Phyllis Gardner

Structural and electrophysiological changes in the epicardial border zone of canine myocardial infarcts during infarct healing.

Structural and electrophysiological properties of the epicardial muscle which survives on the surface of transmural infarcts of the canine heart (epicardial border zone) were studied at different times after occlusion of the left anterior coronary artery (LAD). Isolated preparations were superfused in vitro, transmembrane potentials recorded, and impulse propagation mapped. In preparations from subacute infarcts (1 and 5 days), resting potential, action potential amplitude, upstroke velocity, and duration were all significantly reduced. Well-defined directional differences in propagation occurred. Propagation was more rapid in the direction perpendicular to the left anterior coronary artery than in the direction perpendicular to the base of the heart, because of the uniform anisotropic structure of the surviving muscle fibers which were arranged in tightly packed bundles oriented perpendicular to the left anterior coronary artery. The only ultrastructural abnormalities found in these muscle fibers was an accumulation of large amounts of lipid droplets. As the infarcts healed, resting potential, action potential amplitude, and upstroke velocity returned to normal by 2 weeks, although action potential duration decreased further. Lipid droplets had disappeared, and connective tissue had invaded the epicardial border zone, separating the muscle bundles. By 2 months, action potentials were normal, but the muscle fibers were widely separated and disoriented by the connective tissue (parallel bundles no longer were found). In these regions with a nonuniform anisotropic structure, the well-defined directional differences in impulse propagation were lost. However, activation was very slow, perhaps because of diminished connections between cells. The persistence of slow conduction in healed infarcts may contribute to the occurrence of chronic arrhythmias.

Activation of intestinal CFTR Cl- channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.

Heat‐stable enterotoxins (STa) produced by pathogenic bacteria induce profound salt and water secretion in the gut, leading to diarrhea. Recently, guanylin, an endogenous peptide with properties similar to STa, was identified. While STa and guanylin bind to the same receptor guanylyl cyclase and raise cell cGMP, the signaling mechanism distal to cGMP remains controversial. Here we show that STa, guanylin and cGMP each activate intestinal Cl‐ secretion, and that this is abolished by inhibitors of cAMP‐dependent protein kinase (PKA), suggesting that PKA is a major mediator of this effect. These agents induce Cl‐ secretion only in cells expressing the wild‐type CFTR, indicating that this molecule is the final common effector of the signaling pathway. The involvement of CFTR suggests a possible cystic fibrosis heterozygote advantage against STa‐induced diarrhea.

Calcium and T lymphocyte activation

A prolonged (at least 2-4 hr) elevation of [Ca2+]i accompanies early T cell activation by TCR/CD3-specific ligands. Ca2+ is generally thought to be an essential second messenger for early activation, but the precise molecular events contingent upon the Ca2+ signal remain to be determined. The Ca2+ signal can be separated into an early transient peak due to InsP3-released Ca2+ from intracellular stores, and a sustained plateau due to altered transmembrane Ca2+ flux. Patch clamp studies have identified an InsP3-activated, Ca2+ permeable channel in the plasma membrane of T lymphocytes that may be responsible for the sustained elevation of [Ca2+]i during continuous TCR/CD3 occupancy. The Ca2+ signal can be further resolved at the level of the single cell into a series of repetitive oscillations between peak and trough levels with a period of 16-20 s. The oscillations may be part of a frequency-encoded signaling system. Several nonlinear internal feedback controls may contribute to the periodic nature of the Ca2+ signal: PKC-mediated phosphorylation of the CD3 gamma subunit, which is a feedback inhibitor of TCR/CD3 function; amplification of Ca2+ release from endoplasmic reticulum by a highly cooperative step in the opening of Ca2+ channels by InsP3, and Ca2+-dependent feedback enhancement of PLC function; autoregulatory negative feedback on Ca2+ influx by Ca2+, both by a direct effect on the plasma membrane Ca2+ channel and by induction of membrane hyperpolarization secondary to Ca2+-activated K+ efflux. In addition, several other internal feedback controls on TCR/CD3 function, by CD4-induced tyrosine-specific phosphorylation of the CD3 zeta subunit, or on the Ca2+ signal, by extracellular Cl- or by GM1 gangliosides, are also postulated. The question of whether a G protein couples TCR/CD3 to PI hydrolysis and to Ca2+ mobilization is unresolved, although some indirect evidence for the involvement of GTP binding proteins in T cell activation has recently been obtained with cholera toxin. There is also preliminary evidence that TCR/CD3 may structurally conform to G protein coupled receptors, i.e., having a core structure of seven alpha helical transmembrane spanning segments, a ligand recognition site, loci for regulatory phosphorylation, and a putative nucleotide binding site.

