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1.
J Nurs Adm ; 47(2): 94-100, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28067682

RESUMO

OBJECTIVE: The aim of this study is to increase nurses' time for direct patient care and improve safety via a novel human factors framework for nursing worksystem improvement. BACKGROUND: Time available for direct patient care influences outcomes, yet worksystem barriers prevent nurses adequate time at the bedside. METHODS: A novel human factors framework was developed for worksystem improvement in 3 units at 2 facilities. Objectives included improving nurse efficiency as measured by time-and-motion studies, reducing missing medications and subsequent trips to medication rooms and improving medication safety. RESULTS: Worksystem improvement resulted in time savings of 16% to 32% per nurse per 12-hour shift. Requests for missing medications dropped from 3.2 to 1.3 per day. Nurse medication room trips were reduced by 30% and nurse-reported medication errors fell from a range of 1.2 to 0.8 and 6.3 to 4.0 per month. CONCLUSIONS: An innovative human factors framework for nursing worksystem improvement provided practical and high priority targets for interventions that significantly improved the nursing worksystem.


Assuntos
Eficiência Organizacional , Recursos Humanos de Enfermagem Hospitalar/organização & administração , Sistemas Automatizados de Assistência Junto ao Leito/organização & administração , Melhoria de Qualidade , Gerenciamento do Tempo/organização & administração , Humanos , Relações Enfermeiro-Paciente , Quartos de Pacientes , Estados Unidos
2.
J Nurs Adm ; 44(7/8): 423-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25072233

RESUMO

OBJECTIVE: The aim of this study was to develop a survey tool to assess electronic health record (EHR) implementation to guide improvement initiatives. BACKGROUND: Survey tools are needed for ongoing improvement and have not been developed for aspects of EHR implementation. METHODS: The Baylor EHR User Experience (UX) survey was developed to capture 5 concept domains: training and competency, usability, infrastructure, usefulness, and end-user support. Validation efforts included content validity assessment, a pilot study, and analysis of 606 nurse respondents. The revised tool was sent to randomly sampled EHR nurse-users in 11 acute care facilities. RESULTS: A total of 1,301 nurses responded (37%). Internal consistency of the survey tool was excellent (Cronbach's α = .892). Survey responses including 1,819 open comments were used to identify and prioritize improvement efforts in areas such as education, support, optimization of EHR functions, and vendor change requests. CONCLUSION: The Baylor EHR UX survey was a valid tool that can be useful for prioritizing improvement efforts in relation to EHR implementation.


Assuntos
Registros Eletrônicos de Saúde/normas , Coleta de Dados/métodos , Enfermeiras e Enfermeiros , Reprodutibilidade dos Testes
3.
EGEMS (Wash DC) ; 2(1): 1121, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25848599

RESUMO

CONTEXT: Electronic health records (EHRs) have been promoted as a key driver of improved patient care and outcomes and as an essential component of learning health systems. However, to date, many EHRs are not optimized to support delivery of quality and safety initiatives, particularly in Intensive Care Units (ICUs). Delirium is a common and severe problem for ICU patients that may be prevented or mitigated through the use of evidence-based care processes (daily awakening and breathing trials, formal delirium screening, and early mobility-collectively known as the "ABCDE bundle"). This case study describes how an integrated health care delivery system modified its inpatient EHR to accelerate the implementation and evaluation of ABCDE bundle deployment as a safety and quality initiative. CASE DESCRIPTION: In order to facilitate uptake of the ABCDE bundle and measure delivery of the care processes within the bundle, we worked with clinical and technical experts to create structured data fields for documentation of bundle elements and to identify where these fields should be placed within the EHR to streamline staff workflow. We created an "ABCDE" tab in the existing patient viewer that allowed providers to easily identify which components of the bundle the patient had and had not received. We examined the percentage of ABCDE bundle elements captured in these structured data fields over time to track compliance with data entry procedures and to improve documentation of care processes. MAJOR THEMES: Modifying the EHR to support ABCDE bundle deployment was a complex and time-consuming process. We found that it was critical to gain buy-in from senior leadership on the importance of the ABCDE bundle to secure information technology (IT) resources, understand the different workflows of members of multidisciplinary care teams, and obtain continuous feedback from staff on the EHR revisions during the development cycle. We also observed that it was essential to provide ongoing training to staff on proper use of the new EHR documentation fields. Lastly, timely reporting on ABCDE bundle performance may be essential to improved practice adoption and documentation of care processes. CONCLUSION: The creation of learning health systems is contingent on an ability to modify EHRs to meet emerging care delivery and quality improvement needs. Although this study focuses on the prevention and mitigation of delirium in ICUs, our process for identifying key data elements and making modifications to the EHR, as well as the lessons learned from the IT components of this program, are generalizable to other health care settings and conditions.

