Cathepsin S in tumours, regional lymph nodes and sera of patients with lung cancer: relation to prognosis

Cysteine proteinase cathepsin S (Cat S) is expressed mainly in lymphatic tissues and has been characterised as a key enzyme in major histocompatibility complex class II (MHC-II) mediated antigen presentation. Cat S has been measured in tissue cytosols of lung parenchyma, lung tumours and lymph nodes and in sera of patients with lung tumours and of healthy controls, by specific enzyme-linked immunosorbent assay (ELISA). A difference in Cat S level was found between tumour and adjacent control tissue cytosols of 60 lung cancer patients (median 4.3 vs. 2.8 ng mg−1protein). In lymph nodes obtained from 24 patients of the same group, the level of Cat S was significantly higher than in tumours or lung parenchyma (P< 0.001). Additionally, significantly higher levels were found in non-infiltrated than in infiltrated lymph nodes (median 16.6 vs 7.5 ng mg−1protein). Patients with low levels of Cat S in tumours and lung parenchyma exhibited a significantly higher risk of death than those with high levels of Cat S (P= 0.025 – tumours;P= 0.02 – parenchyma). Immunohistochemical analysis (IHA) of lung parenchyma revealed a staining reaction in alveolar type II cells, macrophages and bronchial epithelial cells. In regional lymph node tissue, strong staining of Cat S was found in lymphocytes and histiocytes. Nevertheless, Cat S was detected also in tumour cells, independently of their origin. Our results provide evidence that Cat S may be involved in malignant progression. Its role, however, differs from that of the related Cats B and L and could be associated with the immune response rather than with remodelling of extracellular matrix. © 2001 Cancer Research Campaign  http://www.bjcancer.co

Whole Transcriptome Profiling of Successful Immune Response to Vibrio Infections in the Oyster Crassostrea gigas by Digital Gene Expression Analysis

The cultivated Pacific oyster Crassostrea gigas has suffered for decades large scale summer mortality phenomenon resulting from the interaction between the environment parameters, the oyster physiological and/or genetic status and the presence of pathogenic microorganisms including Vibrio species. To obtain a general picture of the molecular mechanisms implicated in C. gigas immune responsiveness to circumvent Vibrio infections, we have developed the first deep sequencing study of the transcriptome of hemocytes, the immunocompetent cells. Using Digital Gene Expression (DGE), we generated a transcript catalog of up-regulated genes from oysters surviving infection with virulent Vibrio strains (Vibrio splendidus LGP32 and V. aestuarianus LPi 02/41) compared to an avirulent one, V. tasmaniensis LMG 20012T. For that an original experimental infection protocol was developed in which only animals that were able to survive infections were considered for the DGE approach. We report the identification of cellular and immune functions that characterize the oyster capability to survive pathogenic Vibrio infections. Functional annotations highlight genes related to signal transduction of immune response, cell adhesion and communication as well as cellular processes and defence mechanisms of phagocytosis, actin cytosqueleton reorganization, cell trafficking and autophagy, but also antioxidant and anti-apoptotic reactions. In addition, quantitative PCR analysis reveals the first identification of pathogen-specific signatures in oyster gene regulation, which opens the way for in depth molecular studies of oyster-pathogen interaction and pathogenesis. This work is a prerequisite for the identification of those physiological traits controlling oyster capacity to survive a Vibrio infection and, subsequently, for a better understanding of the phenomenon of summer mortality

A “Crossomics” Study Analysing Variability of Different Components in Peripheral Blood of Healthy Caucasoid Individuals

Background: Different immunotherapy approaches for the treatment of cancer and autoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable. Methodology/Principal Findings: To assess intra- and inter-individual variability of various biomarkers, peripheral blood of 16 age and gender equilibrated healthy volunteers was sampled on 3 different days within a period of one month. Complex "crossomics'' analyses of plasma metabolite profiles, antibody concentrations and lymphocyte subset counts as well as whole genome expression profiling in CD4(+)T and NK cells were performed. Some of the observed age, gender and BMI dependences are in agreement with the existing knowledge, like negative correlation between sex hormone levels and age or BMI related increase in lipids and soluble sugars. Thus we can assume that the distribution of all 39.743 analysed markers is well representing the normal Caucasoid population. All lymphocyte subsets, 20% of metabolites and less than 10% of genes, were identified as highly variable in our dataset. Conclusions/Significance: Our study shows that the intra- individual variability was at least two-fold lower compared to the inter-individual one at all investigated levels, showing the importance of personalised medicine approach from yet another perspective

