Michael Kleinberg

Single-length and double-length channels formed by nystatin in lipid bilayer membranes

SummaryNystatin forms two types of channels in sterol-containing planar bilayer membranes. One type is formed when it is added to onlyone side of the membrane: the other is formed when it is added toboth sides of the membrane. The relative permeability of these channels to nonelectrolytes (urea and glycerol) is identical. The sensitivity of membranes to the one-sided action of nystatin is critically dependent on their thickness; in particular, membranes made from monoglycerides with more than 18 carbon atoms in their acyl chain are insensitive to nystatins one-sided action. These data are consistent with a model in which the two types of channels formed by nystatin have essentially identical structures, except that the channel formed by its two-sided action is twice the length of that formed by its one-sided action, because it is a tail-to-tail dimer of the latter.

BLADDER EPITHELIAL CELLS FROM PATIENTS WITH INTERSTITIAL CYSTITIS PRODUCE AN INHIBITOR OF HEPARIN-BINDING EPIDERMAL GROWTH FACTOR-LIKE GROWTH FACTOR PRODUCTION

PURPOSE The etiology of interstitial cystitis is unknown. We previously identified an interstitial cystitis urine factor, antiproliferative factor, that inhibits proliferation of bladder epithelial cells in vitro and complex changes in epithelial growth factor levels, including profound decreases in heparin-binding epidermal growth factor-like growth factor (HB-EGF). Bladder and renal pelvic catheterization of patients with interstitial cystitis indicated that the antiproliferative factor is made and/or activated in the distal ureter or bladder. Therefore, we determined whether bladder epithelial cells from interstitial cystitis cases produced the antiproliferative factor and whether purified antiproliferative factor could alter production of growth factors known to be abnormal in interstitial cystitis. MATERIALS AND METHODS Antiproliferative factor activity was determined by 3H-thymidine incorporation into primary bladder epithelial cells. The antiproliferative factor was purified by size fractionation followed by sequential chromatography involving ion exchange, hydrophobic interaction and high performance liquid chromatography. HB-EGF, epidermal growth factor, insulin-like growth factor and insulin-like growth factor binding protein 3 levels were determined by enzyme-linked immunosorbent assay. RESULTS Bladder epithelial cells from patients with interstitial cystitis produced a single antiproliferative factor with the same purification profile as that purified from interstitial cystitis urine. Purified antiproliferative factor specifically inhibited HB-EGF production by bladder epithelial cells in vitro, and the effect of interstitial cystitis urine or purified antiproliferative factor on bladder cell proliferation was inhibited by recombinant human HB-EGF in a dose dependent manner. Similar to urine HB-EGF, serum HB-EGF was also significantly lower in interstitial cystitis cases than in controls. CONCLUSIONS Bladder epithelial abnormalities in interstitial cystitis may be caused by a negative autocrine growth factor that inhibits cell proliferation by down-regulating HB-EGF production. Furthermore, decreased levels of urine and serum HB-EGF indicate that interstitial cystitis may be a urinary tract manifestation of a systemic disorder.

Recruitment of Murine Neutrophils in Vivo through Endogenous Sialidase Activity

Upon activation with various noncytokine stimuli, polymorphonuclear leukocytes (PMNs) mobilize intracellular sialidase to the plasma membrane, where the sialidase releases sialic acid from the cell surface. This desialylation enhances PMN adherence, spreading, deformability, and motility, functions critical to diapedesis. We now have examined the role of sialidase activity in PMN adhesion to and migration across the endothelium in vivo. A polyclonal antibody prepared against Clostridium perfringens neuraminidase 1) detected surface expression of sialidase on human PMNs stimulated with IL-8 in vitro and on murine PMNs stimulated in vivo, but not on that of unstimulated cells, 2) recognized proteins in human PMN lysates and granule preparations that were not detected by preimmune antibody, 3) inhibited bacterial neuraminidase and human PMN sialidase activitiesin vitro, and 4) inhibited both pulmonary leukostasis in mice systemically infused with cobra venom factor and intrapulmonary transendothelial migration of PMNs into the bronchoalveolar compartment of mice intranasally challenged with interleukin-8. We conclude that the chemokine interleukin-8, like other PMN agonists, induces the translocation of sialidase to the PMN surface and that surface expression of this sialidase is a prerequisite to PMN recruitmentin vivo. The ability of antibodies raised against a prokaryotic neuraminidase to recognize eukaryotic sialidase extends the concept of the neuraminidase superfamily to mammalian enzymes. Inhibition of mobilized endogenous sialidase may provide a novel strategy for limiting the inflammatory response.

Regulation of hyperoxia-induced NADPH oxidase activation in human lung endothelial cells by the actin cytoskeleton and cortactin

Although the actin cytoskeleton has been implicated in the control of NADPH oxidase in phagocytosis, very little is known about the cytoskeletal regulation of endothelial NADPH oxidase assembly and activation. Here, we report a role for cortactin and the tyrosine phosphorylation of cortactin in hyperoxia-induced NADPH oxidase activation and ROS production in human pulmonary artery ECs (HPAECs). Exposure of HPAECs to hyperoxia for 3 h induced NADPH oxidase activation, as demonstrated by enhanced superoxide production. Hyperoxia also caused a thickening of the subcortical dense peripheral F-actin band and increased the localization of cortactin in the cortical regions and lamellipodia at cell-cell borders that protruded under neighboring cells. Pretreatment of HPAECs with the actin-stabilizing agent phallacidin attenuated hyperoxia-induced cortical actin thickening and ROS production, whereas cytochalasin D and latrunculin A enhanced basal and hyperoxia-induced ROS formation. In HPAECs, a 3-h hyperoxic exposure enhanced the tyrosine phosphorylation of cortactin and interaction between cortactin and p47phox, a subcomponent of the EC NADPH oxidase, when compared with normoxic cells. Furthermore, transfection of HPAECs with cortactin small interfering RNA or myristoylated cortactin Src homology domain 3 blocking peptide attenuated ROS production and the hyperoxia-induced translocation of p47phox to the cell periphery. Similarly, down-regulation of Src with Src small interfering RNA attenuated the hyperoxia-mediated phosphorylation of cortactin tyrosines and blocked the association of cortactin with actin and p47phox. In addition, the hyperoxia-induced generation of ROS was significantly lower in ECs expressing a tyrosine-deficient mutant of cortactin than in vector control or wild-type cells. These data demonstrate a novel function for cortactin and actin in hyperoxia-induced activation of NADPH oxidase and ROS generation in human lung endothelial cells.