A Phase II, double-blind, randomized, placebo-controlled clinical trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with antrostomies

tgAAVCF, an adeno-associated cystic fibrosis transmembrane conductance regulator (CFTR) viral vector/gene construct, was administered to 23 patients in a Phase II, double-blind, randomized, placebo-controlled clinical trial. For each patient, a dose of 100,000 replication units of tgAAVCF was administered to one maxillary sinus, while the contralateral maxillary sinus received a placebo treatment, thereby establishing an inpatient control. Neither the primary efficacy endpoint, defined as the rate of relapse of clinically defined, endoscopically diagnosed recurrent sinusitis, nor several secondary endpoints (sinus transepithelial potential difference [TEPD], histopathology, sinus fluid interleukin [IL]-8 measurements) achieved statistical significance when comparing treated to control sinuses within patients. One secondary endpoint, measurements of the anti-inflammatory cytokine IL-10 in sinus fluid, was significantly (p < 0.03) increased in the tgAAVCF-treated sinus relative to the placebo-treated sinus at day 90 after vector instillation. The tgAAVCF administration was well tolerated, without adverse respiratory events, and there was no evidence of enhanced inflammation in sinus histopathology or alterations in serum-neutralizing antibody titer to adeno-associated virus (AAV) capsid protein after vector administration. In summary, this Phase II trial confirms the safety of tgAAVCF but provides little support of its efficacy in the within-patient controlled sinus study. Various potentially confounding factors are discussed.

Safety and Biological Efficacy of an Adeno‐Associated Virus Vector–Cystic Fibrosis Transmembrane Regulator (AAV‐CFTR) in the Cystic Fibrosis Maxillary Sinus

Objective: The host immune response and low vector efficiency have been key impediments to effective cystic fibrosis transmembrane regulator (CFTR) gene transfer for cystic fibrosis (CF). An adeno‐associated virus vector (AAV‐CFTR) was used in a phase I dose‐escalation study to transfer CFTR cDNA into respiratory epithelial cells of the maxillary sinus of 10 CF patients. Study Design: A prospective, randomized, unblinded, dose‐escalation, within‐subjects, phase I clinical trial of AAV‐CFTR was conducted. Patients: Ten patients with previous bilateral maxillary antrostomies were treated. Main Outcome Measures: Safety, gene transfer as measured by semiquantitative polymerase chain reaction (PCR), and sinus transepithelial potential difference (TEPD) were measured. Results: The highest level of gene transfer was observed in the range of 0.1–1 AAV‐CFTR vector copy per cell in biopsy specimens obtained 2 weeks after treatment. When tested, persistence was observed in one patient for 41 days and in another for 10 weeks. Dose‐dependent changes in TEPD responses to pharmacologic intervention were observed following treatments. Little or no inflammatory or immune responses were observed. Conclusion: AAV‐CFTR administration to the maxillary sinus results in successful, dose‐dependent gene transfer to the maxillary sinus and alterations in sinus TEPD suggestive of a functional effect, with little or no cytopathic or host immune response. Further study is warranted for AAV vectors as they may prove useful for CFTR gene transfer and other in vivo gene transfer therapies. A prospective, randomized, double‐blind, placebo‐controlled, within‐subjects, phase II clinical trial of the effect AAV‐CFTR on clinical recurrence of sinusitis will determine the clinical efficacy of AAV gene therapy for CF.

The Structural Biology of CD2

The CD2 molecule is a 50-55KD transmembrane glycoprotein expressed on the vast majority of thymocytes and virtually all peripheral T lymphocytes. Its functions are two-fold: adhesion and activation. CD2 serves to facilitate conjugate formation between the T-lineage cell and its cognate partner via intermolecular interaction of CD2 and LFA-3 on the former and latter cells, respectively. Perturbation of the CD2 extracellular segment by certain combinations of anti-CD2 MAbs or LFA-3 and a single anti-CD2 MAb activate T-lineage function. These CD2-mediated activation events also synergize with signals mediated through the TCR to augment T-cell response. Based on microchemical analysis of immunoaffinity-purified human CD2 and cDNA and genomic cloning of mouse and human molecules, considerable structural information is now available. The mature surface human CD2 molecule consists of 327 amino acids: a 185 aa extracellular segment; a 25 aa hydrophobic transmembrane segment; and a 117 aa cytoplasmic domain rich in prolines and basic residues. The CD2 gene is comprised of five exons which span approximately 12 Kb on chromosome 1. A similar protein structure and gene exon organization is found for the mouse CD2 homologue. The CD2 adhesion domain is approximately 103 aa in length and is encoded by a single exon (exon 2). This domain is resistant to proteolysis, even though it lacks any intrachain disulfides and, like the entire extracellular segment protein expressed in a baculovirus system, binds to its cellular ligand, LFA-3. The latter occurs with a micromolar Kd. This relatively low affinity suggests that multivalent interactions among CD2 monomers on the T cells and individual LFA-3 structures on the cognate partner are important in enhancing the avidity of the T-cell interaction with its target or stimulator cell. The affinity of the CD2 extracellular segment for LFA-3 is not affected by truncations in the CD2 cytoplasmic domain, implying that ligand binding is not regulated by intracellular mechanisms. Given that CD2 mRNA expression and surface CD2 copy number are increased by more than one order of magnitude post-TCR stimulation, it is more likely that adhesion via CD2 is modulated by alteration in surface copy number. Analysis of early transduction events occurring via CD3-Ti (TCR) and CD2 including single channel Ca2+ patch-clamp recordings on living human T lymphocytes indicate a virtual identity of signals.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition by SK&F 96365 of Ca2+ current, IL‐2 production and activation in T lymphocytes

