Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of <em>1</em> mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤<em>1</em> <em>mL</em>. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.

Fabry disease (FD) is an X-linked lysosomal storage disease caused by alpha-galactosidase A deficiency. The Fabry Registry is a global clinical effort to collect longitudinal data on FD. In the past, most "carrier" females were usually thought to be clinically unaffected. A systematic effort has been made to enroll all FD females, regardless of symptomology. Of the <em>1</em>077 enrolled females in the Registry, 69.4% had symptoms and signs of FD. The median age at symptom onset among females was <em>1</em>3 years, and even though 84.<em>1</em>% had a positive family history, the diagnosis was not made until a median age of 3<em>1</em> years. Twenty percent experienced major cerebrovascular, cardiac, or renal events, at a median age of 46 years. Among adult females with estimated glomerular filtration rate (eGFR) data (N=638), 62.5% had an eGFR <90 <em>ml</em>/min/<em>1</em>.73 m2 and <em>1</em>9.0% had eGFR <60 <em>ml</em>/min/<em>1</em>.73 m2. Proteinuria 300 mg/day was present in 39.0% of females, and 22.2% had>><em>1</em> gram/day. Quality of life (QoL), as measured by the SF-36((R)) survey, was impaired at a later age than in males, but both genders experience significantly impaired QoL from the third decade of life onward. Thus, females with FD have a significant risk for major organ involvement and decreased QoL. Females should be regularly monitored for signs and symptoms of FD, and considered for enzyme replacement therapy.

The balance between proangiogenic and antiangiogenic factors, such as vascular endothelial growth factor, placental growth factor, and soluble fms-like tyrosine kinase-<em>1</em> (sFlt-<em>1</em>), is altered in preeclampsia, and this dysregulation of angiogenic factors may be important in the pathogenesis of preeclampsia. Although sFlt-<em>1</em> is elevated in preeclampsia, the mechanisms responsible for increasing this antiangiogenic factor remain unclear. We hypothesized that the hypertension produced by reduced uterine perfusion pressure (RUPP) is associated with increased sFlt-<em>1</em> expression and decreased plasma vascular endothelial growth factor and placental growth factor concentrations in the pregnant rat. Arterial pressure was increased (<em>1</em>30+/-3 versus <em>1</em>00+/-2 mm Hg; P<0.0<em>1</em>) in the RUPP rats compared with the normal pregnant control rats. Plasma sFlt-<em>1</em> concentration (660+/-270 versus 82+/-26 pg/<em>mL</em>; P<0.05) was increased, whereas plasma free placental growth factor (0.28+/-0.05 versus <em>1</em>.7+/-0.5 pg/<em>mL</em>; P<0.0<em>1</em>) and vascular endothelial growth factor (594+/-34 versus 830+/-33 pg/<em>mL</em>; P<0.0<em>1</em>) concentrations were decreased in the RUPP rats compared with normal pregnant rats. Plasma sFlt-<em>1</em>:placental growth factor (37.2+/-7.8 versus 8.9+/-<em>1</em>.6; P<0.02) and sFlt-<em>1</em>:vascular endothelial growth factor (0.86+/-0.22 versus 0.28+/-0.06; P<0.05) ratios were increased in the RUPP rats compared with normal pregnant rats. Immunoreactive placental sFlt-<em>1</em> was increased (<em>1</em>.<em>1</em>+/-0.<em>1</em> versus 0.3+/-0.<em>1</em>; P<0.0<em>1</em>) in RUPP rats contrasted with the normal pregnant rats. These findings support our hypothesis that RUPP increases the expression of sFlt-<em>1</em> and alters the balance of angiogenic factors in the maternal circulation. These data also indicate that the RUPP model of pregnancy-induced hypertension may provide an invaluable model for mechanistic studies into the role of sFlt-<em>1</em> in the pathogenesis preeclampsia.

Protegrin-<em>1</em> (PG-<em>1</em>) is a cysteine-rich, <em>1</em>8-residue beta-sheet peptide isolated from porcine leukocytes with antimicrobial activity against a broad range of microorganisms. The MICs of PG-<em>1</em> against representative gram-positive and gram-negative bacteria ranged from 0.<em>1</em>2 to 2 microg/<em>ml</em>. At these levels, PG-<em>1</em> was rapidly bactericidal in vitro, reducing the number of viable CFU of either methicillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa by more than three log units in less than <em>1</em>5 min. Resistance to PG-<em>1</em> did not develop after <em>1</em><em>1</em> subculturings of P. aeruginosa or <em>1</em>8 subcultures of MRSA in Mueller-Hinton broth containing PG-<em>1</em> at one-half the MIC. Under similar conditions of serial passage, the MICs of norfloxacin and gentamicin against P. aeruginosa increased <em>1</em>0 and <em>1</em>90 times, respectively. Similarly, the MIC of norfloxacin against MRSA increased 85 times. Immunocompetent mice inoculated intraperitoneally (i.p.) with P. aeruginosa or S. aureus exhibited 93 to <em>1</em>00% mortality in the vehicle control group compared with 0 to 27% mortality in animals that received a single i.p. injection of PG-<em>1</em> (0.5 mg/kg of body weight). Mice inoculated with S. aureus by intravenous (i.v.) injection and dosed 0 to 60 min later with a single i.v. injection of PG-<em>1</em> (5 mg/kg) had a mortality of 7 to 33%, compared to a mortality of 73 to 93% in the vehicle controls. In leukopenic mice inoculated i.v. with vancomycin-resistant Enterococcus faecium, mortality was 87% in the vehicle control group and 33% in animals that received a single i.v. injection of PG-<em>1</em> (2.5 mg/kg). Taken together, these data indicate that PG-<em>1</em> has potential for use as an antimicrobial agent in the treatment of local or systemic infections caused by clinically relevant pathogens.

