Biomedical subjects

Alirio J Melendez

Publications and source records attributed to Alirio J Melendez.

17 recordsLinked to original sources

PubMed2007

Protein i: interference at protein level by intrabodies.

Immunoglobulin molecules have long been used in biomedical research as in vitro tools for identification, purification and functional manipulation of target proteins. The specificity and diversity of immunoglobulins can be exploited to target a wide range of intracellular proteins by expressing them in vivo. Such antibody molecules, which are expressed intracellularly and directed to defined sub-cellular compartments, are termed 'intrabodies'. They represent a new and versatile tool that has the potential to manipulate diverse biological processes. Gene knockout, antisense and small interfering RNA knockdown techniques, are employed to characterize the function of many gene products and to validate these gene products as potential drug targets for novel therapeutics, but they have their limitations. The experimental results to date suggest that intrabodies represent a powerful alternative to modulate protein function and analyze its effects. Moreover, they can also be utilized to target specific domains of a particular target protein. Even though their clinical application might take a long time, they can play an important role for target identification and validation in functional genomics and/or proteomics. Here we review the latest advances in the concept, construction and applications of Intrabodies.

HIV Infections↗

PubMed2006

Genome-wide gene expression profiling of human mast cells stimulated by IgE or FcepsilonRI-aggregation reveals a complex network of genes involved in inflammatory responses.

BACKGROUND: Mast cells are well established effectors of IgE-triggered allergic reactions and immune responses to parasitic infections. Recent studies indicate that mast cells may play roles in adaptive and innate immunity, suggesting an innovative view of the regulation of immune responses. Here, we profiled the transcriptome of human mast cells sensitized with IgE alone, or stimulated by FcepsilonRI aggregation. RESULTS: Our data show that among 8,793 genes examined, 559 genes are differentially regulated in stimulated mast cells when compared with resting/unstimulated mast cells. The major functional categories of upregulated genes include cytokines, chemokines, and other genes involved in innate and adaptive immune-responses. We observed the increased expression of over 63 gene-transcripts following IgE-sensitization alone. Our data was validated using Real-Time-PCR; ELISA and western blot. We confirmed that IgE alone does not trigger mast cell-immediate responses, such as calcium signals, degranulation or protein-phosphorylation. CONCLUSION: This report represents a substantial advance in our understanding of the genome wide effects triggered by "passive sensitization" or active stimulation of human mast cells, supporting mast cells' potential involvement in a wide range of inflammatory responses.

Gene Expression Profiling↗

PubMed2006

The immunogenicity and immunomodulatory function of osteogenic cells differentiated from mesenchymal stem cells.

Multipotent mesenchymal stem cells (MSC) are reported to be immunoprivileged as well as immunosuppressive. Hence, they are ideal candidates for allogeneic transplantation to induce regeneration of diseased tissues and organs. However, it is not known whether MSC would retain their immunoprivileged and immunomodulatory properties after differentiating into the local cell types of the transplantation site. This study sought to investigate this question with a novel New Zealand White rabbit osteogenesis model. Results showed that osteogenic cells differentiated from MSC (DOC) in vitro did not express the MHC class II molecule, were incapable of inducing allogeneic lymphocyte proliferation in mixed lymphocyte culture or generating CTL, were inhibitory in ongoing lymphocyte proliferation, and secreted anti-inflammatory cytokines (IL-10 and TGF-beta). There was a significantly higher secretion of IL-10 by DOC than that by MSC, while there was no significant difference between the TGF-beta secretion of MSC and DOC in vitro. However, after IFN-gamma treatment, TGF-beta secretion by DOC significantly decreased despite the increased production by MSC. Four weeks after local DOC implantation, despite MHC class II expression, second-set allogeneic skin rejection showed similar survival to first-set allogeneic skin rejection and DOC appeared to function as osteoblasts. In conclusion, DOC retained their immunoprivileged and immunomodulatory properties in vitro, but the latter was lost following transplantation.