1 By use of whole cell patch‐clamp and Indo‐1 fluorescence studies of the Jurkat T leukaemic cell line, we show that the new organic antagonist of receptor‐mediated Ca2+ entry, SK&F 96365, inhibits the T cell Ca2+ current in a dose‐dependent fashion, with an IC50 of 12 μm. 2 SK&F 96365 also inhibits [3H]‐thymidine incorporation and interleukin‐2 (IL‐2) synthesis in peripheral blood lymphocytes. 3 SK&F 96365 has no effect on Ca2+ stores release or K+ channels. 4 This is the first account of an organic inhibitor of the T cell Ca2+ current. The ability of SK&F 96365 to inhibit IL‐2 synthesis and cell proliferation suggests that a new class of related Ca2+ channel blockers can be developed as immunosuppressive agents.

Reduced IL‐10 secretion by CD4+ T lymphocytes expressing mutant cystic fibrosis transmembrane conductance regulator (CFTR)

Expression of the CFTR protein is thought to be physiologically important only in exocrine epithelial cells. However, chronic respiratory inflammation and infection remain unexplained phenomena in disease pathogenesis. Non‐transformed, antigen‐responsive CD4+ T cells cloned from healthy controls and CF patients homozygous or heterozygous for the δF508 mutation transcribed CFTR mRNA and expressed immunoreactive cytoplasmic CFTR protein. T cell clones (TCC) from controls and CF patients displayed equivalent Ca2+‐mediated Cl− current; however, TCC from patients with CF but not controls displayed defective cAMP‐mediated Cl− current. Although CF‐derived TCC preserved mitogen and antigen proliferative responses and specificity to tetanus toxoid epitopes, they selectively secreted ≈ 45% less IL‐10 compared with control TCC after activation with concanavalin A (Con A) (624 ± 101 versus 1564 ± 401 pg/ml per 106 cells, respectively; P = 0.04) or anti‐CD3/phorbol ester (5148 ± 1634 versus 11 788 ± 2390 pg/ml; P = 0.05). This difference was independent of atopy. Secretion of interferon‐gamma, IL‐2, and IL‐4 was comparable in CF and control TCC after both forms of activation, while IL‐5 was reduced in CF TCC following anti‐CD3/phorbol myristate acetate (PMA) but not after Con A. We conclude that expression of mutant CFTR in human TCC is accompanied by ion channel dysfunction characteristic of the CF phenotype, and is accompanied by a reduction in IL‐10 secretion after polyclonal activation. It is possible that disruption of IL‐10‐mediated anti‐inflammatory homeostasis may contribute to early onset sustained inflammation in CF airways.

Activation of CFTR chloride current by nitric oxide in human T lymphocytes.

Nitric oxide, which is produced by cytokine‐activated mononuclear cells, is thought to play an important role in inflammation and immunity. While the function of nitric oxide as a direct cytotoxic effector molecule is well established, its function as a transducer molecule in immune cells is not. By use of whole‐cell patch clamp recordings, we show that nitric oxide activates cystic fibrosis transmembrane conductance regulator CI‐ currents in normal human cloned T cells by a cGMP‐dependent mechanism. This pathway is defective in cystic fibrosis‐derived human cloned T cells. These findings not only delineate a novel transduction mechanism for nitric oxide but also support the hypothesis that an intrinsic immune defect may exist in cystic fibrosis.

Simultaneous Multigene Mutation Detection in Patients With Sensorineural Hearing Loss Through a Novel Diagnostic Microarray: A New Approach for Newborn Screening Follow-up

OBJECTIVE. The advent of universal newborn hearing screening in the United States and other countries, together with the identification of genes involved in the process of hearing, have led to an increase in both the need and opportunity for accurate molecular diagnosis of patients with hearing loss. Deafness and hearing impairment have a genetic cause in at least half the cases. The molecular genetic basis for the majority of these patients remains obscure, however, because of the absence of associated clinical features in ∼70% (ie, nonsyndromic hearing loss) of patients, genetic heterogeneity, and the lack of molecular genetic tests that can evaluate a large number of mutations across multiple genes. DESIGN. We report on the development of a diagnostic panel with 198 mutations underlying sensorineural (mostly nonsyndromic) hearing loss. This panel, developed on a microarray, is capable of simultaneous evaluation of multiple mutations in 8 genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5 and the mitochondrial genes encoding 12S rRNA and tRNA-Ser[UCN]). RESULTS. The arrayed primer extension array for sensorineural hearing loss is based on a versatile platform technology and is a robust, cost-effective, and easily modifiable assay. Because hearing loss is a major public health concern and common at all ages, this test is suitable for follow-up after newborn hearing screening and for the detection of a genetic etiology in older children and adults. CONCLUSIONS. Comprehensive and relatively inexpensive genetic testing for sensorineural hearing loss will improve medical management for affected individuals and genetic counseling for their families.