Identification of Anti-Alpha Toxin Monoclonal Antibodies That Reduce the Severity of Staphylococcus aureus Dermonecrosis and Exhibit a Correlation between Affinity and Potency

Staphylococcus aureus alpha toxin (AT) is an important virulence determinant and may be a valid target for immunoprophylaxis against staphylococcal disease. Here we report the identification of potent inhibitory anti-AT monoclonal antibodies (MAbs) derived using B-cell hybridoma technology from VelocImmune mice engineered to produce IgG with a human variable domain. A small panel of inhibitory MAbs blocked AT-mediated lysis of rabbit red blood cells, A549 human lung epithelial cells, and THP-1 human monocytic cells, in a dose-dependent manner. Binding studies indicated that these MAbs recognize a similar epitope on AT and exhibit dissociation constants (K_D) ranging from 0.50 to 15 nM. In an S. aureus dermonecrosis model, mice passively immunized with anti-AT inhibitory MAbs exhibited significant reductions of lesion size relative to mice treated with an irrelevant IgG control. Interestingly, there was a correlation between MAb affinity for a single epitope, the 50% inhibitory concentration (IC₅₀) in the AT hemolytic assay, and lesion size reduction in the dermonecrosis model. A representative high-affinity MAb, 2A3.1, was demonstrated to significantly reduce lesion size following infection with three different clinical isolates (USA300, CC30, and CC5). Taken together, these results indicate that in vitro potency of anti-AT MAbs predicts in vivo potency in this model, supporting their continued preclinical evaluation as molecules for immunoprophylaxis against staphylococcal skin and soft tissue infections caused by diverse clinical isolates.     

Conformational differences of the C8-deoxyguanosine adduct of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) within the NarI recognition sequence

2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a highly mutagenic heterocyclic amine found in cooked meats. The major DNA adduct of IQ is at the C8-position of dGuo. We have previously reported the incorporation of the C8-IQ adduct into oligonucleotides, namely, the G1-position of codon 12 of the N-ras oncogene sequence (G1G2T) and the G3-position of the NarI recognition sequence (G1G2CG3CC) (Elmquist et al. (2004) J. Am. Chem. Soc. 126, 11189-11201). Ultraviolet spectroscopy and circular dichroism studies indicated that the conformation of the adduct in the two oligonucleotides was different, and they were assigned as groove-bound and base-displaced intercalated, respectively. The conformation of the latter was subsequently confirmed through NMR and restrained molecular dynamics studies (Wang et al. (2006) J. Am. Chem. Soc. 128, 10085-10095). We report here the incorporation of the C8-IQ adduct into the G1- and G2-positions of the NarI sequence. A complete analysis of the UV, CD, and NMR chemical shift data for the IQ protons are consistent with the IQ adduct adopting a minor groove-bound conformation at the G1- and G2-positions of the NarI sequence. To further correlate the spectroscopic data with the adduct conformation, the C8-aminofluorene (AF) adduct of dGuo was also incorporated into the NarI sequence; previous NMR studies demonstrated that the AF-modified oligonucleotides were in a sequence-dependent conformational exchange between major groove-bound and base-displaced intercalated conformations. The spectroscopic data for the IQ- and AF-modified oligonucleotides are compared. The sequence-dependent conformational preferences are likely to play a key role in the repair and mutagenicity of C8-arylamine adducts.     

Serum concentrations of DKK‐1 decrease in patients with multiple myeloma responding to anti‐myeloma treatment

Lytic bone destruction is a hallmark of multiple myeloma (MM) and is because of an uncoupling of bone remodeling. Secretion of Dickkopf (DKK)‐1 by myeloma cells is a major factor which causes inhibition of osteoblast precursors. In this study, the effect of different treatment regimens for MM on serum DKK‐1 was evaluated and correlated with the response to treatment in 101 myeloma patients receiving bortezomib, thalidomide, lenalidomide, adriamycin and dexamethasone (AD) or high‐dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT). At baseline, myeloma patients had increased serum DKK‐1 as compared with patients with MGUS (mean 3786 pg/mL vs. 1993 pg/mL). There was no difference between previously untreated MM patients and patients at relapse. A significant decrease of DKK‐1 after therapy was seen in the following groups: Bortezomib (4059 pg/mL vs. 1862 pg/mL, P = 0.016), lenalidomide (11837 pg/mL vs. 4374 pg/mL, P = 0.039), AD (1668 pg/mL vs. 1241 pg/mL, P = 0.016), and AD + HDCT + ASCT (2446 pg/mL vs. 1082 pg/mL, P = 0.001). Thalidomide led to a non‐significant decrease in DKK‐1 (1705 pg/mL vs. 1269 pg/mL, P = 0.081). Within all groups, a significant decrease of DKK‐1 was only seen in responders (i.e. patients achieving complete remission or partial remission), but not in non‐responders. We show for the first time that serum DKK‐1 levels decrease in myeloma patients responding to treatment, irrespective of the regimen chosen. These data suggest that myeloma cells are the main source of circulating DKK‐1 protein and provide a framework for clinical trials on anti‐DKK‐1 treatment in MM.     

Development and Validation of Patient-Reported Outcome Measures for Sleep Disturbance and Sleep-Related Impairments

Study Objectives:

To develop an archive of self-report questions assessing sleep disturbance and sleep-related impairments (SRI), to develop item banks from this archive, and to validate and calibrate the item banks using classic validation techniques and item response theory analyses in a sample of clinical and community participants.

Design:

Cross-sectional self-report study.

Setting:

Academic medical center and participant homes.

Participants:

One thousand nine hundred ninety-three adults recruited from an Internet polling sample and 259 adults recruited from medical, psychiatric, and sleep clinics.

Interventions:

None.

Measurements and Results:

This study was part of PROMIS (Patient-Reported Outcomes Information System), a National Institutes of Health Roadmap initiative. Self-report item banks were developed through an iterative process of literature searches, collecting and sorting items, expert content review, qualitative patient research, and pilot testing. Internal consistency, convergent validity, and exploratory and confirmatory factor analysis were examined in the resulting item banks. Factor analyses identified 2 preliminary item banks, sleep disturbance and SRI. Item response theory analyses and expert content review narrowed the item banks to 27 and 16 items, respectively. Validity of the item banks was supported by moderate to high correlations with existing scales and by significant differences in sleep disturbance and SRI scores between participants with and without sleep disorders.

Conclusions:

The PROMIS sleep disturbance and SRI item banks have excellent measurement properties and may prove to be useful for assessing general aspects of sleep and SRI with various groups of patients and interventions.

Citation:

Buysse DJ; Yu L; Moul DE; Germain A; Stover A; Dodds NE; Johnston KL; Shablesky-Cade MA; Pilkonis PA. Development and validation of patient-reported outcome measures for sleep disturbance and sleep-related impairments. SLEEP 2010;33(6):781-792.