4.
Artigo em Inglês | MEDLINE | ID: mdl-22919628

RESUMO

We previously reported the development of a human monoclonal antibody (CS-D7, IgG(1)) with specificity and affinity for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus. CS-D7 mediates opsonophagocytic killing in vitro and protection in a murine sepsis model. In light of recent data indicating that IsdB specific T cells (CD4+, Th17), not Ab, mediate protection after vaccination with IsdB, it is important to investigate the mechanism of protection mediated by CS-D7. The mAb was examined to determine if it blocked heme binding to IsdB in vitro. The mAb was not found to have heme blocking activity, nor did it prevent bacterial growth under in vivo conditions, in an implanted growth chamber. To assess the role of the mAb Fc a point mutation was introduced at aa 297 (CS-D7·N297A). This point mutation removes Fc effector functions. In vitro analysis of the mutein confirmed that it lacked measurable binding to FcγR, and that it did not fix complement. The mutein had dramatically reduced in vitro opsonic OP activity compared to CS-D7. Nonetheless, the mutein conferred protection equivalent to the wild type mAb in the murine sepsis model. Both wild type and mutein mAbs were efficacious in FcγR deletion mice (including both FcγRII(-/-) mice and FcγRIII(-/-) mice), indicating that these receptors were not essential for mAb mediated protection in vivo. Protection mediated by CS-D7 was lost in Balb/c mice depleted of C3 with cobra venom factor (CFV), was lost in mice depleted of superoxide dismutase (SOD) in P47phox deletion mice, and as previously reported, was absent in SCID mice (Joshi et al., 2012). Enhanced clearance of S. aureus in the liver of CS-D7 treated mice and enhanced production of IFN-γ, but not of IL17, may play a role in the mechanism of protection mediated by the mAb. CS-D7 apparently mediates survival in challenged mice through a mechanism involving complement, phagocytes, and lymphocytes, but which does not depend on interaction with FcγR, or on blocking heme uptake.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Proteínas de Transporte de Cátions/imunologia , Proteínas Opsonizantes/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas do Sistema Complemento/imunologia , Modelos Animais de Doenças , Heme/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Proteínas Mutantes/metabolismo , Proteínas Opsonizantes/metabolismo , Fagocitose , Ligação Proteica , Sepse/imunologia , Sepse/prevenção & controle , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Análise de Sobrevida
5.
Protein Cell ; 2(7): 573-84, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21822802

RESUMO

We have previously described a novel artificial NFEV ß-secretase (BACE1) cleavage site, which when introduced into the amyloid-ß precursor protein (APP), significantly enhances APP cleavage by BACE1 in in vitro and cellular assays. In this study, we describe the identification and characterization of a single chain fragment of variable region (scFv), specific to the EV neo-epitope derived from BACE1 cleavage of the NFEV-containing peptide, and its conversion to IgG1. Both the scFv displayed on phage and EV-IgG1 show exquisite specificity for binding to the EV neoepitope without cross-reactivity to other NFEV containing peptides or WT-APP KMDA cleavage products. EV-IgG1 can detect as little as 0.3 nmol/L of the EV peptide. EV-IgG1 antibody was purified, conjugated with alkaline phosphatase and utilized in various biological assays. In the BACE1 enzymatic assay using NFEV substrate, a BACE1 inhibitor MRK-3 inhibited cleavage with an IC(50) of 2.4 nmol/L with excellent reproducibility. In an APP_NFEV stable SH-SY5Y cellular assay, the EC(50) for inhibition of EV-Aß peptide secretion with MRK-3 was 236 nmol/L, consistent with values derived using an EV polyclonal antibody. In an APP_NFEV knock-in mouse model, both Aß_EV40 and Aß_EV42 peptides in brain homogenate showed excellent gene dosage dependence. In conclusion, the EV neoepitope specific monoclonal antibody is a novel reagent for BACE1 inhibitor discovery for both in vitro, cellular screening assays and in vivo biochemical studies. The methods described herein are generally applicable to novel synthetic substrates and enzyme targets to enable robust screening platforms for enzyme inhibitors.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide , Anticorpos/farmacologia , Química Encefálica/efeitos dos fármacos , Anticorpos de Cadeia Única/farmacologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Técnicas de Introdução de Genes , Humanos , Concentração Inibidora 50 , Camundongos , Dados de Sequência Molecular
6.
Clin Vaccine Immunol ; 16(8): 1095-104, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19553551

