Pseudomonas aeruginosa PcrV and Psl, the Molecular Targets of Bispecific Antibody MEDI3902, Are Conserved Among Diverse Global Clinical Isolates.

Background: Bispecific antibody MEDI3902, targeting the Pseudomonas aeruginosa type 3 secretion system (PcrV) and Psl exopolysaccharide, is currently in phase 2b development for prevention of nosocomial pneumonia in patients undergoing mechanical ventilation. We surveyed a diverse collection of isolates to study MEDI3902 epitope conservation and protective activity. Methods: P. aeruginosa clinical isolates (n = 913) were collected from diverse patients and geographic locations during 2003-2014. We conducted whole-genome sequencing; performed PcrV and Psl expression analyses via immunoblotting and enzyme-linked immunosorbent assay, respectively; performed crystallography to determine the MEDI3902 PcrV epitope, using anti-PcrV Fab and PcrV components (resolved at 2.8 Å); and evaluated MEDI3902 protective activity against select isolates in vitro and in vivo. Results: Intact psl operon and pcrV genes were present in 94% and 99% of isolates, respectively, and 99.9% of isolates contained at least one of the genetic elements. Anti-Psl binding was confirmed in tested isolates harboring a complete Psl operon or lacking nonessential psl genes. We identified 46 PcrV variant sequences, and MEDI3902-PcrV contact residues were preserved. MEDI3902 maintained potent in vivo activity against various strains, including strains expressing only a single target. Conclusions: Psl and PcrV are highly prevalent in global clinical isolates, suggesting MEDI3902 can mediate broad coverage against P. aeruginosa.

Multimechanistic Monoclonal Antibodies (MAbs) Targeting Staphylococcus aureus Alpha-Toxin and Clumping Factor A: Activity and Efficacy Comparisons of a MAb Combination and an Engineered Bispecific Antibody Approach.

Secreted alpha-toxin and surface-localized clumping factor A (ClfA) are key virulence determinants in Staphylococcus aureus bloodstream infections. We previously demonstrated that prophylaxis with a multimechanistic monoclonal antibody (MAb) combination against alpha-toxin (MEDI4893*) and ClfA (11H10) provided greater strain coverage and improved efficacy in an S. aureus lethal bacteremia model. Subsequently, 11H10 was found to exhibit reduced affinity and impaired inhibition of fibrinogen binding to ClfA002 expressed by members of a predominant hospital-associated methicillin-resistant S. aureus (MRSA) clone, ST5. Consequently, we identified another anti-ClfA MAb (SAR114) from human tonsillar B cells with >100-fold increased affinity for three prominent ClfA variants, including ClfA002, and potent inhibition of bacterial agglutination by 112 diverse clinical isolates. We next constructed bispecific Abs (BiSAbs) comprised of 11H10 or SAR114 as IgG scaffolds and grafted anti-alpha-toxin (MEDI4893*) single-chain variable fragment to the amino or carboxy terminus of the anti-ClfA heavy chains. Although the BiSAbs exhibited in vitro potencies similar to those of the parental MAbs, only 11H10-BiSAb, but not SAR114-BiSAb, showed protective activity in murine infection models comparable to the respective MAb combination. In vivo activity with SAR114-BiSAb was observed in infection models with S. aureus lacking ClfA. Our data suggest that high-affinity binding to ClfA sequesters the SAR114-BiSAb to the bacterial surface, thereby reducing both alpha-toxin neutralization and protection in vivo These results indicate that a MAb combination targeting ClfA and alpha-toxin is more promising for future development than the corresponding BiSAb.

Field Determination of Multipollutant, Open Area Combustion Source Emission Factors with a Hexacopter Unmanned Aerial Vehicle.

An emission sensor/sampler system was coupled to a National Aeronautics and Space Administration (NASA) hexacopter unmanned aerial vehicle (UAV) to characterize gases and particles in the plumes emitted from open burning of military ordnance. The UAV/sampler was tested at two field sites with test and sampling flights spanning over 16 hours of flight time. The battery-operated UAV was remotely maneuvered into the plumes at distances from the pilot of over 600 m and at altitudes of up to 122 m above ground level. While the flight duration could be affected by sampler payload (3.2 to 4.6 kg) and meteorological conditions, the 57 sampling flights, ranging from 4 to 12 min, were typically terminated when the plume concentrations of CO2 were diluted to near ambient levels. Two sensor/sampler systems, termed "Kolibri," were variously configured to measure particulate matter, metals, chloride, perchlorate, volatile organic compounds, chlorinated dioxins/furans, and nitrogen-based organics for determination of emission factors. Gas sensors were selected based on their applicable concentration range, light weight, freedom from interferents, and response/recovery times. Samplers were designed, constructed, and operated based on U.S. Environmental Protection Agency (EPA) methods and quality control criteria. Results show agreement with published emission factors and good reproducibility (e.g., 26% relative standard deviation for PM2.5). The UAV/Kolibri represents a significant advance in multipollutant emission characterization capabilities for open area sources, safely and effectively making measurements heretofore deemed too hazardous for personnel or beyond the reach of land-based samplers.