Pharmacoeconomics of antifungal pharmacotherapy – challenges and future directions

The frequency and severity of invasive fungal infections have been increasingly recognised and new antifungal therapies have expanded the therapeutic armamentarium available to manage such infections. Antifungal agents comprise a significant portion of antibiotic expenditures at major medical centres, prompting adoption of cost-containment measures and treatment guidelines. This paper reviews available data regarding the costs associated with managing fungal infections, including pharmacoeconomic analyses that have been performed in the setting of documented fungal infections, as well as prophylactic and empiric use of antifungal agents. The challenges of performing such studies are discussed, as well as the limitations of published investigations. Finally, recommendations are made regarding the design and implementation of future pharmacoeconomic analyses that can help establish the true costs of managing invasive fungal infections in at-risk patient populations.

Granulomatous amebic encephalitis: an under-recognized cause of infectious mortality after hematopoietic stem cell transplantation.

G. Akpek, A. Uslu, T. Huebner, A. Taner, A.P. Rapoport, I. Gojo, Y.T. Akpolat, O. Ioffe, M. Kleinberg, M.R. Baer. Granulomatous amebic encephalitis: an under‐recognized cause of infectious mortality after hematopoietic stem cell transplantation.
Transpl Infect Dis 2011: 13: 366–373. All rights reserved

Implementation of an Infectious Disease Fellow-Managed Penicillin Allergy Skin Testing Service

An inpatient penicillin allergy skin testing program can be successfully managed by infectious diseases fellows under attending supervision offering a novel practice area for infectious diseases practitioners.

Infection Prevention in the Cancer Center

Cancer patients are frequently immunosuppressed and at risk for a wide range of opportunistic and healthcare-associated infections. A good infection prevention program is extremely important to reduce risk of infection. This review focuses on infection prevention measures specific to patients, healthcare personnel, and visitors in the cancer center.

Aspergillosis in the CLEAR outcomes trial: working toward a real-world clinical perspective

Aspergillosis is a potentially lethal infection of immunocompromised patients. Until 10 years ago, antifungal therapy was largely limited to amphotericin B deoxycholate. Perceived poor response rates and inherent toxicities with amphotericin B deoxycholate were a major stimulus for the development of newer antifungals, including lipid-formulated amphotericin B, broad spectrum azoles, and echinocandins. Response rates to antifungals are highly dependent on the underlying diagnosis and degree of immune suppression of the patient. Patients at highest risk of death from aspergillosis also have very high mortality rates from other causes as well. Outcomes reported in historical literature reviews fail to distinguish between overall mortality and death attributable to aspergillosis. While this distinction can often be difficult to assess clinically, the net effect is to underestimate the therapeutic success rates of antifungals. The CLEAR (Collaborative Exchange of Antifungal Research) project started as a post approval survey to monitor clinical use of amphotericin B lipid complex (ABLC). The scope of the CLEAR project included collection of clinical data to assess outcomes in patients with invasive fungal infections treated with ABLC. Clinical data from more than 3500 patients were entered into the CLEAR database. Outcomes were assessed for 509 patients with documented aspergillosis and complete data records. Overall response rate was 63% (cured/improved/stable) with site-specific response rates of 61%, 59%, and 32% for lung, sinus, and central nervous system infections, respectively. Solid organ transplant recipients had higher response rates than patients with hematological malignancies. Bone marrow transplant recipients had the lowest response rates. Clinical response rates with ABLC reported in the CLEAR trial are higher than response rates reported for amphotericin B deoxycholate in other trials. Since it is unlikely we will see any new comparative Phase III trials for aspergillosis, CLEAR-type outcome studies will prove useful for the foreseeable future to guide clinical management of aspergillosis.

Prior colonization is associated with increased risk of antibiotic-resistant Gram-negative bacteremia in cancer patients.

We hypothesized that prior colonization with antibiotic-resistant Gram-negative bacteria is associated with increased risk of subsequent antibiotic-resistant Gram-negative bacteremia among cancer patients. We performed a matched case-control study. Cases were cancer patients with a blood culture positive for antibiotic-resistant Gram-negative bacteria. Controls were cancer patients with a blood culture not positive for antibiotic-resistant Gram-negative bacteria. Prior colonization was defined as any antibiotic-resistant Gram-negative bacteria in surveillance or non-sterile-site cultures obtained 2-365 days before the bacteremia. Thirty-two (37%) of 86 cases and 27 (8%) of 323 matched controls were previously colonized by any antibiotic-resistant Gram-negative bacteria. Prior colonization was strongly associated with antibiotic-resistant Gram-negative bacteremia (odds ratio [OR] 7.2, 95% confidence interval [CI] 3.5-14.7) after controlling for recent treatment with piperacillin-tazobactam (OR 2.5, 95% CI 1.3-4.8). In these patients with suspected bacteremia, prior cultures may predict increased risk of antibiotic-resistant Gram-negative bacteremia.