Cathepsin L Inhibition Prevents Murine Autoimmune Diabetes via Suppression of CD8+ T Cell Activity

Background: Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet b cells. To determine whether specific lysosomal proteases might influence the outcome of a T cell–mediated autoimmune response, we examined the functional significance of cathepsin inhibition on autoimmune T1D-prone non-obese diabetic (NOD) mice. Methods and Findings: Here it was found that specific inhibition of cathepsin L affords strong protection from cyclophosphamide (CY)-induced insulitis and diabetes of NOD mice at the advanced stage of CD8 + T cell infiltration via inhibiting granzyme activity. It was discovered that cathepsin L inhibition prevents cytotoxic activity of CD8 + T cells in the pancreatic islets through controlling dipeptidyl peptidase I activity. Moreover, the gene targeting for cathepsin L with application of in vivo siRNA administration successfully prevented CY-induced diabetes of NOD mice. Finally, cathepsin L mRNA expression of peripheral CD8 + T cells from NOD mice developing spontaneous T1D was significantly increased compared with that from control mice. Conclusions: Our results identified a novel function of cathepsin L as an enzyme whose activity is essential for the progression of CD8 + T cell-mediated autoimmune diabetes, and inhibition of cathepsin L as a powerful therapeutic strateg

A Helminth Immunomodulator Exploits Host Signaling Events to Regulate Cytokine Production in Macrophages

Parasitic worms alter their host's immune system to diminish the inflammatory responses directed against them, using very efficient immunomodulating molecules. We have previously shown that the helminth immunomodulator cystatin (AvCystatin) profoundly reduces the progression of inflammatory diseases via modulation of macrophages. Here we elucidate the signaling events in macrophages triggered by AvCystatin. Labeled AvCystatin was predominantly taken up by macrophages and subsequently induced the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK1/2 and p38. IL-10 expression induced by AvCystatin in macrophages was tyrosine kinase sensitive and dependent on activation of both MAP kinases, in clear contrast to expression of IL-12/23p40. In addition, phosphorylation of the transcription factors CREB and STAT3 was induced by AvCystatin and regulated by phospho-ERK. Chemical inhibition of phosphoinositide 3-kinase (PI3K) reduced AvCystatin-induced cytokine release; however, AKT, the downstream target of PI3K, was not activated following AvCystatin exposure. To characterize signaling elements involved in alteration of the macrophage phenotype we applied mathematical modeling. Experimental testing of the in silico generated hypotheses identified dual specificity phosphatase (DUSP) 1 and 2, as regulators in AvCystatin triggered macrophages in vitro and in vivo. In particular, DUSP1 was subsequently found to be responsible for regulation of ERK- and p38-phosphorylation and controlled the IL-10 expression in macrophages by AvCystatin. Thus, we show that AvCystatin exploits activation and deactivation pathways of MAP kinases to induce regulatory macrophages. This study provides insights into molecular mechanisms of macrophage manipulation by parasites and highlights the utility of mathematical modeling for the elucidation of regulatory circuits of immune cells

Photoactive assemblies of organic compounds and biomolecules: drug-protein supramolecular systems

