Quantitative Proteomics for the Identification of Differentially Expressed Proteins in the Extracellular Vesicles of Cervical Cancer Cells.

The extracellular vesicles (EVs) in a tumoral microenvironment can exert different functions by transferring their content, which has been poorly described in cervical cancer. Here, we tried to clarify the proteomic content of these EVs, comparing those derived from cancerous HPV (+) keratinocytes (HeLa) versus those derived from normal HPV (-) keratinocytes (HaCaT). We performed a quantitative proteomic analysis, using LC-MS/MS, of the EVs from HeLa and HaCaT cell lines. The up- and downregulated proteins in the EVs from the HeLa cell line were established, along with the cellular component, molecular function, biological processes, and signaling pathways in which they participate. The biological processes with the highest number of upregulated proteins are cell adhesion, proteolysis, lipid metabolic process, and immune system processes. Interestingly, three of the top five signaling pathways with more up- and downregulated proteins are part of the immune response. Due to their content, we can infer that EVs can have a significant role in migration, invasion, metastasis, and the activation or suppression of immune system cells in cancer.

Recent Advances in the Discovery and Function of Antimicrobial Molecules in Platelets.

The conventional function described for platelets is maintaining vascular integrity. Nevertheless, increasing evidence reveals that platelets can additionally play a crucial role in responding against microorganisms. Activated platelets release molecules with antimicrobial activity. This ability was first demonstrated in rabbit serum after coagulation and later in rabbit platelets stimulated with thrombin. Currently, multiple discoveries have allowed the identification and characterization of PMPs (platelet microbicidal proteins) and opened the way to identify kinocidins and CHDPs (cationic host defense peptides) in human platelets. These molecules are endowed with microbicidal activity through different mechanisms that broaden the platelet participation in normal and pathologic conditions. Therefore, this review aims to integrate the currently described platelet molecules with antimicrobial properties by summarizing the pathways towards their identification, characterization, and functional evaluation that have promoted new avenues for studying platelets based on kinocidins and CHDPs secretion.

Extracellular Vesicles in Cervical Cancer and HPV Infection.

Since their description, extracellular vesicles (EVs) have shown growing relevance in cancer progression. These cell structures contain and transfer molecules such as nucleic acids (including DNA and RNA), proteins, and lipids. Despite the rising information about EVs' relationship with cancer, there is still scarce evidence about their content and function in cervical cancer. Interestingly, the composition and purposes of some cellular molecules and the expression of oncogenic proteins packaged in EVs seem modified in HPV-infected cells; and, although only the E6 oncogenic protein has been detected in exosomes from HPV-positive cells, both E6/E7 oncogenes mRNA has been identified in EVs; however, their role still needs to be clarified. Given that EVs internalizing into adjacent or distant cells could modify their cellular behavior or promote cancer-associated events like apoptosis, proliferation, migration, or angiogenesis in receptor cells, their comprehensive study will reveal EV-associated mechanisms in cervical cancer. This review summarizes the current knowledge in composition and functions of cervical cancer and HPV Infection-derived EVs.

Evaluation of the Interference of Lipemia and Hemolysis in the Detection Limit of Anti-HIV-1 Antibodies.

BACKGROUND: The instructions of manufacturers of methodologies for anti-HIV-1/2 antibodies screening tests re-commend avoiding analyzing blood samples with hemolysis or lipemia, but they do not mention references about scientific studies evaluating their interference. The increased need for an opportune detection of HIV infection to avoid its spread has led to public health institutions including routine HIV screening even in internal medicine and emergency rooms. Nevertheless, these blood samples are usually associated with the presence of lipemia and/ or hemolysis, leaving doubt for probable misinterpretations. This fact highlights the need for applying verification techniques, established under the internal methodological conditions of each laboratory, in order to increase the coverage of HIV screening and to ensure the reliability of their results. METHODS: Following the ethics committee approval and patient's informed consent, a confirmed anti-HIV-1 positive human serum (undetectable viral load and p24 antigen, and stable total lymphocytes > 30%) was obtained. This work describes techniques for the semiquantitative analysis of anti-HIV antibodies of three commercial HIV-screening methodologies (immunochromatography, enzyme-immunoassay and chemiluminescence) and to deter-mine the detection limit of these screening tests, as well as evaluating the maximum concentration of total lipids and of free hemoglobin that do not interfere in the detection limits. RESULTS: The highest analyzed concentration of total lipid (870 mg/dL) did not interfere with the detection limits of anti-HIV-1 antibodies in any of the evaluated methodologies. Free hemoglobin presented interference at different concentrations depending on the methodology: immunochromatography (0.57 g/dL)), enzyme-immunoassay (8.6 g/dL), and chemiluminescence (11.5 g/dL)). CONCLUSIONS: Concentrations of lipemia above postprandial levels or hemolysis induced by experimental manipulation might not interfere with HIV-serological screening. Determining the maximum permissible limits of lipemia and hemolysis by each manufacturer or laboratory based on an internal evaluation of their serological methodology would increase the reliability of HIV-diagnosis in internal medicine and emergency rooms and in patients with dyslipidemia or physiological hemolysis.

Infections as triggers of flares in systemic autoimmune diseases: novel innate immunity mechanisms.