RESUMO

In an effort to characterize important epitopes of Staphylococcus aureus iron-regulated surface determinant B (IsdB), murine IsdB-specific monoclonal antibodies (MAbs) were isolated and characterized. A panel of 12 MAbs was isolated. All 12 MAbs recognized IsdB in enzyme-linked immunosorbent assays and Western blots; 10 recognized native IsdB expressed by S. aureus. The antigen epitope binding of eight of the MAbs was examined further. Three methods were used to assess binding diversity: MAb binding to IsdB muteins, pairwise binding to recombinant IsdB, and pairwise binding to IsdB-expressing bacteria. Data from these analyses indicated that MAbs could be grouped based on distinct or nonoverlapping epitope recognition. Also, MAb binding to recombinant IsdB required a significant portion of intact antigen, implying conformational epitope recognition. Four MAbs with nonoverlapping epitopes were evaluated for in vitro opsonophagocytic killing (OPK) activity and efficacy in murine challenge models. These were isotype switched from immunoglobulin G1 (IgG1) to IgG2b to potentially enhance activity; however, this isotype switch did not appear to enhance functional activity. MAb 2H2 exhibited OPK activity (> or =50% killing in the in vitro OPK assay) and was protective in two lethal challenge models and a sublethal indwelling catheter model. MAb 13C7 did not exhibit OPK (<50% killing in the in vitro assay) and was protective in one lethal challenge model. Neither MAb 13G11 nor MAb 1G3 exhibited OPK activity in vitro or was active in a lethal challenge model. The data suggest that several nonoverlapping epitopes are recognized by the IsdB-specific MAbs, but not all of these epitopes induce protective antibodies.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/isolamento & purificação , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Proteínas de Transporte de Cátions/imunologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/imunologia , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Camundongos , Viabilidade Microbiana , Proteínas Opsonizantes/imunologia , Infecções Estafilocócicas/imunologia , Análise de Sobrevida
7.
MAbs ; 1(6): 572-9, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20073128

RESUMO

The Fc region of an antibody mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and plays a key role in the in vivo half-life of an antibody. In designing antibody therapeutics, it is sometimes desirable that the antibody has altered Fc-mediated properties. In the case of a "benign blocker" antibody, it is often desirable to diminish or abolish the ADCC and CDC functions while retaining its PK profile. Here, we report a novel engineered IgG isotype, IgG2m4, with reduced Fc functionality. IgG2m4 is based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S). An IgG2m4 antibody has an overall reduction in complement and Fc gamma receptor binding in in vitro binding analyses while maintaining the normal in vivo serum half-life in rhesus.


Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Complemento C1q/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Engenharia de Proteínas , Receptores de IgG/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Complemento C1q/genética , Complemento C1q/imunologia , Cricetinae , Meia-Vida , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptores de IgG/genética , Receptores de IgG/imunologia
8.
Hum Vaccin ; 5(4): 254-63, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18787395

RESUMO

Staphylococcus aureus is a clinically important capsule-forming bacterium. The capsule polysaccharide (CPs) occurs as different chemical structures depending on the serotype of the organism, but one form, capsular polysaccharide type 8 (CPs8) found in clinical isolates, is largely unstudied. The potential of CPs8 as a vaccine target was evaluated using two approaches. The first approach used a conjugate vaccine, made by chemically linking purified CPs8 to the outer membrane protein complex of N. meningitidis serotype B (OMPC). In efficacy studies, the CPs8-OMPC conjugate vaccine was immunogenic in Balb/c mice, however the immune response gave no protection from death after a lethal intravenous (IV) challenge with S. aureus Becker. In the second approach, two monoclonal antibodies were produced against CPs8 (mAbs 8E8 and 1C10). These were found to have functional activity in an opsonophagocytic killing assay (OPA), and provided protection from a lethal challenge when bacteria were pre-opsonized ex vivo before intra-peritoneal (IP) challenge. However, mAb 8E8 was not efficacious in the lethal challenge model, in which antibodies were passively transferred to the peritoneum and the animals were infected via the tail vein 18-24 h later. Additionally, the monoclonal antibodies did not opsonize capsule-expressing S. aureus Becker obtained from in vivo growth conditions. These results indicated that functional capsule antibodies may not be sufficient for protection from S. aureus under all in vivo conditions.


Assuntos
Anticorpos Antibacterianos/uso terapêutico , Cápsulas Bacterianas/imunologia , Infecções Estafilocócicas/prevenção & controle , Infecções Estafilocócicas/terapia , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Feminino , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Opsinas/imunologia , Análise de Sobrevida , Vacinas Conjugadas/imunologia
9.
MAbs ; 1(5): 462-74, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20065653

RESUMO

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.


Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Fragmentos de Imunoglobulinas/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Regiões Determinantes de Complementaridade/química , Anticorpos Anti-HIV/química , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Testes de Neutralização , Biblioteca de Peptídeos
10.
AIDS Res Hum Retroviruses ; 24(12): 1537-44, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19102685

RESUMO

Class 1 and class 2 fusion peptides bind to the trimeric N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions of HIV-1 envelope glycoprotein gp41, respectively, and block its intramolecular folding required for Env-mediated viral and host cell membrane fusion and subsequent viral entry. Using a combination of T-20 (class 1) and (CCIZN17)(3) (class 2), we provide evidence that these classes of fusion peptides work synergistically in an in vitro infectivity assay in inhibiting the entry of primary HIV-1 isolate 89.6 with combination indexes reaching 0.37 and 0.32 at IC(50) and IC(90), respectively. We further demonstrate a similar degree of neutralization synergy between a monoclonal antibody (MAb), D5, targeting the hydrophobic pocket region of the NHR, and 2F5, a well-characterized MAb that targets the C-terminal end of CHR and the membrane-proximal external region (MPER), providing a rational basis for developing combination vaccines targeting these two highly conserved regions of gp41.


Assuntos
Antivirais/farmacologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/antagonistas & inibidores , Proteína gp41 do Envelope de HIV/farmacologia , HIV-1/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Internalização do Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antivirais/metabolismo , Sinergismo Farmacológico , Enfuvirtida , Anticorpos Anti-HIV/metabolismo , Proteína gp41 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/metabolismo , Humanos , Concentração Inibidora 50 , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo
11.
J Immunol Methods ; 322(1-2): 94-103, 2007 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-17362979

RESUMO

Screening antibodies from phage displayed in vitro libraries and from affinity maturation of lead antibodies requires testing of antibody fragments (scFvs and Fabs) for function and binding affinities. Crude scFv or Fab periplasmic preparations from Escherichia coli are often not pure and/or concentrated enough for use in functional and affinity assays. We have developed an automated high-throughput approach for small and large-scale expression and purification of His-tagged scFvs and Fabs using the Qiagen BioRobot 3000 LS with optimized application software. This automated procedure enabled us to rapidly evaluate antibody fragments in functional and surface plasmon resonance (SPR) assays. We have used these procedures to make thousands of purified scFv/Fabs for several antibody maturation campaigns and significantly decreased the time needed to select the best candidates. The assay results from these purified samples were used to prioritize candidates before converting them to IgG. This protocol can process up to 300 small-scale and up to 72 large-scale scFvs or Fabs per week per full-time employee (FTE).