[EN] The properties of singlet and triplet excited states are strongly medium-dependent. Hence, these species constitute valuable tools as reporters to probe compartmentalised microenvironments, including drug@protein supramolecular systems. In the present review, the attention is focused on the photophysical properties of the probe drugs (rather than those of the protein chromophores) using transport proteins (serum albumins and 1-acid glycoproteins) as hosts. Specifically, fluorescence measurements allow investigating the structural and dynamic properties of biomolecules or their complexes. Thus, the emission quantum yields and the decay kinetics of the drug singlet excited states provide key information to determine important parameters such as the stoichiometry of the complex, the binding constant, the relative degrees of occupancy of the different compartments, etc. Application of the FRET concept allows determining donor-acceptor interchromophoric distances. In addition, anisotropy measurements can be related to the orientation of the drug within the binding sites, where the degrees of freedom for conformational relaxation are restricted. Transient absorption spectroscopy is also a potentially powerful tool to investigate the binding of drugs to proteins, where formation of encapsulated triplet excited states is favoured over other possible processes leading to ionic species (i. e. radical ions), and their photophysical properties are markedly sensitive to the microenvironment experienced within the protein binding sites. Even under aerobic conditions, the triplet lifetimes of protein-complexed drugs are remarkably long, which provides a broad dynamic range for identification of distinct triplet populations or for chiral discrimination. Specific applications of the laser flash photolysis technique include the determination of drug distribution among the bulk solution and the protein binding sites, competition of two types of proteins to bind a 3 drug, occurrence of drug-drug interactions within protein binding sites, enzymatic-like activity of the protein or determination of enantiomeric compositions. The use of proteins as supramolecular hosts modifies the photoreactivity of encapsulated substrates by providing protection against oxygen or other external reagents, by imposing conformational restrictions in the binding pockets, or by influencing the stereochemical outcome. In this review, a selected group of examples is presented including decarboxylation, dehalogenation, nucleophilic addition, dimerisation, oxidation, Norrish type II reaction, photo-Fries rearrangement and 6 electrocyclisationFinancial support from the Spanish Government (CTQ2010-14882, JCI-2011-09926, RyC-2007-00476), from the EU (PCIG12-GA-2012-334257), from the Universitat Politènica de València (SP20120757) and from the Consellería de Educació, Cultura i Esport (PROMETEOII/2013/005, GV/2013/051) is gratefully acknowledged.Vayá Pérez, I.; Lhiaubet-Vallet, VL.; Jiménez Molero, MC.; Miranda Alonso, MÁ. (2014). Photoactive assemblies of organic compounds and biomolecules: drug-protein supramolecular systems. Chemical Society Reviews. 43:4102-4122. https://doi.org/10.1039/C3CS60413FS410241224

Dysregulation of Macrophage-Secreted Cathepsin B Contributes to HIV-1-Linked Neuronal Apoptosis

Chronic HIV infection leads to the development of cognitive impairments, designated as HIV-associated neurocognitive disorders (HAND). The secretion of soluble neurotoxic factors by HIV-infected macrophages plays a central role in the neuronal dysfunction and cell death associated with HAND. One potentially neurotoxic protein secreted by HIV-1 infected macrophages is cathepsin B. To explore the potential role of cathepsin B in neuronal cell death after HIV infection, we cultured HIV-1ADA infected human monocyte-derived macrophages (MDM) and assayed them for expression and activity of cathepsin B and its inhibitors, cystatins B and C. The neurotoxic activity of the secreted cathepsin B was determined by incubating cells from the neuronal cell line SK-N-SH with MDM conditioned media (MCM) from HIV-1 infected cultures. We found that HIV-1 infected MDM secreted significantly higher levels of cathepsin B than did uninfected cells. Moreover, the activity of secreted cathepsin B was significantly increased in HIV-infected MDM at the peak of viral production. Incubation of neuronal cells with supernatants from HIV-infected MDM resulted in a significant increase in the numbers of apoptotic neurons, and this increase was reversed by the addition of either the cathepsin B inhibitor CA-074 or a monoclonal antibody to cathepsin B. In situ proximity ligation assays indicated that the increased neurotoxic activity of the cathepsin B secreted by HIV-infected MDM resulted from decreased interactions between the enzyme and its inhibitors, cystatins B and C. Furthermore, preliminary in vivo studies of human post-mortem brain tissue suggested an upregulation of cathepsin B immunoreactivity in the hippocampus and basal ganglia in individuals with HAND. Our results demonstrate that HIV-1 infection upregulates cathepsin B in macrophages, increases cathepsin B activity, and reduces cystatin-cathepsin interactions, contributing to neuronal apoptosis. These findings provide new evidence for the role of cathepsin B in neuronal cell death induced by HIV-infected macrophages