PURPOSE OF REVIEW: The innate immune response (IIR) has to be immediate facing pathogens, and effective to induce a long-lasting adaptive immunity and immune memory. In genetically susceptible individuals, beyond a first defense, a chronically activated by infections IIR may represent a trigger for the onset or flares in systemic autoimmune diseases. This article reviews the recent scientific literature in this regard and highlights the key issues needing investigation. RECENT FINDINGS: Thanks to its high specificity mediated by pattern recognition receptors, the IIR is not called unspecific anymore. The discovery of these increasingly accurate recognizing molecular mechanisms has also evidenced their involvement in breaking self-immune tolerance and to maintain chronic inflammation in autoimmune responses. Neutrophil extracellular traps (NETS) as the main source of antinuclear antibodies; the 'neutrophils-pDC activation loop' theory; and the Th1/Th2/Th17 misbalances induced by microbial products because of chronically activated innate immune cells, are some of the recent uncovered IIR origins involved in infectious-induced systemic autoimmune diseases. SUMMARY: A deeper understanding of the genetic predisposition and the pathogen-derived factors responsible to exacerbate the IIR might potentially provide therapeutic targets to counteract flares in systemic autoimmune diseases. VIDEO ABSTRACT.

Human CD4(+) effector T lymphocytes generated upon TCR engagement with self-peptides respond defectively to IL-7 in their transition to memory cells.

The peripheral repertoire of CD4(+) T lymphocytes contains autoreactive cells that remain tolerant through several mechanisms. However, nonspecific CD4(+) T cells can be activated in physiological conditions as in the course of an ongoing immune response, and their outcome is not yet fully understood. Here, we investigate the fate of human naive CD4(+) lymphocytes activated by dendritic cells (DCs) presenting endogenous self-peptides in comparison with lymphocytes involved in alloresponses. We generated memory cells (Tmem) from primary effectors activated with mature autologous DCs plus interleukin (IL)-2 (Tmauto), simulating the circumstances of an active immune response, or allogeneic DCs (Tmallo). Tmem were generated from effector cells that were rested in the absence of antigenic stimuli, with or without IL-7. Tmem were less activated than effectors (demonstrated by CD25 downregulation) particularly with IL-7, suggesting that this cytokine may favour the transition to quiescence. Tmauto and Tmallo showed an effector memory phenotype, and responded similarly to polyclonal and antigen-specific stimuli. Biochemically, IL-7-treated Tmallo were closely related to conventional memory lymphocytes based on Erk-1/2 activation, whereas Tmauto were more similar to effectors. Autologous effectors exhibited lower responses to IL-7 than allogeneic cells, which were reflected in their reduced proliferation and higher cell death. This was not related to IL-7 receptor expression but rather to signalling deficiencies, according to STAT5 activation These results suggest that ineffective responses to IL-7 could impair the transition to memory cells of naive CD4(+) T lymphocytes recognizing self-peptides in the setting of strong costimulation.

Human CD16+ and CD16- monocyte subsets display unique effector properties in inflammatory conditions in vivo.

Two major subsets of human Mo are identified based on CD14 and CD16 expression: the classical CD16(-) Mo and the minor CD14(+)CD16(+) Mo. In vitro studies suggested distinct function and differentiation potential for each cell population. However, the in vivo relevance of these findings remains unclear. To evaluate the development and function of human Mo in an in vivo model, we transferred both Mo subpopulations into the peritoneum of immunocompromised mice in homeostatic or inflammatory conditions. Inflammation was induced with soluble LPS or particulate zymosan. CD16(+) were more phagocytic and produced higher amounts of TNF and IL-6 than CD16(-) Mo early after transfer with zymosan. They also produced higher levels of ß2-defensin in any condition evaluated, which could represent a new marker for this subpopulation. In contrast, differentiating CD16(-) Mo (24 h after transfer) acquired greater APC capacity in LPS-induced peritonitis, whereas none of the Mo subsets attained this ability with zymosan. CX(3)CL1 supported the survival of both Mo subsets in vivo. Similar Mo subpopulations were present in human peritonitis. These results support the idea of specialized roles of the Mo subset, where CD16(+) might act in an immediate innate immune response, whereas CD16(-) could have a major role as APCs.

Tolerogenic dendritic cells generated with different immunosuppressive cytokines induce antigen-specific anergy and regulatory properties in memory CD4+ T cells.

Dendritic cells (DCs) are professional APCs involved in the initiation of both immunity and immunological tolerance. In autoimmune diseases or graft rejections, most reactive lymphocytes are effector/memory cells. It is believed that memory T cells are more resistant to tolerance induction than naive lymphocytes; however, studies on mechanisms for their efficient tolerization are still scarce. In this study, we generated human monocyte-derived DCs by culture with GM-CSF and IL-4 (control DCs), as well as tolerogenic DCs (tDCs) by adding IL-10, IL-10/TGF-beta1, or IL-10/IL-6. Cells were maturated with TNF-alpha/PGE(2). Compared with control DCs, tDCs had similar expression of HLA-DR, CD80, and CD86, lower expression of CD40, higher levels of macrophage markers, enhanced endocytic ability, increased secretion of IL-6, IL-10 (only tDCs generated with IL-10 and tDCs generated with IL-10/IL-6), and PGE(2), and lower secretion of IL-12 and IL-23. In vitro, tDCs had the capacity to induce anergy in tetanus toxoid-specific memory CD4(+) T cells, whereas the proliferative response to an unrelated Ag was intact. Anergy could be reverted upon exposure to IL-2. tDC-primed T cells have low suppressive ability. Nevertheless, the generation of both anergic and regulatory T cells was more efficient with tDCs generated with IL-10/TGF-beta1. Microarray-based gene expression profiling reflected modulated expression of several transcripts in tDCs. Surface CLIP-HLA-DR complexes and intracellular thrombospondin-1 were increased in the three tDCs. CD39 was highly expressed only in tDC-TGF, which correlated with increased adenosine production. We propose that these molecules, together with IL-10 and prostanoids, are key factors to induce Ag-specific tolerance in memory T cells.