<AbstractText>Most patients with small-cell lung cancer (SCLC) have extensive-stage disease at presentation, and prognosis remains poor. Recently, immunotherapy has demonstrated clinical activity in extensive-stage SCLC (ES-SCLC). The CASPIAN trial assessed durvalumab, with or without tremelimumab, in combination with etoposide plus either cisplatin or carboplatin (platinum-etoposide) in treatment-naive patients with ES-SCLC.</AbstractText><p><div><b>METHODS</b></div>This randomised, open-label, phase 3 trial was done at 209 sites across 23 countries. Eligible patients were adults with untreated ES-SCLC, with WHO performance status 0 or <em>1</em> and measurable disease as per Response Evaluation Criteria in Solid Tumors, version <em>1</em>.<em>1</em>. Patients were randomly assigned (in a <em>1</em>:<em>1</em>:<em>1</em> ratio) to durvalumab plus platinum-etoposide; durvalumab plus tremelimumab plus platinum-etoposide; or platinum-etoposide alone. All drugs were administered intravenously. Platinum-etoposide consisted of etoposide 80-<em>1</em>00 mg/m² on days <em>1</em>-3 of each cycle with investigator's choice of either carboplatin area under the curve 5-6 mg/<em>mL</em> per min or cisplatin 75-80 mg/m² (administered on day <em>1</em> of each cycle). Patients received up to four cycles of platinum-etoposide plus durvalumab <em>1</em>500 mg with or without tremelimumab 75 mg every 3 weeks followed by maintenance durvalumab <em>1</em>500 mg every 4 weeks in the immunotherapy groups and up to six cycles of platinum-etoposide every 3 weeks plus prophylactic cranial irradiation (investigator's discretion) in the platinum-etoposide group. The primary endpoint was overall survival in the intention-to-treat population. We report results for the durvalumab plus platinum-etoposide group versus the platinum-etoposide group from a planned interim analysis. Safety was assessed in all patients who received at least one dose of their assigned study treatment. This study is registered at ClinicalTrials.gov, NCT03043872, and is ongoing.</p><AbstractText>Patients were enrolled between March 27, 20<em>1</em>7, and May 29, 20<em>1</em>8. 268 patients were allocated to the durvalumab plus platinum-etoposide group and 269 to the platinum-etoposide group. Durvalumab plus platinum-etoposide was associated with a significant improvement in overall survival, with a hazard ratio of 0·73 (95% CI 0·59-0·9<em>1</em>; p=0·0047]); median overall survival was <em>1</em>3·0 months (95% CI <em>1</em><em>1</em>·5-<em>1</em>4·8) in the durvalumab plus platinum-etoposide group versus <em>1</em>0·3 months (9·3-<em>1</em><em>1</em>·2) in the platinum-etoposide group, with 34% (26·9-4<em>1</em>·0) versus 25% (<em>1</em>8·4-3<em>1</em>·6) of patients alive at <em>1</em>8 months. Any-cause adverse events of grade 3 or 4 occurred in <em>1</em>63 (62%) of 265 treated patients in the durvalumab plus platinum-etoposide group and <em>1</em>66 (62%) of 266 in the platinum-etoposide group; adverse events leading to death occurred in <em>1</em>3 (5%) and <em>1</em>5 (6%) patients.</AbstractText><AbstractText>First-line durvalumab plus platinum-etoposide significantly improved overall survival in patients with ES-SCLC versus a clinically relevant control group. Safety findings were consistent with the known safety profiles of all drugs received.</AbstractText><AbstractText>AstraZeneca.</AbstractText>

A human mAb (HmAb) termed F<em>1</em>05 was obtained by fusion of antibody-producing EBV-transformed cells with the HMMA2.<em>1</em><em>1</em>TG/O cell line. F<em>1</em>05 is an IgG<em>1</em> kappa antibody that binds to the surfaces of cells infected with all HIV-<em>1</em> strains tested: MN, RF, IIIB, and SF2, but not uninfected cells. The HmAb immunoprecipitates GP<em>1</em>20 from all four strains. F<em>1</em>05 does not react with denatured GP<em>1</em>20 on Western blots, but does react with viral lysates and purified GP<em>1</em>20 dotted onto nitrocellulose filter paper under nondenaturing conditions. rGP<em>1</em>20 from SF2 and soluble rCD4 inhibit antibody binding to infected cells in a dose-dependent manner. F<em>1</em>05 inhibits the binding of free, infectious virions to uninfected HT-H9 cells with 50% of maximal (<em>1</em>00%) inhibition at approximately <em>1</em> microgram/<em>ml</em>. F<em>1</em>05 inhibits infection of HT-H9 cells by <em>1</em>00 tissue culture infective dose 50% units of MN and IIIB strains with 50% inhibition at concentrations of HmAb readily achievable in man. It appears that the F<em>1</em>05 HmAb reacts with a conformationally defined epitope on HIV-<em>1</em>/GP<em>1</em>20 that is exposed on the free virion and is important for binding to the cell surface by the virion. The epitope, which is immunogenic in humans, appears to be within, or topographically near, the CD4-binding site. F<em>1</em>05 and the F<em>1</em>05 epitope are potentially useful in therapy and in the design of peptide or anti-Id based vaccines; monitoring of the expression of the Id may prove useful in evaluating immune responses in infected individuals or vaccinated volunteers.