Emissions from prescribed burning of agricultural fields in the Pacific Northwest.

Prescribed burns of winter wheat stubble and Kentucky bluegrass fields in northern Idaho and eastern Washington states (U.S.A.) were sampled using ground-, aerostat-, airplane-, and laboratory-based measurement platforms to determine emission factors, compare methods, and provide a current and comprehensive set of emissions data for air quality models, climate models, and emission inventories. Batch measurements of PM2.5, volatile organic compounds (VOCs), polycyclic aromatic hydrocarbons (PAHs), and polychlorinated dibenzodioxins/dibenzofurans (PCDDs/PCDFs), and continuous measurements of black carbon (BC), particle mass by size, CO, CO2, CH4, and aerosol characteristics were taken at ground level, on an aerostat-lofted instrument package, and from an airplane. Biomass samples gathered from the field were burned in a laboratory combustion facility for comparison with these ground and aerial field measurements. Emission factors for PM2.5, organic carbon (OC), CH4, and CO measured in the field study platforms were typically higher than those measured in the laboratory combustion facility. Field data for Kentucky bluegrass suggest that biomass residue loading is directly proportional to the PM2.5 emission factor; no such relationship was found with the limited wheat data. CO2 and BC emissions were higher in laboratory burn tests than in the field, reflecting greater carbon oxidation and flaming combustion conditions. These distinctions between field and laboratory results can be explained by measurements of the modified combustion efficiency (MCE). Higher MCEs were recorded in the laboratory burns than from the airplane platform. These MCE/emission factor trends are supported by 1-2 min grab samples from the ground and aerostat platforms. Emission factors measured here are similar to other studies measuring comparable fuels, pollutants, and combustion conditions. The size distribution of refractory BC (rBC) was single modal with a log-normal shape, which was consistent among fuel types when normalized by total rBC mass. The field and laboratory measurements of the Angstrom exponent (α) and single scattering albedo (ω) exhibit a strong decreasing trend with increasing MCEs in the range of 0.9-0.99. Field measurements of α and ω were consistently higher than laboratory burns, which is likely due to less complete combustion. When VOC emissions are compared with MCE, the results are consistent for both fuel types: emission factors increase as MCE decreases.

Detection of human papillomavirus DNA in cytologically normal women and subsequent cervical squamous intraepithelial lesions.

BACKGROUND: Human papillomavirus (HPV) infection has been strongly associated with cervical carcinoma and its cytologic precursors, squamous intraepithelial lesions (SIL). We investigated the risk of SIL prospectively following polymerase chain reaction (PCR)-based DNA testing for a wide range of genital HPV types in a cohort of initially cytologically normal women, to clarify the role of HPV in the etiology of SIL. METHODS: Starting in April 1989, 17,654 women who were receiving routine cytologic screening at Kaiser Permanente (Portland, OR) were followed for the development of incident SIL. During follow-up, 380 incident case patients and 1037 matched control subjects were eligible for this nested case-control study. Cervical lavages collected at enrollment and, later, at the time of case diagnosis (or the corresponding time for selection of control subjects) were tested for HPV DNA using a PCR-based method. The data were analyzed as contingency tables with two-sided P values or, for multivariable analyses, using odds ratios (ORs) with 95% confidence intervals (CIs). RESULTS: In comparison with initially HPV-negative women, women who tested positive for HPV DNA at enrollment were 3.8 times (95% CI = 2.6-5.5) more likely to have low-grade SIL subsequently diagnosed for the first time during follow-up and 12.7 times more likely (95% CI = 6.2-25.9) to develop high-grade SIL. At the time of diagnosis, the cross-sectional association of HPV DNA and SIL was extremely strong (OR = 44.4 and 95% CI = 24.2-81.5 for low-grade SIL and OR = 67.1 and 95% CI = 19.3-233.7 for high-grade SIL). HPV16 was the virus type most predictive of SIL, even low-grade SIL. CONCLUSIONS: These findings are consistent with the hypothesis that HPV infection is the primary cause of cervical neoplasia. Furthermore, they support HPV vaccine research to prevent cervical cancer and efforts to develop HPV DNA diagnostic tests.