Animals↗

PubMed2006

Sphingosine kinase 1 regulates pro-inflammatory responses triggered by TNFalpha in primary human monocytes.

Monocytes play an important role in inflammation, angiogenesis, and atherosclerosis. During these processes monocytes release pre-formed proinflammatory mediators from granules, and synthesize de novo cytokines and chemokines important in the amplification of the inflammatory response. One of the most prominent triggers of inflammatory responses is the cytokine TNFalpha. However, the intracellular signaling cascades triggered by TNFalpha are not fully understood. In this study we investigated the roles of SPHK on the TNFalpha-triggered responses on human primary monocytes. We show that TNFalpha rapidly triggers S1P generation and activation of SPHK. Moreover, our data shows that SPHK1 is the isoform activated by TNFalpha, and plays an essential role on the TNFalpha-triggered intracellular Ca2+ signals, degranulation, cytokine production, and activation of NFkappaB, thus suggesting a pivotal role for SPHK1 on the proinflammatory responses triggered by TNFalpha.

Blotting, Western↗

PubMed2005

Proteasome inhibition by lactacystin in primary neuronal cells induces both potentially neuroprotective and pro-apoptotic transcriptional responses: a microarray analysis.

Although inhibition of the ubiquitin proteasome system has been postulated to play a key role in the pathogenesis of neurodegenerative diseases, studies have also shown that proteasome inhibition can induce increased expression of neuroprotective heat-shock proteins (HSPs). The global gene expression of primary neurons in response to treatment with the proteasome inhibitor lactacystin was studied to identify the widest range of possible pathways affected. Our results showed changes in mRNA abundance, both at different time points after lactacystin treatment and at different lactacystin concentrations. Genes that were differentially up-regulated at the early time point but not when most cells were undergoing apoptosis might be involved in an attempt to reverse proteasome inhibitor-mediated apoptosis and include HSP70, HSP22 and cell cycle inhibitors. The up-regulation of HSP70 and HSP22 appeared specific towards proteasome inhibitor-mediated cell death. Overexpression of HSP22 was found to protect against proteasome inhibitor-mediated loss of viability by up to 25%. Genes involved in oxidative stress and the inflammatory response were also up-regulated. These data suggest an initial neuroprotective pathway involving HSPs, antioxidants and cell cycle inhibitors, followed by a pro-apoptotic response possibly mediated by inflammation, oxidative stress and aberrant activation of cell cycle proteins.

Acetylcysteine↗

PubMed2005

A critical role for sphingosine kinase in anaphylatoxin-induced neutropenia, peritonitis, and cytokine production in vivo.

The aim of our study was to investigate the roles played by sphingosine kinase (SPHK) in the anaphylatoxin C5a-triggered responses in vivo. Our data show that i.v. administration of C5a triggers a rapid neutropenic response, but pretreating mice with the SPHK inhibitor, N,N-dimethylsphingosine (DMS), 10 min before the C5a i.v. administration substantially inhibited the C5a-triggered neutropenia. Similarly the i.v. administration of C5a caused a rapid increase in the serum levels of TNF-alpha and IL-6, and this increase in cytokine levels was blocked by DMS. We then induced acute peritonitis with C5a. The C5a i.p. injection triggered a fast recruitment of neutrophils, later followed by monocytes, into the peritoneal cavity. Vascular permeability was also observed: when we i.v. injected Evans blue before C5a i.p. injection, we could observe a continued influx of the dye into the peritoneum. In mice pretreated with DMS, there was a significant reduction on the C5a-triggered neutrophil and monocyte infiltration, as well as a marked reduction on the Evans blue influx. Our data also show that the i.p. administration of C5a caused a rapid increase in TNF-alpha and IL-6 levels in the peritoneal cavity, and this increase in cytokine levels was substantially inhibited in mice pretreated with the SPHK inhibitor. Taken together, these observations suggest a potential role for SPHK in the C5a-triggered inflammatory responses in vivo.