<em>1</em>. Fifty to ninety per cent of the Na efflux from axons of Loligo forbesi is inhibited by ouabain. The properties of the ouabain-sensitive component of the Na efflux are different from those of the ouabain-insensitive component.2. In unpoisoned axons with an average Na content of 75 m-mole/kg axoplasm the bulk of the ouabain-sensitive Na efflux is dependent on external K.3. In the presence of 460 mM Na in the external medium, raising the external K concentration from 0 to <em>1</em>00 mM increases the ouabain-sensitive Na efflux along a sigmoid curve which shows signs of saturating at high K concentrations.4. The curve relating ouabain-sensitive K influx to external K concentration is similar in shape to that for the ouabain-sensitive Na efflux. At all K concentrations examined the ouabain-sensitive K influx was less than the ouabain-sensitive Na efflux.5. Potassium-free sea water acts rapidly in reducing the Na efflux. There is no appreciable difference between the rates of action of K-free sea water on the Na pump and Na-free sea water on the action potential.6. Caesium and Rb can replace external K in activating the ouabain-sensitive Na efflux. Both the affinity and maximum rate of the Na efflux mechanism are lower when Cs replaces K as the activating cation.7. Isosmotic replacement of external Na by either choline or dextrose, but not Li, increases the affinity of the ouabain-sensitive Na efflux mechanism for external K without appreciably affecting the maximum rate of pumping. External Li behaves like external Na and exerts an inhibitory action on the Na efflux.8. There is a large ouabain-sensitive Na efflux into K-free choline or dextrose sea waters. Addition of either Na or Li to the external medium reduces this efflux along a section of a rectangular hyperbola. The properties of this efflux suggest that there is a residual K concentration of up to 2 mM immediately external to the pumping sites in the axolemma.9. Over the range of internal Na concentrations studied (<em>1</em>6-<em>1</em>40 m-mole/kg axoplasm) the ouabain-sensitive Na efflux increased linearly with Na concentration.<em>1</em>0. Tetrodotoxin (<em>1</em>0(-6) g/<em>ml</em>.) reduces the Na influx by about half, but does not affect the ouabain-sensitive Na efflux.<em>1</em><em>1</em>. Isobutanol (<em>1</em>% v/v) reversibly decreases both the ouabain-sensitive and ouabain-insensitive components of the Na efflux.<em>1</em>2. Application of 2 mM cyanide to axons immersed in K-free sea water produces a transient rise in the Na efflux. This rise is not seen if ouabain is included in the sea water. The rise in efflux occurs at a time when the axons are partially poisoned and contain adenosine triphosphate (ATP) but no arginine phosphate (ArgP). A similar, but maintained rise can be obtained after application of dinitrophenol (DNP) at pH 8.0. The increased Na efflux in these partially poisoned axons is also inhibited by ouabain.<em>1</em>3. Under conditions of partial-poisoning by alkaline DNP, there is a ouabain-sensitive Na influx from K-free sea water. The ouabain-sensitive Na influx is of similar size to the ouabain-sensitive Na efflux. These results show that in partially-poisoned axons immersed in K-free sea water intracellular Na exchanges with extracellular Na in a one-for-one manner by a ouabain-sensitive route. External Li cannot replace external Na in maintaining this process.<em>1</em>4. Axons partially poisoned with alkaline DNP are not insensitive to external K. In the absence of external Na their response to external K is essentially the same as that seen in unpoisoned axons.<em>1</em>5. Possible mechanisms are discussed for the appearance of Na-Na exchange in partially poisoned axons.

The enteric pathogen, Entamoeba histolytica, appears to cause disease by adhering to and then destroying mucosal barriers. Using an in vitro method of studying the interaction of E. histolytica with target cells (Chinese hamster ovary [CHO] and human erythrocytes [RBC]), we examined the mechanism of amebic adherence and its role in lysis of target cells. Killing and phagocytosis of target cells by amebas ceases at 4 degrees C, allowing observation of adherence. Amebas adhere to CHO cells at 4 degrees C, 78.9% formed rosettes (amebas with>>/=3 adherent CHO cells each) at 2 h. At 37 degrees C, cytochalasins B and D inhibit adherence of amebas to CHO cells (P < 0.0005). Amebas adhere to and kill CHO cells in media with <0.<em>1</em> muM calcium and magnesium plus <em>1</em>0 mM EDTA, indicating that divalent cations are not required in the medium. Adherence of amebas to human RBC was not ABO blood group specific and showed greater adherence to human than bovine or sheep RBC (P < 0.005). Neither Fc nor complement receptors were found on amebas by standard rosette studies. The amebic adherence receptor is not trypsin (0.<em>1</em>25%) sensitive nor inhibited by trypan blue (<em>1</em> mM). N-acetyl-d-galactosamine (GALNAc) inhibited the adherence of amebas to CHO cells and human RBC (0.<em>1</em> g/<em>1</em>00 <em>ml</em> or 4.5 mM GALNAc, P < 0.005) by binding to a receptor on the amebic surface. GALNAc abolishes amebic cytolysis of target CHO cells (determined by (<em>1</em><em>1</em><em>1</em>)Indium oxine release from CHO cells, P < 0.00<em>1</em>) but not amebic phagocytosis of CHO cells. By suspending ameba-CHO cells rosettes in dextran, we found that GALNAc (<em>1</em>%) reversibly inhibits amebic adherence (P < 0.0005) and that cytochalasins decrease amebic killing of adherent CHO cells (P < 0.025). These findings indicate that the adherence of E. histolytica to target cells requires microfilament function, is via a specific amebic receptor that has affinity for GALNAc, and is required to lyse cells. Inhibition of the adherence of E. histolytica may alter the pathogenicity of this organism.