Regulation of gene expression by SREBP and SCAP.

Sterol regulatory element binding proteins (SREBPs) function as transcription factors that activate specific genes involved in cholesterol synthesis, endocytosis of low density lipoproteins, the synthesis of both saturated and unsaturated fatty acids and glucose metabolism. As such, these proteins provide a link between lipid and carbohydrate metabolism. There are three SREBPs, SREBP-1a, SREBP-1c and SREBP-2, that are encoded by two genes. SREBPs are synthesized as 125 kDa precursor proteins that are localized to the endoplasmic reticulum. The precursor is transported to the Golgi by a chaperone protein (SREBP-cleavage activating protein) and then cleaved by two proteases to release the mature, transcriptionally active 68 kDa amino terminal domain. Recent studies have shown that formation of mature SREBP is controlled at multiple levels in response to changes in the levels of oxysterols, insulin/glucose and polyunsaturated fatty acids. These recent findings have important clinical implications relevant to hyperlipidemia and diabetes and are the topic of this review.

Irradiation- and cyclophosphamide-induced alterations in Syrian hamster T-cell population activity.

The treatment of hamsters with either irradiation (IR) or cyclophosphamide (CYP) markedly alters select aspects of their cellular immune functions in a dose-related manner. One mechanism that may be responsible for this activity appears to be the dimunition of a T-lymphocyte subpopulation that exerts suppressive influence upon the B-lymphocyte reactivity toward antigens. This study shows that in the hamster, immune susceptibility is affected by the magnitude and orientation of these agents (ie, IR, CYP) as they temporally relate to immunization and/or challenge with the antigen. Moreover, there is evidence that T-independent as well as T-dependent responses are affected by these treatments. Therefore, cyclophosphamide and irradiation modalities can be employed to modify the cellular immune responses in the hamster.

The participation of activated peritoneal macrophages in Treponema pallidum subspecies pertenue infection in Syrian hamsters.

The role of cell-mediated immunity in hamsters during treponemal infection appears to involve the activated macrophage. To date, studies have been hindered by the inability to confirm that macrophages exhibit enhanced treponemicidal activity at the infection site. We show that lipopolysaccharide and thioglycollate-treated animals, when inoculated with Treponema pallidum subsp. pertenue, exhibit enhanced clearance of these organisms compared with controls. Macrophages from these infected groups display an enhanced respiratory burst, as detected by NBT reduction, as well as a marked increase in C3b receptor-mediated ingestion activity. Significant changes in these parameters indicate that alterations in macrophage activation are occurring in the infected compartment. Thus the stimulatory agents apparently modify the host's immune responses to promote subsequent reduction of treponemal infection. In addition, hamster peritoneal macrophages demonstrate enhanced activation behavior as a result of exposure to at least two signals, which may be prerequisite for processing this organism efficiently.

Receptor-mediated ingestion responses by lung macrophages from a canine model of ARDS.

Receptor-mediated ingestion was examined in macrophages derived from a canine model of the adult respiratory distress syndrome (ARDS). The results showed that Fc-mediated ingestion by alveolar macrophages (AM) and macrophages from lung parenchyma (PM) was significantly diminished when compared with their respective controls. Pulsing all the experimental groups with lipopolysaccharide (LPS) for 1 hr in vitro failed to either enhance the response or return the activity to levels achieved by control cells. In parallel studies, an analysis of C3b-mediated ingestion showed that both the experimental AM and PM performed this function only at a magnitude equal to the control cells. Similar responses were observed when an LPS pulse was performed. Although there was a reduction in Fc-mediated ingestion and an apparent restraint of the C3b-mediated ingestion, both AM and PM expressed a significantly enhanced ability to spread. These results suggested that the canine model of ARDS alters at least one select macrophage function that may be important to subsequently protect the host. Such disturbances in the cellular immune response may contribute to the progression of infection and lung pathology associated with this disease process.

Differential induction of macrophage GSIB4-binding activity.

