Clinical and diagnostic laboratory immunology, Mar 1, 2001
We have developed a novel plasmid-based, quantitative, in vitro screen to test the protease-inhib... more We have developed a novel plasmid-based, quantitative, in vitro screen to test the protease-inhibiting activities of existing and newly discovered agents.
SUMMARYDuring multicellular organization, individual cells need to constantly respond to environm... more SUMMARYDuring multicellular organization, individual cells need to constantly respond to environmental cues and adjust contractile and adhesive forces in order to maintain tissue integrity. The signaling pathways linking biochemical cues and tissue mechanics are unclear. Here, we show that Plexin-B2 regulates mechanochemical integration during multicellular organization. In human embryonic stem cells (hESCs), Plexin-B2 controls cell shape and tissue geometry in both 2D epithelial colony and 3D spheroid aggregates by regulating actomyosin contractility and junctional/cell-matrix adhesive properties. Atomic force microscopy (AFM) directly demonstrates that Plexin-B2 modulates cell stiffness in hESC colonies, which in turn impacts cell proliferation and cell fate specification through β-catenin signaling and YAP mechanosensing. YAP also functions as a mechanoregulator downstream of Plexin-B2, thus forming a mechanochemical integrative loop. In human neuroprogenitor cells (hNPCs), Plexi...
We studied the constitutive and lipopolysaccharide (LPS)-induced expression of nuclear protein bi... more We studied the constitutive and lipopolysaccharide (LPS)-induced expression of nuclear protein binding to the negative regulatory element (NRE) of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in fresh human monocytes. We demonstrated the existence of a constitutive factor binding to the NRE 73-bp HpaII/HpaII fragment (–216 to –143) whose expression is up-regulated by LPS treatment. Competition experiments with overlapping oligonucleotides covering the HpaII/HpaII fragment and with mutated oligonucleotides mapped the binding within the TTTCATCAC region (–171 to –163). This binding pattern is unique to human monocytes. J. Leukoc. Biol. 56: 21–26; 1994.
The important role of CD8 + T cells in controlling HIV-1 infection through the innate as well as ... more The important role of CD8 + T cells in controlling HIV-1 infection through the innate as well as the adaptive immune system is well established. In addition to the major histocompatibility complex (MHC)dependent cytotoxic activity of CD8 + T cells, they produce soluble factors that suppress HIV-1 replication in an MHC-independent manner. Several of those factors have been identified, including -chemokines, Rantes, MIP-1␣, MIP-1, and MDC. We previously identified that prothymosin alpha (ProT␣) in the conditioned medium of HVS transformed CD8 + T cells was a potent inhibitor of HIV-1 replication following proviral integration. In this report we further characterize the anti-HIV-1 activity of ProT␣ by demonstrating its target-cell specificity, distinction from additional inhibitors of HIV-1 transcription in CD8 + T cell supernatants, as well as the differential regulation of host cell antiviral genes that could impact HIV-1 replication. These genes include a number of transcription factors as well IFN-␣-inducible genes including PKR, IRF1, and Rantes, in the absence of induction of IFN-␣. These data suggest that the anti-HIV-1 activity of ProT␣ is mediated through the modulation of a number of genes that have been reported to suppress HIV-1 replication including the dysregulation of transcription factors and the induction of PKR and Rantes mRNA.
IL-2 has pleiotropic properties and is a potent activator of monocytic functions. Since monocytes... more IL-2 has pleiotropic properties and is a potent activator of monocytic functions. Since monocytes are an important source of the chemoattractant cytokine IL-8, we studied the effects of IL-2 on the expression of IL-8 in human monocytes. IL-8 mRNA expression was detectable in resting human monocytes. Treatment of monocytes with IL-2 increased IL-8 mRNA expression by a protein synthesis-independent process. The augmentation of IL-8 mRNA by IL-2 was associated with an increase in IL-8 secretion. The expression of IL-8 mRNA was not a nonspecific response to any stimulus of monocyte activation. In fact, IFN-gamma, which is also a potent monocyte activator, not only failed to induce IL-8 expression but inhibited the stimulation of IL-8 by IL-2. Nuclear run-on experiments demonstrated that both the enhancement of IL-8 mRNA expression and its down-regulation by IFN-gamma occurred at the transcriptional level. These results show for the first time that in fresh human monocytes, IL-8 expressi...
Recently purified and molecularly cloned monocyte chemotactic and activating factor (MCAF) may pl... more Recently purified and molecularly cloned monocyte chemotactic and activating factor (MCAF) may play a major role in recruiting and activating monocytes in the inflammatory process. We examined the effects of a potent anti-inflammatory agent, dexamethasone (DXS), on the production of this factor. Over a wide range of concentrations (10(-5) to 10(-8) M), DXS inhibited the production of MCAF at the mRNA and protein level in a human fibrosarcoma cell line, which was stimulated with either IL-1 or TNF-alpha. We examined the turn-over of synthesized MCAF mRNA that showed DXS decreased the stability of MCAF mRNA. Furthermore, the addition of actinomycin D and cycloheximide abolished this effect of DXS, indicating that de novo mRNA and protein synthesis were required for this process. In addition, a nuclear run-off analysis revealed that DXS also inhibited the transcription of IL-1- or TNF-activated MCAF genes. Therefore, both the destabilization of MCAF mRNA and the inhibition of transcrip...