This chapter about antithrombotic therapy in neonates and children is part of the Antithrombotic and Thrombolytic Therapy: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Grade <em>1</em> recommendations are strong and indicate that the benefits do, or do not, outweigh risks, burden, and costs, and Grade 2 suggests that individual patient values may lead to different choices (for a full understanding of the grading, see Guyatt et al in this supplement, pages <em>1</em>23S-<em>1</em>3<em>1</em>S). In this chapter, many recommendations are based on extrapolation of adult data, and the reader is referred to the appropriate chapters relating to guidelines for adult populations. Within this chapter, the majority of recommendations are separate for neonates and children, reflecting the significant differences in epidemiology of thrombosis and safety and efficacy of therapy in these two populations. Among the key recommendations in this chapter are the following: In children with first episode of venous thromboembolism (VTE), we recommend anticoagulant therapy with either unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) [Grade <em>1</em>B]. Dosing of IV UFH should prolong the activated partial thromboplastin time (aPTT) to a range that corresponds to an anti-factor Xa assay (anti-FXa) level of 0.35 to 0.7 U/<em>mL</em>, whereas LMWH should achieve an anti-FXa level of 0.5 to <em>1</em>.0 U/<em>mL</em> 4 h after an injection for twice-daily dosing. In neonates with first VTE, we suggest either anticoagulation or supportive care with radiologic monitoring and subsequent anticoagulation if extension of the thrombosis occurs during supportive care (Grade 2C). We recommend against the use of routine systemic thromboprophylaxis for children with central venous lines (Grade <em>1</em>B). For children with cerebral sinovenous thrombosis (CSVT) without significant intracranial hemorrhage (ICH), we recommend anticoagulation initially with UFH, or LMWH and subsequently with LMWH or vitamin K antagonists (VKAs) for a minimum of 3 months (Grade <em>1</em>B). For children with non-sickle-cell disease-related acute arterial ischemic stroke (AIS), we recommend UFH or LMWH or aspirin (<em>1</em> to 5 mg/kg/d) as initial therapy until dissection and embolic causes have been excluded (Grade <em>1</em>B). For neonates with a first AIS, in the absence of a documented ongoing cardioembolic source, we recommend against anticoagulation or aspirin therapy (Grade <em>1</em>B).

We have examined elongation by RNA polymerase II initiated at a promoter and have identified two classes of elongation complexes. Following initiation at a promoter, all polymerase molecules enter an abortive mode of elongation. Abortive elongation is characterized by the rapid generation of short transcripts due to pausing of the polymerase followed by termination of transcription. Termination of the early elongation complexes can be suppressed by the addition of 250 mM KCl or <em>1</em> mg of heparin per <em>ml</em> soon after initiation. Elongation complexes of the second class carry out productive elongation in which long transcripts can be synthesized. Productive elongation complexes are derived from early paused elongation complexes by the action of a factor which we call P-TEF (positive transcription elongation factor). P-TEF is inhibited by 5,6-dichloro-<em>1</em>-beta-D-ribofuranosylbenzimidazole at concentrations which have no effect on the initiation of transcription. By using templates immobilized on paramagnetic particles, we show that isolated preinitiation complexes lack P-TEF and give rise to transcription complexes which can carry out only abortive elongation. The ability to carry out productive elongation can be restored to isolated transcription complexes by the addition of P-TEF after initiation. A model is presented which describes the role of elongation factors in the formation and maintenance of elongation complexes. The model is consistent with the available in vivo data concerning control of elongation and is used to predict the outcome of other potential in vitro and in vivo experiments.

OBJECTIVE

Cryptococcus neoformans is an important cause of central nervous system infection in adults with acquired immunodeficiency syndrome (AIDS) but an unusual cause of disease in children with AIDS. The basis for this age-related difference in incidence is not known but may be caused by differences in exposure or immune response. The objective of this study was to determine whether the low prevalence of cryptococcal disease among children is related to a lack of exposure to C neoformans.

METHODS

Sera were obtained from <em>1</em>85 immunocompetent individuals ranging in age from <em>1</em> week to 2<em>1</em> years who were being evaluated in an urban emergency department. Sera were analyzed for antibodies to C neoformans and Candida albicans proteins by immunoblotting. Immunoblot patterns were compared with those obtained from sera of patients with cryptococcosis (n = <em>1</em>0) and workers in a laboratory devoted to the study of C neoformans. The specificity of our results was confirmed by several approaches, including antibody absorption and blocking studies. Sera were also analyzed for the presence of cryptococcal polysaccharide by both enzyme-linked immunosorbent assay and latex agglutination assays.

RESULTS

Sera from children <em>1</em>.<em>1</em> to 2 years old demonstrated minimal reactivity to C neoformans proteins. In contrast, the majority of sera from children >2 years old recognized many >>/=6) C neoformans proteins. For children between 2.<em>1</em> and 5 years old, 56% of sera (n = 25) reacted with many proteins, whereas for children >5 years old (n = <em>1</em>20), 70% of samples reacted with many proteins. Reactivity was decreased by absorbing sera with C neoformans extracts or by preincubating blots with sera from experimentally infected but not from control rats. Reactivity to C neoformans proteins did not correlate with reactivity to C albicans proteins, which was common in sera from children between the ages of <em>1</em>.<em>1</em> and 2 years. Cryptococcal polysaccharide was detected at a titer of <em>1</em>:<em>1</em>6 (~<em>1</em>0 ng/mL) in the sera of <em>1</em> child, a 5.6-year-old boy who presented to the emergency department with vomiting.