Activated macrophages display a terminal galactopyranosyl group on their membrane surface that binds the lectin Griffonia simplicifolia-IB4 (GSIB4). Using FITC-conjugated GSIB4, we examined the induction and subsequent expression of this corresponding marker on peritoneal macrophages from normal (NMO) and LPS-treated (LPS MO) mice. Although the percentage of fresh LPS MO explants that bound GSIB4 was always higher when compared to the NMO counterparts, marker expression on the latter was readily enhanced by culturing the cells in vitro either alone or with stimuli. Moreover, we found that an increase in this activity was promoted by either nonspecific phagocytosis of latex beads, or gamma-interferon (gamma-IFN) treatment. Further investigation showed that a prerequisite sequence of signal delivery to the macrophages was associated with maximal expression of the GSIB4 binding. When gamma-IFN treatment preceded latex bead ingestion, maximum GSIB4 binding occurred. Data obtained from using short-term (1 hr) and long-term (24 hr) exposure to latex beads showed that metabolic processing of induction signals was required to enhance the response over time. This yielded better GSIB4-binding activity when responses to these pulses were analyzed in freshly explanted macrophages. The overall results of this study demonstrated that macrophage binding of GSIB4 was differentially associated with stimuli induction. Moreover, select signals in the form of soluble mediators, or the mechanical events characteristic of internalization were capable of eliciting an increase in the percentage of macrophages that were positive for binding GSIB4. Thus, the enhanced affinity for binding this lectin may serve as a useful marker to determine the magnitude of macrophage responsiveness when these cells are examined following their exposure to different stimuli.

Fc-gamma receptor-ligand interactions enhance macrophage GSIB4-binding activity.

Activated macrophages have an increased ability to bind the lectin Griffonia simplicifolia-IB4 (GSIB4). Since macrophages readily use the Fc-receptor (FcR) during several immunologic and inflammatory processes, it is important to determine whether interactions with this moiety affect GSIB4-binding ability. Peritoneal macrophages cultured in vitro with Fc fragments of immunoglobulin G (IgG), whole IgG, or heat-aggregated IgG demonstrate an increase in this function. Conversely, treatment of macrophages with (Fab')2 fractions alone has no direct effect on this activity. Although the GSIB4-binding response is minimally expressed by normal macrophages, it is more markedly apparent on macrophages from LPS-treated animals. In both cases, however, pretrypsinization of the cells renders them refractory to IgG-mediated induction of the GSIB4-binding response. Moreover, macrophages cultured independently with IgG subclasses 1, 2a, or 3 demonstrate that the magnitude of their response to this signal is directly associated with the type of subclass used. Although each subclass enhanced the response, in this study interactions with IgG2a produced the best results. Overall, however, the greatest GSIB4-binding activity is generated when FcRs are crosslinked by aggregated IgG rather than simply bound by independent monomeric interactions at the FcRs. This suggests that the event of appropriately interacting with the FcRs amplifies the GSIB4-binding function. Such a mechanism could play a key role in coordinating the humoral, cell-mediated, and innate responses of the immune system.

In vivo prenatal chlordane exposure induces development of endogenous inflammatory macrophages.

Macrophages (m phi s), important cells in host resistance, undergo a series of biochemical changes during their progression from the resident to the fully activated stage. Both resident and inflammatory m phi s are characterized by some unique properties. In the present study, female BALB/c mice were prenatally treated with 8 mg/kg body weight of chlordane, a cyclodiene poly-chlorinated hydrocarbon that appears to reduce immunocompetence by selectively impairing m phi function. Therefore, we examined functions in m phi s from chlordane-treated mice that had been stimulated with thioglycollate. The 5'-nucleotidase activity, present in high levels in resident m phi s but low levels in inflammatory m phi s was elevated in resident m phi s from vehicle-exposed animals. Conversely, inflammatory m phi s from these animals showed significantly diminished levels of this function. Moreover, chlordane-exposed m phi s, regardless of whether they were resident or inflammatory, exhibited decreased 5'-nucleotidase responses. When a second function, transferrin receptor binding, was analyzed, vehicle-treated inflammatory m phi s displayed high levels of activity whereas the resident m phi s showed very little transferrin binding. However, both resident and inflammatory m phi s from the chlordane-exposed group demonstrated transferrin binding activity similar in magnitude to that of the vehicle-treated inflammatory m phi s. Finally, two-dimensional polyacrylamide gel electrophoresis analysis of m phi s from chlordane-exposed mice have characteristics of normal m phi s that have advanced to the inflammatory stage.

Intraductal "noninfiltrating" carcinoma of the breast.

We are reviewing 40 patients with noninvasive intraductal carcients; 10% developed oppostie breast cancer of the invasive variety. Twenty-one patients underwent radical mastectomy in which one patient was found to have a positive axillary node. The 19 remaining patients had total mastectomy alone. No recurrence of death attributed to intraductal carcinoma was found in 39 patients available for follow-up.

