosol and binding of the peptide to the preassembled ER resident heavy chain/₂m dimers. Association of mouse MHC class I heavy chain with ₂m is characterized by allelic differences in the number and/or positions of amino acid interactions. It is unclear, however, whether all alleles follow common binding patterns with minimal contributions by allele-specific contacts, or whether essential contacts with ₂m are different for each allele. While searching for the PLC binding site in the ₃ domain of the mouse MHC class I molecule H-2D^b, we unexpectedly discovered a site critical for binding mouse, but not human, ₂m. Interestingly, amino acids in the corresponding region of another MHC class I heavy chain allele do not make contacts with the mouse ₂m. Thus, there are allelic differences in the modes of binding of ₂m to the heavy chain of MHC class I.     

Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans

An animal's ability to detect and avoid toxic compounds in the environment is crucial for survival. We show that the nematode Caenorhabditis elegans avoids many water-soluble substances that are toxic and that taste bitter to humans. We have used laser ablation and a genetic cell rescue strategy to identify sensory neurons involved in the avoidance of the bitter substance quinine, and found that ASH, a polymodal nociceptive neuron that senses many aversive stimuli, is the principal player in this response. Two G protein alpha subunits GPA-3 and ODR-3, expressed in ASH and in different, nonoverlapping sets of sensory neurons, are necessary for the response to quinine, although the effect of odr-3 can only be appreciated in the absence of gpa-3. We identified and cloned a new gene, qui-1, necessary for quinine and SDS avoidance. qui-1 codes for a novel protein with WD-40 domains and which is expressed in the avoidance sensory neurons ASH and ADL.     

Effect of collagen substrates on proteomic modulation of breast cancer cells

We have previously described the occurrence, in breast and colon cancer extra-cellular matrix, of an oncofoetal form of collagen, OF/LB, able to induce an increase in cell proliferation and motility in the breast cancer cell line 8701-BC. It also caused an increased amount of type V collagen which appears to exert an anti-proliferative effect on the same cells. The aim of the present study was to investigate, at the proteomic level, the effect of OF/LB and type V collagens used as substrates for neoplastic cell growth. Due to the complexity of a whole proteomic profile, a subset of significant protein classes was used to assess variations in protein expression levels. For this study we adopted a multivariate statistical procedure that allows a global view of the variations induced by different growth conditions, when several variables have to be analyzed simultaneously. The results of this research indicate that in response to different growth substrates, chaperons and heat shock proteins contributed most to the dissimilarity in levels of expression of the selected protein spots. Moreover, we observed that different isoforms of the same protein showed independent levels of expression from one another in relation to the different collagen treatments.     

Analyzing a randomized trial on breast self-examination with noncompliance and missing outcomes

Recently, instrumental variables methods have been used to address non-compliance in randomized experiments. Complicating such analyses is often the presence of missing data. The standard model for missing data, missing at random (MAR), has some unattractive features in this context. In this paper we compare MAR-based estimates of the complier average causal effect (CACE) with an estimator based on an alternative, nonignorable model for the missing data process, developed by Frangakis and Rubin (1999, Biometrika, 86, 365-379). We also introduce a new missing data model that, like the Frangakis-Rubin model, is specially suited for models with instrumental variables, but makes different substantive assumptions. We analyze these issues in the context of a randomized trial of breast self-examination (BSE). In the study two methods of teaching BSE, consisting of either mailed information about BSE (the standard treatment) or the attendance of a course involving theoretical and practical sessions (the new treatment), were compared with the aim of assessing whether teaching programs could increase BSE practice and improve examination skills. The study was affected by the two sources of bias mentioned above: only 55% of women assigned to receive the new treatment complied with their assignment and 35% of the women did not respond to the post-test questionnaire. Comparing the causal estimand of the new treatment using the MAR, Frangakis-Rubin, and our new approach, the results suggest that for these data the MAR assumption appears least plausible, and that the new model appears most plausible among the three choices.     