CONCLUSIONS

Our findings provide both indirect and direct evidence of C neoformans infection in immunocompetent children. Our results indicate that C neoformans infects a majority of children living in the Bronx after 2 years old. These results are consistent with several observations: the ubiquitous nature of C neoformans in the environment, including its association with pigeon excreta; the large number of pigeons in urban areas; and the increased likelihood of environmental exposure for children once they have learned to walk. The signs and symptoms associated with C neoformans infection in immunocompetent children remained to be determined. Primary pulmonary cryptococcosis may be asymptomatic or produce symptoms confused with viral infections and, therefore, not recognized as a fungal infection. Our results suggest that the low incidence of symptomatic cryptococcal disease in children with AIDS is not a result of lack of exposure to C neoformans. These findings have important implications for C neoformans pathogenesis and the development of vaccine strategies.

We recently discovered that a ubiquitous protein, high mobility group box <em>1</em> protein (HMGB<em>1</em>), is released by activated macrophages, and functions as a late mediator of lethal systemic inflammation. To elucidate mechanisms underlying the regulation of HMGB<em>1</em> release, we examined the roles of other cytokines in induction of HMGB<em>1</em> release in macrophage cell cultures. Macrophage migration inhibitory factor, macrophage-inflammatory protein <em>1</em>beta, and IL-6 each failed to significantly induce the release of HMGB<em>1</em> even at supraphysiological levels (up to 200 ng/<em>ml</em>). IFN-gamma, an immunoregulatory cytokine known to mediate the innate immune response, dose-dependently induced the release of HMGB<em>1</em>, TNF, and NO, but not other cytokines such as IL-<em>1</em>alpha, IL-<em>1</em>beta, or IL-6. Pharmacological suppression of TNF activity with neutralizing Abs, or genetic disruption of TNF expression (TNF knockout) partially (50-60%) inhibited IFN-gamma-mediated HMGB<em>1</em> release. AG490, a specific inhibitor for Janus kinase 2 of the IFN-gamma signaling pathway, dose-dependently attenuated IFN-gamma-induced HMGB<em>1</em> release. These data suggest that IFN-gamma plays an important role in the regulation of HMGB<em>1</em> release through a TNF- and Janus kinase 2-dependent mechanism.

BACKGROUND

This report compares the clinical characteristics, epidemiologic investigations, infection-control evaluations, and microbiologic findings of all 7 of the cases of vancomycin-resistant Staphylococcus aureus (VRSA) infection in the United States during the period 2002-2006.

METHODS

Epidemiologic, clinical, and infection-control information was collected. VRSA isolates underwent confirmatory identification, antimicrobial susceptibility testing, pulsed-field gel electrophoresis, and typing of the resistance genes. To assess VRSA transmission, case patients and their contacts were screened for VRSA carriage.

RESULTS

Seven cases were identified from 2002 through 2006; 5 were reported from Michigan, <em>1</em> was reported from Pennsylvania, and <em>1</em> was reported from New York. All VRSA isolates were vanA positive and had a median vancomycin minimum inhibitory concentration of 5<em>1</em>2 microg/<em>mL</em>. All case patients had a history of prior methicillin-resistant S. aureus and enterococcal infection or colonization; all had several underlying conditions, including chronic skin ulcers; and most had received vancomycin therapy prior to their VRSA infection. Person-to-person transmission of VRSA was not identified beyond any of the case patients. Infection-control precautions were evaluated and were consistent with established guidelines.

CONCLUSIONS

Seven patients with vanA-positive VRSA have been identified in the United States. Prompt detection by microbiology laboratories and adherence to recommended infection control measures for multidrug-resistant organisms appear to have prevented transmission to other patients.

BACKGROUND

Overdiagnosis and subsequent overtreatment are important side effects of screening for, and early detection of, prostate cancer (PCa). Active surveillance (AS) is of growing interest as an alternative to radical treatment of low-risk PCa.

OBJECTIVE

To update our experience in the largest worldwide prospective AS cohort.

METHODS

Eligible patients had clinical stage T<em>1</em>/T2 PCa, prostate-specific antigen (PSA) ≤ <em>1</em>0 ng/<em>ml</em>, PSA density <0.2 ng/<em>ml</em> per milliliter, one or two positive biopsy cores, and Gleason score ≤ 6. PSA was measured every 3-6 mo, and volume-based repeat biopsies were scheduled after <em>1</em>, 4, and 7 yr. Reclassification was defined as more than two positive cores or Gleason >6 at repeat biopsy. Recommendation for treatment was triggered in case of PSA doubling time <3 yr or reclassification.

METHODS

Multivariate regression analysis was used to evaluate predictors for reclassification at repeat biopsy. Active therapy-free survival (ATFS) was assessed with a Kaplan-Meier analysis, and Cox regression was used to evaluate the association of clinical characteristics with active therapy over time.

CONCLUSIONS

In total, 2494 patients were included and followed for a median of <em>1</em>.6 yr. One or more repeat biopsies were performed in <em>1</em>480 men, of whom 4<em>1</em>5 men (28%) showed reclassification. Compliance with the first repeat biopsy was estimated to be 8<em>1</em>%. During follow-up, 527 patients (2<em>1</em>.<em>1</em>%) underwent active therapy. ATFS at 2 yr was 77.3%. The strongest predictors for reclassification and switching to deferred treatment were the number of positive cores (two cores compared with one core) and PSA density. The disease-specific survival rate was <em>1</em>00%. Follow-up was too short to draw definitive conclusions about the safety of AS.

CONCLUSIONS

Our short-term data support AS as a feasible strategy to reduce overtreatment. Clinical characteristics and PSA kinetics during follow-up can be used for risk stratification. Strict monitoring is even more essential in men with high-risk features to enable timely recognition of potentially aggressive disease and offer curative intervention. Limitations of using surrogate end points and markers in AS should be recognized.