During morphogenesis, molecular mechanisms that orchestrate biomechanical dynamics across cells r... more During morphogenesis, molecular mechanisms that orchestrate biomechanical dynamics across cells remain unclear. Here, we show a role of guidance receptor Plexin-B2 in organizing actomyosin network and adhesion complexes during multicellular development of human embryonic stem cells and neuroprogenitor cells. Plexin-B2 manipulations affect actomyosin contractility, leading to changes in cell stiffness and cytoskeletal tension, as well as cell-cell and cell-matrix adhesion. We have delineated the functional domains of Plexin-B2, RAP1/2 effectors, and the signaling association with ERK1/2, calcium activation, and YAP mechanosensor, thus providing a mechanistic link between Plexin-B2-mediated cytoskeletal tension and stem cell physiology. Plexin-B2-deficient stem cells exhibit premature lineage commitment, and a balanced level of Plexin-B2 activity is critical for maintaining cytoarchitectural integrity of the developing neuroepithelium, as modeled in cerebral organoids. Our studies thu...
Public reporting burden for this collection of information is estimated to average 1 hour per res... more Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number.
Prothymosin α (ProTα) (isoform 2: iso2) is a widely distributed, small acidic protein with intrac... more Prothymosin α (ProTα) (isoform 2: iso2) is a widely distributed, small acidic protein with intracellular and extracellular-associated functions. Recently, we identified two new ProTα variants with potent anti-HIV activity from CD8+ T cells and cervicovaginal lavage. The first is a splice variant of the ProTα gene known as isoB and the second is the product of ProTα pseudogene 7 (p7). Similarly to iso2, the anti-HIV activity of both variants is mediated by type I IFN. Here we tested whether the immunomodulatory activity of isoB and p7 are also TLR4 dependent and determined their kinetic of release in response to HIV-1 infection. Type I, type III, TNF-α and IL-6 mRNA inducing activity was determined in macrophages from wild type and TLR4 knockout mice treated with recombinant ProTα variants. Supernatants from mock and HIV infected cells were analyzed by mass spectrometry in positive and negative modes for the presence of ProTα variants. In silico structural and functional analysis of ...
Journal of the American Society of Nephrology : JASN, Jan 21, 2014
Dysregulation of polycystin-1 (PC1) leads to autosomal dominant polycystic kidney disease (ADPKD)... more Dysregulation of polycystin-1 (PC1) leads to autosomal dominant polycystic kidney disease (ADPKD), a disorder characterized by the formation of multiple bilateral renal cysts, the progressive accumulation of extracellular matrix (ECM), and the development of tubulointerstitial fibrosis. Correspondingly, cystic epithelia express higher levels of integrins (ECM receptors that control various cellular responses, such as cell proliferation, migration, and survival) that are characteristically altered in cystic cells. To determine whether the altered expression of ECM and integrins could establish a pathologic autostimulatory loop, we tested the role of integrin-β1 in vitro and on the cystic development of ADPKD in vivo. Compared with wild-type cells, PC1-depleted immortalized renal collecting duct cells had higher levels of integrin-β1 and fibronectin and displayed increased integrin-mediated signaling in the presence of Mn(2+). In mice, conditional inactivation of integrin-β1 in collec...
Soluble factors from CD8(+) T cells and cervicovaginal mucosa of women are recognized as importan... more Soluble factors from CD8(+) T cells and cervicovaginal mucosa of women are recognized as important in controlling human immunodeficiency virus type 1 (HIV-1) infection and transmission. Previously, we have shown the strong anti-HIV-1 activity of prothymosin α (ProTα) derived from CD8(+) T cells. ProTα is a small acidic protein with wide cell distribution, to which several functions have been ascribed, depending on its intracellular or extracellular localization. To date, activities of ProTα have been attributed to a single protein known as isoform 2. Here we report the isolation and identification of 2 new ProTα variants from CD8(+) T cells and cervicovaginal lavage with potent anti-HIV-1 activity. The first is a splice variant of the ProTα gene, known as isoform CRA_b, and the second is the product of a ProTα gene, thus far classified as a pseudogene 7. Native or recombinant ProTα variants potently restrict HIV-1 replication in macrophages through the induction of type I interferon...