Animals↗

PubMed2005

Expression profile of immune response genes in patients with Severe Acute Respiratory Syndrome.

BACKGROUND: Severe acute respiratory syndrome (SARS) emerged in later February 2003, as a new epidemic form of life-threatening infection caused by a novel coronavirus. However, the immune-pathogenesis of SARS is poorly understood. To understand the host response to this pathogen, we investigated the gene expression profiles of peripheral blood mononuclear cells (PBMCs) derived from SARS patients, and compared with healthy controls. RESULTS: The number of differentially expressed genes was found to be 186 under stringent filtering criteria of microarray data analysis. Several genes were highly up-regulated in patients with SARS, such as, the genes coding for Lactoferrin, S100A9 and Lipocalin 2. The real-time PCR method verified the results of the gene array analysis and showed that those genes that were up-regulated as determined by microarray analysis were also found to be comparatively up-regulated by real-time PCR analysis. CONCLUSIONS: This differential gene expression profiling of PBMCs from patients with SARS strongly suggests that the response of SARS affected patients seems to be mainly an innate inflammatory response, rather than a specific immune response against a viral infection, as we observed a complete lack of cytokine genes usually triggered during a viral infection. Our study shows for the first time how the immune system responds to the SARS infection, and opens new possibilities for designing new diagnostics and treatments for this new life-threatening disease.

Antigens, Viral↗

PubMed2005

Sphingosine kinase signalling in immune cells.

1. Sphingolipids are potent second messengers modulating biochemical intracellular events and acting as ligands to mediate extracellular systems. Sphingosine kinase (SPHK) is the enzyme that phosphorylates sphingosine into sphingosine-1-phosphate (S1P), a potent bioactive sphingolipid. 2. The fact that SPHK is highly conserved from protozoa to mammals and is ubiquitous in living tissues reveals important roles of the SPHK pathway for the maintenance of health maintenance. This is also supported by comprehensive reviews on features of its main product, S1P, as having intracellular as well as extracellular roles, inducing a wide range of physiological responses from triggering Ca2+ release from internal stores to promoting growth and cell motility. 3. Immune cell activities have been shown to be modulated by the dynamic balance between ceramide, sphingosine and S1P, conceptualized as a rheostat. Cell proliferation, differentiation, motility and survival have been attributed to the regulatory actions of S1P. The properties of SPHK activity in immune cells are linked to the functions of triggered growth and survival factors, phorbol esters, hormones, cytokines and chemokines, as well as antigen receptors, such as FcgammaRI and FcepsilonRI. 4. Mechanisms of the SPHK signalling pathway are explored as new targets for drug development to suppress inflammation and other pathological conditions.

Animals↗

PubMed2004

Anaphylatoxin signaling in human neutrophils. A key role for sphingosine kinase.

Anaphylatoxins activate immune cells to trigger the release of proinflammatory mediators that can lead to the pathology of several immune-inflammatory diseases. However, the intracellular signaling pathways triggered by anaphylatoxins are not well understood. Here we report for the first time that sphingosine kinase (SPHK) plays a key role in C5a-triggered signaling, leading to physiological responses of human neutrophils. We demonstrate that C5a rapidly stimulates SPHK activity in neutrophils and differentiated HL-60 cells. Using the SPHK inhibitor N,N-dimethylsphingosine (DMS), we show that inhibition of SPHK abolishes the Ca2+ release from internal stores without inhibiting phospholipase C or protein kinase C activation triggered by C5a but has no effect on calcium signals triggered by other stimuli (FcgammaRII). We also show that DMS inhibits degranulation, activation of the NADPH oxidase, and chemotaxis triggered by C5a. Moreover, an antisense oligonucleotide against SPHK1, in neutrophil-differentiated HL-60 cells, had similar inhibitory properties as DMS, suggesting that the SPHK utilized by C5a is SPHK1. Our data indicate that C5a stimulation decreases cellular sphingosine levels and increases the formation of sphingosine-1-phosphate. Exogenously added sphingosine has a dual effect on C5a-stimulated oxidative burst: it has a priming effect at lower concentrations but a dose-dependent inhibitory effect at higher concentrations; however, C5a-triggered protein kinase C activity was only reduced at high concentration of sphingosine. In contrast, C5a-triggered Ca2+ signals, chemotaxis, and degranulation were not affected by sphingosine at all. Exogenous sphingosine-1-phosphate, by itself, did not induce degranulation or chemotaxis, but it did marginally induce Ca2+ signals and oxidative burst and had a priming effect, enhancing all the C5a-triggered responses. Taken together, these results suggest that SPHK plays an important role in the immune-inflammatory pathologies triggered by anaphylatoxins in human neutrophils and point out SPHK as a potential therapeutic target for the treatment of diseases associated with neutrophil hyperactivation.

