Direct induction of lymphocyte killing by calcium ionophore and phorbol ester treatment

To analyze transduction mechanisms in human lymphocyte killing, intracellular Ca2+ levels were increased by ionophore A23187 treatment and protein kinase C activated by phorbol ester 12-O-tetradecanoylphorbol-acetate (TPA). Drugs were tested either alone or in combinations on effector cells active in natural, antibody-dependent, and lectin-dependent killing. TPA suppressed killing in all systems at 100 ng/ml whereas A23187 was only suppressive for NK killing at concentrations higher than 0.1 microM. TPA combined with A23187, above 10 ng/ml and 0.5 microM, respectively, induced killing of all tested target cell lines with a slower kinetic than NK killing of K562 cells. Drug-induced killing did not increase optimal lectin and antibody-dependent killing and was demonstrated most easily on NK-resistant target cell lines. Fractionation of effector lymphocytes into NK cell-depleted, T3-positive and NK cell-enriched, T3-negative cells demonstrated that similar levels of TPA/A23187-dependent killing could be induced in both fractions. It is concluded that TPA/A23187 induce normal lymphocytes to nonselective killing of different target cells in similarity to the triggering effect these drugs have in many other cell systems. Whether the induced killing is representative of NK killing is discussed in relation to the presence of other potential effector cells and effector molecules in peripheral blood lymphocytes.     

Effects of natalizumab,an α4 integrin inhibitor,on the development of Hartley guinea pigs

BACKGROUND : Natalizumab is a humanized monoclonal immunoglobulin G4 antibody directed against the human α4 integrin subunit, disrupting interaction with its ligands. Natalizumab inhibits the interaction of α4 integrins with fibronectin, vascular cell adhesion molecule-1, and mucosal addressin cellular adhesion molecule-1, which are of potential importance in development. Two studies were undertaken to evaluate the effects of natalizumab on embryo/fetal development in guinea pigs. METHODS : In the first study, pregnant guinea pigs were treated with intravenous injections of 3, 10, or 30 mg/kg natalizumab or vehicle every other day from gestational day (GD) 4 to 30. In the second study, females were treated on alternate days starting at least 28 days prior to mating through GD 30. Fetal examinations and histopathologic examination of the liver, heart, thymus, spleen, and intestinal tract were performed following maternal euthanasia on GD 59–62. RESULTS : Natalizumab had no significant effect on embryo/fetal development in either study. Exposure to natalizumab during organogenesis did not result in treatment-related external, visceral, or skeletal variations or malformations or histopathologic changes. CONCLUSION : No fetotoxicity or teratogenic effects were attributable to natalizumab in these studies. Birth Defects Res (Part B)86: 98-107, 2009. © 2009 Wiley-Liss, Inc.     

Postnatal development in cynomolgus monkeys following prenatal exposure to natalizumab,an α4 integrin inhibitor

BACKGROUND : Natalizumab is a humanized monoclonal IgG4 antibody to human α4 integrin that blocks the interaction of α4β1 and α4β7 integrins with their ligands, including fibronectin, vascular cell adhesion molecule-1, and mucosal addressin cellular adhesion molecule-1. Because α4 integrins and their ligands are widely involved in mammalian development, lymphopoeisis, and hematopoiesis, natalizumab may interfere with these processes. METHODS : The effects of prenatal exposure to natalizumab on postnatal development were assessed in cynomolgus monkeys at doses of 0 and 30 mg/kg administered intravenously every other day from gestational day (GD) 20 to 70 or GD 20 to term. Infants were delivered by natural birth and evaluated for general health, survival, development, and immunological structure and function at 12 or 18 months. RESULTS : An increase in abortions was seen in the first cohort of natalizumab-treated dams (39.3 vs. 7.1% in the controls) but not in the second cohort (33.3, 37.5%). Infants in the term treatment group had elevated lymphocyte (∼150%) and nucleated red blood cell counts (∼400%), consistent with the pharmacological effect of natalizumab, and reductions in platelet counts (∼28%), which were reversible following clearance of natalizumab. No anemia was observed. Infants in the term treatment group had significantly increased spleen weights at 12 months but not at 18 months. All other experimental observations in infants from natalizumab-treated dams were comparable with those of controls. CONCLUSION : Natalizumab had no adverse effects on the general health, survival, development, or immunological structure and function of infants born to dams treated with natalizumab during pregnancy. Birth Defects Res (Part B) 86: 144-156, 2009. © 2009 Wiley-Liss, Inc.     