Helicobacter pylori associated antigastric autoantibodies: role in Sjögren's syndrome gastritis

Background. Previous studies have shown that Helicobacter pylori seroprevalence in Sjögren's syndrome is comparable with that of the general population. However, the origin of the chronic gastropathy associated with this syndrome and the role of local autoimmunity – possibly triggered by bacterial infection – in its pathogenesis remain unclear. Materials and Methods. We initially determined the prevalence of IgG anti H. pylori in dyspeptic subjects with and without Sjögren's syndrome. In subsets of both groups we then determined anti CagA and human tissue‐tested anticanalicular/antifoveolar autoantibodies. We also compared activity, atrophy and Mucosa Associated Lymphoid Tissue (MALT) scores, as well as symptoms, before and after bacterial eradication. Results. Prevalence of H. pylori in Sjögren's syndrome patients was similar to controls: 31/54 (57%) vs. 93/150 (62%). Anti CagA prevalence was also similar in the two groups. Twenty weeks after H. pylori eradication, histological activity decreased in both groups, however, atrophy and MALT decreased significantly only in controls. Sixteen months after H. pylori eradication, 75% of Sjögren's syndrome patients still complained of dyspepsia compared with 13% of controls. Finally, antigastric autoantibodies were present in 29% of tested Sjögren's syndrome patients vs. 28% of controls. Conclusions. H. pylori infection was equally prevalent among dyspeptic Sjögren's syndrome patients and dyspeptic controls. Likewise, there were no differences regarding anti CagA prevalence or antigastric autoantibodies among the two groups. The persistence of symptoms as well as of the lymphocytic infiltration and atrophy after H. pylori eradication in Sjögren's syndrome may underlie the ‘endogenous’ and still unknown nature of the gastropathy in this condition.     

Celiac disease association with CD8+ T cell responses: identification of a novel gliadin-derived HLA-A2-restricted epitope

One of the diagnostic hallmarks of the histological lesions associated with celiac disease is the extensive infiltration of the small intestinal epithelium by CD8(+) T cells of unknown Ag specificity. In this study, we report recognition of the gliadin-derived peptide (A-gliadin 123-132) by CD8(+) T lymphocytes from celiac patients. A-gliadin 123-132-specific IFN-gamma production and cytotoxic activity were detected in PBMCs derived from patients on gluten-free diet, but not from either celiac patients on gluten-containing diet or healthy controls. In contrast, A-gliadin 123-132-specific cells were isolated from small intestine biopsies of patients on either gluten-free or gluten-containing diets. Short-term T cell lines derived from the small intestinal mucosa and specific for the 123-132 epitope recognized human APC pulsed with either whole recombinant alpha-gliadin or a partial pepsin-trypsin gliadin digest. Finally, we speculate on a possible mechanism leading to processing and presentation of class I-restricted gliadin-derived epitopes in celiac disease patients.     

Evidence for an active transport of morphine-6-beta-d-glucuronide but not P-glycoprotein-mediated at the blood-brain barrier

Morphine-6-beta-d-glucuronide (M6G) is an active metabolite of morphine with high analgesic potency despite a low blood-brain barrier (BBB) permeability. The aim of the study was to elucidate its transport mechanism across the BBB. We first checked if M6G was effluxed by the P-glycoprotein (P-gp), as previously reported by others. Second, we investigated the role of anionic transporters like the multidrug resistance-associated protein mrp1 and the glucose transporter GLUT-1. The brain uptake of [14C]M6G was measured by the in situ brain perfusion technique in wild-type and deficient mice [mdr1a(-/-) and mrp1(-/-)], with and without probenecid, digoxin, PSC833 or d-glucose. No difference was found between P-gp and mrp1 competent and deficient mice. The brain uptake of [14C]M6G co-perfused with probenecid in wild-type mice was not significantly different from that found in group perfused with [14C]M6G alone. The co-perfusion of [14C]M6G with digoxin or PSC833 was responsible of a threefold decrease of its uptake in mdr1a competent and deficient mice, suggesting that another transporter than P-gp and sensitive to digoxin and PSC833, may be involved. The co-perfusion of [14C]M6G with d-glucose revealed a threefold decrease in M6G uptake. In conclusion, P-gp and mrp1 are not involved in the transport of M6G at the BBB level in contrast to GLUT-1 and a digoxin-sensitive transporter (probably oatp2), which can actively transport M6G but with a weak capacity.