Anaphylatoxins↗

PubMed2004

Antisense knockdown of sphingosine kinase 1 in human macrophages inhibits C5a receptor-dependent signal transduction, Ca2+ signals, enzyme release, cytokine production, and chemotaxis.

The anaphylatoxin C5a is produced following the activation of the complement system and is associated with a variety of pathologies, including septic shock and adult respiratory distress syndrome, and with immune complex-dependent diseases such as rheumatoid arthritis. C5a has been shown to regulate inflammatory functions by interacting with its receptor, C5aR, which belong to the rhodopsin family of seven-transmembrane GPCRs. However, the intracellular signaling pathways triggered by C5aR on immune-effector cells are not well understood. In this report we present data showing that, in human monocyte-derived macrophages, C5aR uses the intracellular signaling molecule sphingosine kinase (SPHK)1 to trigger various physiological responses. Our data show that C5a rapidly stimulates the generation of sphingosine-1-phosphate, SPHK activity, and membrane translocation of SPHK1. Using an antisense oligonucleotide against SPHK1, we show that knockdown of SPHK1 abolishes the C5a-triggered intracellular Ca(2+) signals, degranulation, cytokine generation, and chemotaxis. Our study shows for the first time that SPHK1 not only plays a key role in the generation and release of proinflammatory mediators triggered by anaphylatoxins from human macrophages but is also involved in the process of immune cell motility, thus pointing out SPHK1 as a potential therapeutic target for the treatment of inflammatory and autoimmune diseases.

Calcium Signaling↗

PubMed2004

Fcgamma RI-triggered generation of arachidonic acid and eicosanoids requires iPLA2 but not cPLA2 in human monocytic cells.

Aggregation of receptors for immunoglobulin G (FcgammaRs) on myeloid cells activates a series of events that are key in the inflammatory response and that can ultimately lead to targeted cell killing by antibody-directed cellular cytotoxicity. Generation of lipid-derived proinflammatory mediators is an important component of the integrated cellular response mediated by receptors for the constant region of immunoglobulins (Fc). We have demonstrated previously that, in interferon-gamma-primed U937 cells, the high affinity receptor for IgG, FcgammaRI, is coupled to a novel intracellular signaling pathway that involves the sequential activation of phospholipase D, sphingosine kinase, calcium transients, and protein kinase C isoforms, leading to the activation of the NADPH-oxidative burst. Here, we investigate the nature of the phospholipase that regulates arachidonic acid and eicosanoid production. Our data show that FcgammaRI couples to iPLA(2)beta for the release of arachidonic acid and the generation of leukotriene B(4) and prostaglandin E(2). Activation of iPLA(2)beta was protein kinase C-dependent; on the other hand, platelet-activating factor triggered cPLA(2)alpha by means of the mitogen-activated protein kinase pathway. These studies demonstrate that intracellular PLA(2)s can be selectively regulated by different stimuli and suggest a critical role for iPLA(2)beta in the intracellular signaling cascades initiated by FcgammaRI and its functional role in the generation of key inflammatory mediators.