Cloning and sequencing of genomic DNA extracted from archival human temporal bone sections.

Cloning techniques allow the engineering and production of highly purified DNA. Further advances in molecular biology have provided the means to identify DNA sequences in a rapid fashion. Sequencing methods can identify mutations, deletions, polymorphisms, or confirm a known genetic sequence. The use of these techniques in clinical medicine has made it possible to accurately diagnose infectious diseases and determine the molecular etiology of many genetic disorders and malignancies. In this study, DNA extracted from archival, celloidin-embedded temporal bone sections has been cloned and sequenced using these techniques. We amplified, cloned, and sequenced varicella-zoster viral DNA extracted from archival temporal bone sections from patients who had herpes zoster oticus. The application of cloning and sequencing techniques to DNA extracted from archival temporal bones provides the methodology to study temporal bone pathology at the molecular level.

Immune cellular interactions during sepsis and septic injury.

The cellular activity generated by PMNs and macrophages in association with diverse cytokines has a profound impact on all major functional responses of host cellular components during sepsis and septic injury. It is the modulation of these cellular interactions and their effect on the continuum between appropriate and inappropriate responses during inflammation that will dictate the outcome of humans with sepsis.

Inclusion of 13C12-labelled mono-, di-, and tri-chlorinated dibenzo-p-dioxin and dibenzofuran standards in US EPA methods 0023A/8290.

13C12-Labelled mono-, di-, and tri-chlorinated dibenzo-p-dioxin (CDD) and chlorinated dibenzofuran (CDF) standards have been tested for their applicability to standard EPA sampling and analytical Methods 0023A/8290. These methods target for analysis only the tetra- through octa-CDD/CDF homologues. Extension of the isotope dilution method to include those lower chlorinated homologues is important toward obtaining reliable species concentration data on the complete, mono- to octa-chlorinated homologue profile. These data will improve our ability to model poly-CDD/CDF concentrations through understanding mechanisms of poly-CDD/CDF formation, chlorination, and dechlorination.

Cancer-related depression: Part II--Neurologic alterations and evolving approaches to psychopharmacology.

PURPOSE/OBJECTIVES: To describe evolving psychopharmacologic options for managing depression related to cancer. DATA SOURCES: Literature review and case study. DATA SYNTHESIS: Optimal recovery from depression requires early detection and selection of psychopharmacologic agents--singly or in combination--to address each depressive symptom. CONCLUSIONS: Adequate psychopharmacologic management of depression requires consistent, informed involvement of healthcare professionals, patients, and caregivers. Patients and caregivers need to be prepared to assess responses to therapy, recognize adverse effects, manage minor side effects and threats to adherence, and seek appropriate assistance for adverse events, including drug-drug interactions. IMPLICATIONS FOR NURSING PRACTICE: Oncology nurses need to take an active role in assessing, documenting, and monitoring patients' responses to antidepressants, particularly during key phases (e.g., induction, substitutions, augmentation, withdrawal). Nurses also need to take an active role in screening patients for depression, preparing patients and caregivers for psychopharmacologic management of depression, and reinforcing these competencies regularly. Oncology nurses also can be instrumental in recognizing signs and symptoms of treatment resistance and addressing these issues appropriately. Because of the rapid pace of psychopharmacologic research, oncology nurses need to update knowledge continuously to provide safe care to depressed patients with cancer.

Cancer-related depression: Part I--Neurologic alterations and cognitive-behavioral therapy.

PURPOSE/OBJECTIVES: To describe neurologic and cognitive alterations underlying symptoms of depression and to explore cognitive-behavioral approaches to promoting recovery from cancer-related depression. DATA SOURCES: Published literature, unpublished raw data, and clinical observations. DATA SYNTHESIS: Depression is a progressive condition that is most responsive to treatment in its earliest stages because of the progressive nature of alterations in neurologic circuits and neurotransmitters. Aggressive screening and management using cognitive-behavioral therapy (CBT) techniques can promote recovery from cancer-related depression and improve patients' quality of life. Application of CBT techniques to patient environments also holds promise of relieving and preventing depression. IMPLICATIONS FOR NURSING PRACTICE: By placing more emphasis on screening for cancer-related depression among newly diagnosed and treated patients, oncology nurses can expedite treatment of cancer-related depression. Working within psychiatric liaison teams or guidelines for routine psychiatric care, oncology nurses can promote recovery and create therapeutic environments that are conducive to promoting patients' mental health along the cancer trajectory.