BACKGROUND

The current program is registered at the Dutch Trial Register with ID NTR<em>1</em>7<em>1</em>8 (http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=<em>1</em>7<em>1</em>8).

Postulating favorable antileukemic effect with improved safety, we used intravenous busulfan and fludarabine as conditioning therapy for allogeneic hematopoietic stem cell transplantation (HSCT) for acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). Fludarabine 40 mg/m2 and intravenous busulfan <em>1</em>30 mg/m2 were given once daily for 4 days, with tacrolimus-methotrexate as graft-versus-host disease (GVHD) prophylaxis. We treated 74 patients with AML and 22 patients with MDS; patients had a median age of 45 years (range, <em>1</em>9-66 years). Only 20% of the patients were in first complete remission (CR) at transplantation. Donors were HLA-compatible related (n = 60) or matched unrelated (n = 36). The CR rate for 54 patients with active disease was 85%. At a median follow-up of <em>1</em>2 months, <em>1</em>-year regimen-related and treatment-related mortalities were <em>1</em>% and 3%, respectively. Two patients had reversible hepatic veno-occlusive disease. Actuarial <em>1</em>-year overall survival (OS) and event-free survival (EFS) were 65% and 52% for all patients, and 8<em>1</em>% and 75% for patients receiving transplants in CR. Recipient age and donor type did not influence OS or EFS. Median busulfan clearance was <em>1</em>09 <em>mL</em>/min/m2 and median daily area-under-the-plasma-concentration-versus-time-curve was 487<em>1</em> micromol-min, with negligible interdose variability in pharmacokinetic parameters. The results suggest that intravenous busulfan-fludarabine is an efficacious, reduced-toxicity, myeloablative-conditioning regimen for patients with AML or MDS undergoing HSCT.

BACKGROUND

Mechanically overloaded cardiomyocytes secrete a soluble interleukin-<em>1</em> receptor family member called ST2. Serum levels of ST2 are associated with prognosis in nonischemic heart failure, but the predictive value of ST2 in patients with acute myocardial infarction is unknown.

RESULTS

ST2 levels were measured in serum from 8<em>1</em>0 patients with acute myocardial infarction in the Thrombolysis In Myocardial Infarction (TIMI) <em>1</em>4 (362 patients) and Enoxaparin and TNK-tPA With or Without GPIIb/IIIa Inhibitor as Reperfusion Strategy in STEMI (ENTIRE)-TIMI 23 (448 patients) clinical trials. Baseline levels of ST2 were significantly higher in those patients who died (0.379 versus 0.233 ng/mL, P=0.000<em>1</em>) or developed new congestive heart failure (0.287 versus 0.233 ng/mL, P=0.009) by 30 days. In an analysis of outcomes at 30 days by ST2 quartiles, both death (P=0.00<em>1</em>) and the combined death/heart failure end point (P=0.00<em>1</em>) showed a significant graded association with levels of ST2; furthermore, in-hospital death (P=0.003) and death/heart failure (P=0.004) were also significantly associated with higher ST2 levels. In a logistic regression analysis that controlled for important clinical factors, increasing levels of ST2 remained associated with death at 30 days (P=0.047). ST2 levels rose during the first day after infarction and were maximal at <em>1</em>2 hours; ST2 levels at <em>1</em>2 hours were also independently associated with death at 30 days (P<0.00<em>1</em>).

CONCLUSIONS

Serum levels of the interleukin-<em>1</em> receptor family member ST2 predict mortality and heart failure in patients with acute myocardial infarction. These data suggest that ST2 may be a useful biomarker and that this novel inflammatory receptor may play a role in cardiac pathophysiology.

Emerging data indicate that neurotrophic factors and cytokines utilize similar signal transduction mechanisms. Although neurotrophic factors can protect CNS neurons against a variety of insults, the role of cytokines in the injury response is unclear. We now report that TNF beta and TNF alpha (<em>1</em>-<em>1</em>00 ng/<em>ml</em>) can protect cultured embryonic rat hippocampal, septal, and cortical neurons against glucose deprivation-induced injury and excitatory amino acid toxicity. The elevation of intracellular calcium concentration ([Ca2+]i) induced by glucose deprivation, glutamate, NMDA, or AMPA was attenuated in neurons pretreated with TNF beta. The mechanism whereby TNFs stabilize [Ca2+]i may involve regulation of the expression of proteins involved in maintaining [Ca2+]i homeostasis, since both TNF beta and TNF alpha caused a 4- to 8-fold increase in the number of neurons expressing the calcium-binding protein calbindin-D28k. These data suggest a neuroprotective role for TNFs in the brain's response to injury.

Macrophage responses to Francisella infection have been characterized previously by subdued proinflammatory responses; however, these studies have generally focused on macrophage cell lines or monocyte-derived macrophages. Therefore, we studied the ability of fresh human blood monocytes to engulf and respond to Francisella by using the live vaccine strain variant and Francisella novicida. Because Francisella organisms have been reported to escape from the phagolysosome into the cytosol, we hypothesized that this escape may trigger the activation of caspase-<em>1</em>. Francisella tularensis variants were readily taken up by fresh human CD<em>1</em>4(+) monocytes, inducing the release of IL-<em>1</em>beta, as well as IL-8, in a time- and dose-dependent fashion. Importantly, whereas live and dead Escherichia coli, F. novicida, and live vaccine strain, as well as the LPS of E. coli, were able to induce abundant IL-<em>1</em>beta mRNA synthesis and intracellular pro-IL-<em>1</em>beta production, only live Francisella induced enhanced IL-<em>1</em>beta processing and release (5<em>1</em> +/- <em>1</em>0 vs. 7.<em>1</em> +/- 2.<em>1</em> ng/<em>ml</em>, for F. novicida vs. E. coli LPS; P = 0.0032). Cytochalasin D blocked the Francisella internalization and the Francisella-induced monocyte IL-<em>1</em>beta processing and release but not that induced by the exogenous stimulus E. coli LPS. Also, killing bacteria did not block uptake but significantly diminished the IL-<em>1</em>beta processing and release that was induced by Francisella. Blocking bacterial escape from the phagosome into the cytosol also decreased IL-<em>1</em>beta but not IL-8 release. These findings demonstrate that Francisella organisms efficiently induce IL-<em>1</em>beta processing and release in fresh monocytes by means of a sensing system that requires the uptake of live bacteria capable of phagosome escape.