Arachidonic Acid↗

PubMed2002

Peritoneal CD5+ B-1 cells have signaling properties similar to tolerant B cells.

CD5(+) B (or B-1) cells are the normal precursors of B cell chronic lymphocytic leukemia. They differ from conventional B (B-2) cells with respect to their phenotype and mitogenic responses and are often secretors of the natural polyreactive antibodies in the serum. The origin of B-1 cells remains controversial, and the relationship between B-1 cells and autoreactive B cells is unclear. Here, we compare the signaling pathways that are activated by the engagement of the B cell antigen receptor (BCR) in B-1 and B-2 cells. Stimulation of the BCR leads to the induced activation of the three major classes of mitogen-activated protein kinases (MAPKs), ERK, JNK, and p38 MAPK, as well as the Akt kinase and the transcription factors nuclear factor of activated T cells (NF-AT) and NF-kappaB in B-2 cells. In contrast, B-1 cells have constitutive activation of ERK and NF-AT but exhibit delayed JNK and lack p38 MAPK and NF-kappaB induction upon BCR cross-linking. The lack of NF-kappaB activation in B-1 cells may be due to a lack of Akt activation in these cells. Furthermore, our study using specific inhibitors reveals that the extended survival of B-1 cells in culture is not due to the constitutive activation of ERK; nor is it due to Akt signaling or Bcl-x(L) up-regulation, since these are not induced in B-1 cells. The current findings of altered MAPK and NF-AT activation and lack of NF-kappaB induction in B-1 cells indicate that these cells have signaling properties similar to tolerant B cells that are chronically exposed to self-antigens. Indeed, BCR stimulation of B-1 cells does not lead to their full activation as indicated by their lack of maximal up-regulation of specific markers such as CD25, CD69, and CD86.

Animals↗

PubMed2002

Dichotomy of Ca2+ signals triggered by different phospholipid pathways in antigen stimulation of human mast cells.

Mast cell activation triggers Ca(2+) signals and the release of enzyme-containing granules, events that play a major role in allergic/hypersensitivity reactions. However, the precise molecular mechanisms that regulate antigen-triggered degranulation and Ca(2+) fluxes in human mast cells are still poorly understood. Here we show, for the first time, that a receptor can trigger Ca(2+) via two separate molecular mechanisms. Using an antisense approach, we show that IgE-antigen stimulation of human bone marrow-derived mast cells triggers a sphingosine kinase (SPHK) 1-mediated fast and transient Ca(2+) release from intracellular stores. However, phospholipase C (PLC) gamma1 triggers a second (slower) wave of calcium release from intracellular stores, and it is this PLCgamma1-generated signal that is responsible for Ca(2+) entry. Surprisingly, FcepsilonRI (a high affinity receptor for IgE)-triggered mast cell degranulation depends on the first, sphingosine kinase-mediated Ca(2+) signal. These two pathways act independently because antisense knock down of either enzyme does not interfere with the activity of the other enzyme. Of interest, similar to PLCgamma1, SPHK1 translocates rapidly to the membrane after FcepsilonRI cross-linking. Here we also show that SPHK1 activity depends on phospholipase D1 and that FcepsilonRI-triggered mast cell degranulation depends primarily on the activation of both phospholipase D1 and SPHK1.

Animals↗

PubMed2002

Phospholipase D and immune receptor signalling.

Immune receptors are coupled to the activation of phosphatidylcholine phospholipase D (PC-PLD) that hydrolyses phosphatidylcholine to generate phosphatidic acid and choline. As these receptors are also coupled to other signalling cascades, it has been difficult to define the precise cell activation events resulting from PLD activation in the absence of specific inhibitors. There is increasing evidence that phosphatidic acid acts as an intracellular signalling molecule regulating release of calcium from intracellular stores, sphingosine kinase and protein kinase C activation and membrane budding. Phosphatidic acid can also be rapidly converted into lysophosphatidic acid, diacylglycerol and arachidonates.