Molecular Mechanism of BST2/Tetherin Downregulation by K5/MIR2 of Kaposi's Sarcoma-Associated Herpesvirus

K3/MIR1 and K5/MIR2 of Kaposi''s sarcoma-associated herpesvirus (KSHV) are viral members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family and contribute to viral immune evasion by directing the conjugation of ubiquitin to immunostimulatory transmembrane proteins. In a quantitative proteomic screen for novel host cell proteins downregulated by viral immunomodulators, we previously observed that K5, as well as the human immunodeficiency virus type 1 (HIV-1) immunomodulator VPU, reduced steady-state levels of bone marrow stromal cell antigen 2 (BST2; also called CD317 or tetherin), suggesting that BST2 might be a novel substrate of K5 and VPU. Recent work revealed that in the absence of VPU, HIV-1 virions are tethered to the plasma membrane in BST2-expressing HeLa cells. By targeting BST2, K5 might thus similarly overcome an innate antiviral host defense mechanism. Here we establish that despite its type II transmembrane topology and carboxy-terminal glycosylphosphatidylinositol (GPI) anchor, BST2 represents a bona fide target of K5 that is downregulated during primary infection by and reactivation of KSHV. Upon exit of the protein from the endoplasmic reticulum, lysines in the short amino-terminal domain of BST2 are ubiquitinated by K5, resulting in rapid degradation of BST2. Ubiquitination of BST2 is required for degradation, since BST2 lacking cytosolic lysines was K5 resistant and ubiquitin depletion by proteasome inhibitors restored BST2 surface expression. Thus, BST2 represents the first type II transmembrane protein targeted by K5 and the first example of a protein that is both ubiquitinated and GPI linked. We further demonstrate that KSHV release is decreased in the absence of K5 in a BST2-dependent manner, suggesting that K5 contributes to the evasion of intracellular antiviral defense programs.Bone marrow stromal cell antigen 2 (BST2) was recently identified as a host cell restriction factor that prevents the release of retroviral and filoviral particles from infected host cells (23). Human immunodeficiency virus type 1 (HIV-1) counteracts this antiviral function of BST2 by expressing the viral auxiliary protein VPU (41, 53). In the absence of VPU, virus particles are prevented from budding off the cellular membrane in cells that express BST2, resulting in virions being tethered to the plasma membrane. BST2 was therefore renamed tetherin (41), although questions still remain as to whether BST2 acts as the actual tether and whether BST2-dependent tethering occurs in all BST2-expressing cell types (36). Independently, BST2 was shown to be induced by type I and type II interferons (IFNs) (7), suggesting that BST2 is part of the innate antiviral response triggered in infected cells.Using a quantitative membrane proteomic approach, we observed that BST2 is underrepresented in plasma membranes from cells expressing not only VPU (14) but also the K5 protein of Kaposi''s sarcoma-associated herpesvirus (KSHV) (4). K5 is a viral homologue of a family of cellular transmembrane ubiquitin ligases, termed membrane-associated RING-CH (MARCH) proteins (3), that mediate the ubiquitination of the cytoplasmic portion of transmembrane proteins (reviewed in reference 40). Each member of this family targets a subset of cellular membrane proteins with both unique and shared specificities (4, 56). One of the functions of cellular MARCH proteins is to modulate antigen presentation by mediating the ubiquitin-dependent turnover of major histocompatibility complex (MHC) class II molecules in dendritic cells, B cells, and monocytes/macrophages (43, 52). In contrast, viral homologues of MARCH proteins encoded by KSHV, murine herpesvirus 68, and the leporipoxvirus myxomavirus all share the ability to mediate the destruction of MHC-I (reviewed in reference 16) but not MHC-II molecules. Thus, one of the functions of the viral proteins is to promote viral escape from immune clearance by CD8⁺ T lymphocytes (50). Furthermore, each viral MARCH homologue specifically eliminates additional host cell proteins, so each plays multiple roles in viral pathogenesis. KSHV carries two viral MARCH proteins, K3 and K5, also known as MIR1 and MIR2, which both support viral escape from T-cell, NK-cell, and NKT-cell recognition by eliminating the corresponding ligands from the surfaces of infected cells (reviewed in reference 10). In endothelial cells (ECs), K5 additionally downregulates EC-specific adhesion molecules that play an essential role in the formation of adhesive platforms and adherens junctions (31, 32). Since Kaposi''s sarcoma is a tumor of EC origin, K5 might thus also contribute to tumorigenesis by disrupting normal EC barrier function and by modulating the interaction of ECs with inflammatory leukocytes.The downregulation of BST2 by K5 further suggests that K5 also counteracts innate antiviral responses, which might benefit KSHV. However, most transmembrane proteins targeted by viral or cellular MARCH proteins are type I transmembrane proteins that belong to the immunoglobulin superfamily. In contrast, BST2 is a type II transmembrane protein that is also glycosylphosphatidylinositol (GPI) anchored (25). Thus, BST2 has a short cytoplasmic amino terminus followed by an outside-in transmembrane domain, a large glycosylated extracellular portion, and a GPI anchor. The additional propensity of BST2 to form homodimers (44) was speculated to be crucial for the tethering function of BST2 in that self-association of BST2 molecules in the viral envelope with plasma membrane BST2 could prevent viral exit (19). The unusual topology of BST2 and its multimerization raised the question of whether BST2 is a bona fide target of K5 or whether its downregulation is a downstream effect of K5 eliminating other transmembrane proteins. Additionally, it is not clear whether BST2 would be downregulated in the context of a normal viral infection and, particularly, whether virally expressed K5 would be able to overcome the high expression levels of BST2 observed upon IFN induction. We now demonstrate that KSHV efficiently downregulates IFN-induced BST2 both during primary infection and upon reactivation from latency in ECs. IFN-induced BST2 is ubiquitinated by K5 upon exiting the endoplasmic reticulum (ER) and is rapidly degraded by a pathway that is sensitive to proteasome inhibitors but resistant to inhibitors of lysosomal acidification. These data suggest that despite its unusual topology, BST2 is directly targeted by K5. We further demonstrate that BST2 reduces KSHV release upon inhibition of K5 expression by small interfering RNA (siRNA), suggesting that BST2 is part of the IFN-induced innate immune response to KSHV. Thus, in addition to contributing to viral evasion of cellular immune responses and remodeling EC function, K5 also counteracts the innate immune defense of the host cell.     