The possibility that the high frequency of treatment failures in Indian kala-azar might be due to infection with antimony-resistant strains of Leishmania donovani has not been experimentally addressed. L. donovani isolates were obtained from splenic aspiration smears of 24 patients in Bihar, India, who either did not respond (<em>1</em>5) or did respond (9) to <em>1</em> or more full courses of treatment with sodium antimony gluconate (SAG). A strong correlation (P<.00<em>1</em>) between clinical response and SAG sensitivity in vitro was observed only when strains were assayed as intracellular amastigotes: responsive isolates ED50=2.4+/-2.6, ED90=6.4+/-7.8 microgram SAG/<em>mL</em>; unresponsive isolates ED50=7.4+/-3.7 microgram SAG/<em>mL</em>, ED90=29.<em>1</em>+/-<em>1</em><em>1</em>.<em>1</em> SAG/<em>mL</em>. No correlation with clinical response was found by use of extracellular promastigotes (ED50=48+/-22 vs. 52+/-29 microgram/<em>mL</em>). The emergence of antimony-resistant L. donovani strains appears to be a cause of treatment failures in India.

BACKGROUND

The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus involved in cases and case clusters of severe acute respiratory infection in the Arabian Peninsula, Tunisia, Morocco, France, Italy, Germany, and the UK. We provide a full description of a fatal case of MERS-CoV infection and associated phylogenetic analyses.

METHODS

We report data for a patient who was admitted to the Klinikum Schwabing (Munich, Germany) for severe acute respiratory infection. We did diagnostic RT-PCR and indirect immunofluorescence. From time of diagnosis, respiratory, faecal, and urine samples were obtained for virus quantification. We constructed a maximum likelihood tree of the five available complete MERS-CoV genomes.

RESULTS

A 73-year-old man from Abu Dhabi, United Arab Emirates, was transferred to Klinikum Schwabing on March <em>1</em>9, 20<em>1</em>3, on day <em>1</em><em>1</em> of illness. He had been diagnosed with multiple myeloma in 2008, and had received several lines of treatment. The patient died on day <em>1</em>8, due to septic shock. MERS-CoV was detected in two samples of bronchoalveolar fluid. Viral loads were highest in samples from the lower respiratory tract (up to <em>1</em>·2 × <em>1</em>0(6) copies per <em>mL</em>). Maximum virus concentration in urine samples was 269<em>1</em> RNA copies per <em>mL</em> on day <em>1</em>3; the virus was not present in the urine after renal failure on day <em>1</em>4. Stool samples obtained on days <em>1</em>2 and <em>1</em>6 contained the virus, with up to <em>1</em>03<em>1</em> RNA copies per g (close to the lowest detection limit of the assay). One of two oronasal swabs obtained on day <em>1</em>6 were positive, but yielded little viral RNA (5370 copies per <em>mL</em>). No virus was detected in blood. The full virus genome was combined with four other available full genome sequences in a maximum likelihood phylogeny, correlating branch lengths with dates of isolation. The time of the common ancestor was halfway through 20<em>1</em><em>1</em>. Addition of novel genome data from an unlinked case treated 6 months previously in Essen, Germany, showed a clustering of viruses derived from Qatar and the United Arab Emirates.

CONCLUSIONS

We have provided the first complete viral load profile in a case of MERS-CoV infection. MERS-CoV might have shedding patterns that are different from those of severe acute respiratory syndrome and so might need alternative diagnostic approaches.

BACKGROUND

European Union; German Centre for Infection Research; German Research Council; and German Ministry for Education and Research.

Cultured lung epithelial cells release antibacterial activity upon contact with Pseudomonas aeruginosa (PA), which is impaired in cystic fibrosis (CF). In order to identify the factors responsible for killing PA by a biochemical approach, we purified antimicrobial activity from supernatants of the A549 lung epithelial cell line, previously stimulated with PA bacteria, by subsequent high performance liquid chromatography. NH(2)-terminal sequencing of a major bactericidal compound revealed it to be identical with human beta-defensin-2 (hBD-2). A mucoid phenotype of PA, but not two nonmucoid PA strains, high concentrations >> <em>1</em>0 microg/<em>ml</em>) of PA lipopolysaccharide, tumor necrosis factor alpha, and interleukin (IL)-<em>1</em>beta, but not IL-6, dose-dependently induced hBD-2 messenger RNA in cultured normal bronchial, tracheal, as well as normal and CF-derived nasal epithelial cells. Genomic analysis of hBD-2 revealed a promoter region containing several putative transcription factor binding sites, including nuclear factor (NF) kappaB, activator protein (AP)-<em>1</em>, AP-2, and NF-IL-6, known to be involved in the regulation of inflammatory responses. Thus, hBD-2 represents a major inducible antimicrobial factor released by airway epithelial cells either on contact with mucoid PA or by endogenously produced primary cytokines. Therefore, it might be important in lung infections caused by mucoid PA, including those seen in patients with CF.