Animals↗

PubMed2002

C-reactive protein-mediated phagocytosis and phospholipase D signalling through the high-affinity receptor for immunoglobulin G (FcgammaRI).

C-reactive protein (CRP) is the prototypic acute-phase protein in man which performs innate immune functions. CRP-mediated phagocytosis may be indirect, through activation of complement and complement receptors, or direct, through receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) or even a putative CRP-specific receptor. No strong evidence has been shown to indicate which receptors may be responsible for phagocytosis or signalling responses. Using BIAcore technology, we confirm that CRP binds directly to the extracellular portion of FcgammaRI with a threefold higher affinity than IgG (KD = 0.81 x 10-9 m). Binding is Ca2+ dependent and is inhibited by IgG1 but not by phosphorylcholine (PC). CRP opsonization (using CRP concentrations within the normal human serum range) of PC-conjugated sheep erythrocytes increased phagocytosis of these particles by COS-7 cells transfected with FcgammaRI-II chimaera or FcgammaRI/gamma-chain. Interferon-gamma-treated U937 cells, which signal through FcgammaRI to activate phospholipase D (PLD) in response to cross-linked IgG, were also activated by CRP without any requirement for further cross-linking. These studies indicate that CRP is capable of binding to and cross-linking FcgammaRI thereby resulting in PLD activation and increased phagocytosis. Uptake by FcgammaRI has been reported to promote various acquired immune responses suggesting that CRP could act in a similar way.

Animals↗

PubMedDate not supplied

DNA microarray technology for target identification and validation.

1. Microarrays, a recent development, provide a revolutionary platform to analyse thousands of genes at once. They have enormous potential in the study of biological processes in health and disease and, perhaps, microarrays have become crucial tools in diagnostic applications and drug discovery. 2. Microarray based studies have provided the essential impetus for biomedical experiments, such as identification of disease-causing genes in malignancies and regulatory genes in the cell cycle mechanism. Microarrays can identify genes for new and unique potential drug targets, predict drug responsiveness for individual patients and, finally, initiate gene therapy and prevention strategies. 3. The present article reviews the principles and technological concerns, as well as the steps involved in obtaining and analysing of data. Furthermore, applications of microarray based experiments in drug target identifications and validation strategies are discussed. 4. To exemplify how this tool can be useful, in the present review we provide an overview of some of the past and potential future aspects of microarray technology and present a broad overview of this rapidly growing field.

Animals↗

PubMedDate not supplied

Short interfering RNA (siRNA) as a novel therapeutic.

1. RNA interference (RNAi) is a robust method of post-transcriptional silencing of genes using double-stranded RNA (dsRNA) with sequence homology driven specificity. The dsRNA can be between 21 and 23 nucleotides long: this is converted to small interfering RNA (siRNA), which then mediates gene silencing by degradation/blocking of translation of the target mRNA. 2. RNA interference provides a simple, fast and cost-effective alternative to existing gene targeting approaches both in vitro and in vivo. The discovery of siRNAs that cause RNAi in mammalian cells opened the door to the therapeutic use of siRNAs. Highly intense research efforts are now aimed at developing siRNAs for therapeutic purposes. 3. Recent advances in the design and delivery of targeting molecules now allow efficient and highly specific gene silencing in mammalian systems. Synthetic siRNA libraries targeting thousands of mammalian genes are publicly available for high-throughput genetic screens for target discovery and validation. Recent studies have demonstrated the clinical potential of aptly designed siRNAs in various types of viral infections, cancer and renal and neurodegenerative disorders. 4. The present review provides insight into the novel therapeutic strategies of siRNA technology, which is the latest development in nucleic acid-based tools for knocking down gene expression, and its potential for silencing genes associated with various human diseases.

Amyloid Precursor Protein Secretases↗