Intraocular iron injection induces oxidative stress followed by elements of geographic atrophy and sympathetic ophthalmia

Iron has been implicated in the pathogenesis of age‐related retinal diseases, including age‐related macular degeneration (AMD). Previous work showed that intravitreal (IVT) injection of iron induces acute photoreceptor death, lipid peroxidation, and autofluorescence (AF). Herein, we extend this work, finding surprising chronic features of the model: geographic atrophy and sympathetic ophthalmia. We provide new mechanistic insights derived from focal AF in the photoreceptors, quantification of bisretinoids, and localization of carboxyethyl pyrrole, an oxidized adduct of docosahexaenoic acid associated with AMD. In mice given IVT ferric ammonium citrate (FAC), RPE died in patches that slowly expanded at their borders, like human geographic atrophy. There was green AF in the photoreceptor ellipsoid, a mitochondria‐rich region, 4 h after injection, followed later by gold AF in rod outer segments, RPE and subretinal myeloid cells. The green AF signature is consistent with flavin adenine dinucleotide, while measured increases in the bisretinoid all‐trans‐retinal dimer are consistent with the gold AF. FAC induced formation carboxyethyl pyrrole accumulation first in photoreceptors, then in RPE and myeloid cells. Quantitative PCR on neural retina and RPE indicated antioxidant upregulation and inflammation. Unexpectedly, reminiscent of sympathetic ophthalmia, autofluorescent myeloid cells containing abundant iron infiltrated the saline‐injected fellow eyes only if the contralateral eye had received IVT FAC. These findings provide mechanistic insights into the potential toxicity caused by AMD‐associated retinal iron accumulation. The mouse model will be useful for testing antioxidants, iron chelators, ferroptosis inhibitors, anti‐inflammatory medications, and choroidal neovascularization inhibitors.     

Distinctive Profile of IsomiR Expression and Novel MicroRNAs in Rat Heart Left Ventricle

MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486.     

Should symptomatic menopausal women be offered hormone therapy?

Many physicians remain uncertain about prescribing hormone therapy for symptomatic women at the onset of menopause. The American Society for Reproductive Medicine (ASRM) convened a multidisciplinary group of healthcare providers to discuss the efficacy and risks of hormone therapy for symptomatic women, and to determine whether it would be appropriate to treat women at the onset of menopause who were complaining of menopausal symptoms. MAJOR FINDINGS: Numerous controlled clinical trials consistently demonstrate that hormone therapy, administered via oral, transdermal, or vaginal routes, is the most effective treatment for vasomotor symptoms. Topical vaginal formulations of hormone therapy should be preferred when prescribing solely for the treatment of symptoms of vulvar and vaginal atrophy. Data from the Women's Health Initiative indicate that the overall attributable risk of invasive breast cancer in women receiving estrogen plus progestin was 8 more cases per 10,000 women-years. No increased risk for invasive breast cancer was detected for women who never used hormone therapy in the past or for those receiving estrogen only. Hormone therapy is not effective for the treatment of cardiovascular disease and that the risk of cardiovascular disease with hormone therapy is principally in older women who are considerably postmenopause. CONCLUSIONS: Healthy symptomatic women should be offered the option of hormone therapy for menopausal symptoms. Symptom relief with hormone therapy for many younger women (at the onset of menopause) with menopausal symptoms outweighs the risks and may provide an overall improvement in quality of life. Hormone therapy should be individualized for symptomatic women. This involves tailoring the regimen and dose to individual needs.