We report a retroviral expression vector (PINCO) that allows high-efficiency gene transfer and selection of hemopoietic progenitor cells (HPCs). The main characteristics of this vector are the presence outside the two long terminal repeats of the EBV origin of replication and the EBNA-<em>1</em> gene and the presence in the retrovirus of the cDNA that encodes for the enhanced green fluorescence protein (GFP), controlled by a cytomegalovirus promoter. Transient transfection of PINCO in Phoenix packaging cells results in episomal propagation of the plasmid and generates viral titers as high as <em>1</em>0(7) colony-forming units/<em>ml</em>. Infection of established cell lines with the PINCO retrovirus yields more than 95% GFP-expressing cells. GFP expression remains stable for months in infected cell cultures and can easily be monitored by fluorescent microscopy or fluorescence-activated cell-sorting (FACS) analysis of living cells. The PINCO vector allows efficient expression of a second gene (thymidine kinase, Shc, and PML), and there is strict correlation between GFP and second gene expression levels in the infected cells. PINCO was used to infect human HPCs; infection efficiency was about 50%. GFP-positive cells can be FACS sorted to yield a homogeneous population of infected cells. FACS-sorted GFP-positive HPC cells have, with respect to unfractionated HPC cells, the same frequency of long-term culture initiating cells and an identical capacity to undergo multilineage and unilineage differentiation. The entire gene transfer procedure, from the transfection of the packaging cell line to the infection of target cells, requires less than a week. The high viral titer and the easy obtainment of homogeneously infected cell populations without drug selection procedures make PINCO an ideal vector for gene transfer of human primary hemopoietic cells.

Hyperglycemia is associated with increased susceptibility to atherothrombotic stimuli. The glycocalyx, a layer of proteoglycans covering the endothelium, is involved in the protective capacity of the vessel wall. We therefore evaluated whether hyperglycemia affects the glycocalyx, thereby increasing vascular vulnerability. The systemic glycocalyx volume was estimated by comparing the distribution volume of a glycocalyx permeable tracer (dextran 40) with that of a glycocalyx impermeable tracer (labeled erythrocytes) in <em>1</em>0 healthy male subjects. Measurements were performed in random order on five occasions: two control measurements, two measurements during normoinsulinemic hyperglycemia with or without N-acetylcysteine (NAC) infusion, and one during mannitol infusion. Glycocalyx measurements were reproducible (<em>1</em>.7 +/- 0.2 vs. <em>1</em>.7 +/- 0.3 l). Hyperglycemia reduced glycocalyx volume (to 0.8 +/- 0.2 l; P < 0.05), and NAC was able to prevent the reduction (<em>1</em>.4 +/- 0.2 l). Mannitol infusion had no effect on glycocalyx volume (<em>1</em>.6 +/- 0.<em>1</em> l). Hyperglycemia resulted in endothelial dysfunction, increased plasma hyaluronan levels (from 70 +/- 6 to <em>1</em><em>1</em>2 +/- <em>1</em>6 ng/<em>ml</em>; P < 0.05) and coagulation activation (prothrombin activation fragment <em>1</em> + 2: from 0.4 +/- 0.<em>1</em> to <em>1</em>.<em>1</em> +/- 0.2 nmol/l; d-dimer: from 0.27 +/- 0.<em>1</em> to 0.55 +/- 0.2 g/l; P < 0.05). Taken together, these data indicate a potential role for glycocalyx perturbation in mediating vascular dysfunction during hyperglycemia.

BACKGROUND

Inflammation has a pathogenetic role in acute myocardial infarction (MI). Pentraxin-3 (PTX3), a long pentraxin produced in response to inflammatory stimuli and highly expressed in the heart, was shown to peak in plasma approximately 7 hours after MI. The aim of this study was to assess the prognostic value of PTX3 in MI compared with the best-known and clinically relevant biological markers.

RESULTS

In 724 patients with MI and ST elevation, PTX3, C-reactive protein (CRP), creatine kinase (CK), troponin T (TnT), and N-terminal pro-brain natriuretic peptide (NT-proBNP) were assayed at entry, a median of 3 hours, and the following morning, a median of 22 hours from symptom onset. With respect to outcome events occurring over 3 months after the index event, median PTX3 values were 7.08 ng/<em>mL</em> in event-free patients, <em>1</em>6.<em>1</em>2 ng/<em>mL</em> in patients who died, 9.<em>1</em>2 ng/<em>mL</em> in patients with nonfatal heart failure, and 6.88 ng/<em>mL</em> in patients with nonfatal residual ischemia (overall P<0.000<em>1</em>). Multivariate analysis including CRP, CK, TnT, and NT-proBNP showed that only age>> or =70 years (OR, 2.<em>1</em><em>1</em>; 95% CI, <em>1</em>.04 to 4.3<em>1</em>), Killip class>><em>1</em> at entry (OR, 2.20; 95% CI, <em>1</em>.<em>1</em>4 to 4.25), and PTX3 >><em>1</em>0.73 ng/<em>mL</em>) (OR, 3.55; 95% CI, <em>1</em>.43 to 8.83) independently predicted 3-month mortality. Biomarkers predicting the combined end point of death and heart failure in survivors were the highest tertile of PTX3 and of NT-proBNP and a CK ratio >6.

CONCLUSIONS

In a representative contemporary sample of patients with MI with ST elevation, the acute-phase protein PTX3 but not the liver-derived short pentraxin CRP or other cardiac biomarkers (NT-proBNP, TnT, CK) predicted 3-month mortality after adjustment for major risk factors and other acute-phase prognostic markers.