Background: The cilium and cilial proteins have emerged as principal mechanosensors of renal epit... more Background: The cilium and cilial proteins have emerged as principal mechanosensors of renal epithelial cells responsible for translating mechanical forces into intracellular signals. Polycystin-2 (PC-2), a cilial protein, regulates flow/shear-induced changes in intracellular Ca2+ ([Ca2+]i) and recently has been implicated in the regulation of mitogen-activated protein (MAP) kinases. We hypothesize that fluid shear stress (FSS) activates PC-2 which regulates MAP kinase and, in turn, induces MAP kinase-dependent gene expression, specifically, monocyte chemoattractant protein-1 (MCP-1). Methods: To test this, PC-2 expression was constitutively reduced in a murine inner medullary collecting duct (IMCD3) cell line, and the expression of FSS-induced MCP-1 expression and MAP kinase signaling compared between the parental (PC-2-expressing) and PC-2-deficient IMCD3 cells. Results: FSS induces MAP kinase signaling and downstream MCP-1 mRNA expression in wild-type IMCD3 cells, while inhibitor...
Proceedings of the National Academy of Sciences, 2011
Primary cilia are required for several signaling pathways, but their function in cellular morphog... more Primary cilia are required for several signaling pathways, but their function in cellular morphogenesis is poorly understood. Here we show that emergence of an hexagonal cellular pattern during development of the corneal endothelium (CE), a monolayer of neural crest-derived cells that maintains corneal transparency, depends on a precise temporal control of assembly of primary cilia that subsequently disassemble in adult corneal endothelial cells (CECs). However, cilia reassembly occurs rapidly in response to an in vivo mechanical injury and precedes basal body polarization and cellular elongation in mature CECs neighboring the wound. In contrast, CE from hypomorphic IFT88 mutants ( Tg737 orpk ) or following in vivo lentiviral-mediated IFT88 knockdown display dysfunctional cilia and show disorganized patterning, mislocalization of junctional markers, and accumulation of cytoplasmic acetylated tubulin. Our results indicate an active role of cilia in orchestrating coordinated morphogen...
Proceedings of the National Academy of Sciences, 2010
Induction of type I interferons (IFN) is a central feature of innate immune responses to microbia... more Induction of type I interferons (IFN) is a central feature of innate immune responses to microbial pathogens and is mediated via Toll-like receptor (TLR)-dependent and -independent pathways. Prothymosin-α (ProTα), a small acidic protein produced and released by CD8 + T cells, inhibits HIV-1, although the mechanism for its antiviral activity was not known. We demonstrate that exogenous ProTα acts as a ligand for TLR4 and stimulates type I IFN production to potently suppress HIV-1 after entry into cells. These activities are induced by native and recombinant ProTα, retained by an acidic peptide derived from ProTα, and lost in the absence of TLR4. Furthermore, we demonstrate that ProTα accounts for some of the soluble postintegration HIV-1 inhibitory activity long ascribed to CD8 + cells. Thus, a protein produced by CD8 + T cells of the adaptive immune system can exert potent viral suppressive activity through an innate immune response. Understanding the mechanism of IFN induction by P...
Proceedings of the National Academy of Sciences, 1993
Interleukin 1 (IL-1) induces pleiotropic effects in many cell types during inflammation and immun... more Interleukin 1 (IL-1) induces pleiotropic effects in many cell types during inflammation and immunity. We have recently shown how the IL-1 signal is transmitted to the nucleus: In T cells and in pituitary cells, IL-1 induced genes via activation of the nuclear factor AP-1. We now demonstrate how IL-1 activates the AP-1 factor in liver cells, which are a major target for IL-1 during the acute phase response in vivo. IL-1 induced gene transcription of both AP-1 components, c-jun and c-fos. IL-1 also increased the stability of c-jun mRNA. We define two enhancer sites in the jun promoter that are required for induction by IL-1. Although the binding sites share some similarity with the AP-1 binding site, the nuclear factors binding the jun motifs are not composed of Jun or Fos proteins. Thus these data identify two binding proteins that serve as one of the first nuclear targets for IL-1 signal transduction.
Viral interference is characterized by the resistance of infected cells to infection by a challen... more Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine wheth...
Journal of the American Society of Nephrology, 2002
ABSTRACT. HIV-associated nephropathy (HIVAN) is now the third leading cause of end-stage renal di... more ABSTRACT. HIV-associated nephropathy (HIVAN) is now the third leading cause of end-stage renal disease in the African American population. HIV-1 infects renal tubular and glomerular epithelial cells or podocytes, cells that are a critical part of the filtration barrier. HIV-1 infection induces the loss of podocyte differentiation markers and increases podocyte proliferation. It has been previously shown that HIV-infection induces loss of contact inhibition. Here, the HIV-1 gene responsible for proliferative changes is identified by using cultured podocytesin vitro. The HIV-1 proviral construct, pNL4-3 was rendered noninfectious by replacing the HIV-1gag/polsequences with an EGFP reporter gene (pNL4-3: ΔG/P-EGFP). This construct was then pseudotyped with VSV.G envelope to infect podocytes that were conditionally immortalized with SV-40 T antigen. In addition, mutated constructs were engineered with premature stop codons in the HIV-1env,vif,vpr,vpu,nef, orrevgenes. The parental constr...
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Papers by Luca Gusella