Assuntos
Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Robótica/instrumentação , Animais , Escherichia coli/genética , Humanos , Imunoensaio , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Cinética , Biblioteca de Peptídeos , Software , Ressonância de Plasmônio de Superfície
12.
Methods Mol Med ; 127: 11-22, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16988443

RESUMO

For more than three decades, plasmids have been widely used in the biotechnology arena. Historically, they have been most often employed for the expression of heterologous proteins in a variety of microorganisms. More recently, plasmids have been used as vectors for the delivery of antigen encoding genes in order to elicit immune responses in higher order animals. In this chapter, we discuss methods for constructing vectors with this unique purpose. Considerations for choosing the replicon, antigen, expression elements, and host cells are discussed within the context of developing a commercially viable vaccine vector.


Assuntos
Expressão Gênica , Plasmídeos/genética , Replicon/genética , Vacinas de DNA/genética , Animais , Vetores Genéticos , Humanos , Plasmídeos/imunologia , Replicon/imunologia , Vacinas de DNA/imunologia
13.
Infect Immun ; 74(4): 2215-23, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16552052

RESUMO

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.


Assuntos
Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Proteínas de Transporte de Cátions/imunologia , Macaca mulatta/imunologia , Sepse/imunologia , Infecções Estafilocócicas/imunologia , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/química , Proteínas de Transporte de Cátions/administração & dosagem , Proteínas de Transporte de Cátions/química , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Sepse/mortalidade , Sepse/prevenção & controle , Homologia de Sequência de Aminoácidos , Infecções Estafilocócicas/mortalidade , Infecções Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/administração & dosagem , Staphylococcus aureus/isolamento & purificação , Taxa de Sobrevida
14.
Antimicrob Agents Chemother ; 47(9): 2740-8, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12936968

RESUMO

Staphylococcal infections associated with catheter and prosthetic implants are difficult to eradicate and often lead to chronic infections. Development of novel antibacterial therapies requires simple, reliable, and relevant models for infection. Using bioluminescent Staphylococcus aureus, we have adapted the existing foreign-body and deep-wound mouse models of staphylococcal infection to allow real-time monitoring of the bacterial colonization of catheters or tissues. This approach also enables kinetic measurements of bacterial growth and clearance in each infected animal. Persistence of infection was observed throughout the course of the study until termination of the experiment at day 16 in a deep-wound model and day 21 in the foreign-body model, providing sufficient time to test the effects of antibacterial compounds. The usefulness of both animal models was assessed by using linezolid as a test compound and comparing bioluminescent measurements to bacterial counts. In the foreign-body model, a three-dose antibiotic regimen (2, 5, and 24 h after infection) resulted in a decrease in both luminescence and bacterial counts recovered from the implant compared to those of the mock-treated infected mice. In addition, linezolid treatment prevented the formation of subcutaneous abscesses, although it did not completely resolve the infection. In the thigh model, the same treatment regimen resulted in complete resolution of the luminescent signal, which correlated with clearance of the bacteria from the thighs.


Assuntos
Corpos Estranhos/microbiologia , Infecções Estafilocócicas/microbiologia , Infecção dos Ferimentos/microbiologia , Abscesso/microbiologia , Acetamidas/uso terapêutico , Animais , Antibacterianos/uso terapêutico , Cateterismo , Contagem de Colônia Microbiana , Relação Dose-Resposta a Droga , Feminino , Corpos Estranhos/tratamento farmacológico , Corpos Estranhos/patologia , Linezolida , Medições Luminescentes , Camundongos , Camundongos Endogâmicos BALB C , Músculo Esquelético/patologia , Oxazolidinonas/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Coxa da Perna/patologia , Fatores de Tempo , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/patologia
15.
Carbohydr Res ; 338(9): 903-22, 2003 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-12681914

RESUMO

Colonization of implanted medical devices by coagulase-negative staphylococci such as Staphylococcus epidermidis is mediated by the bacterial polysaccharide intercellular adhesin (PIA), a polymer of beta-(1-->6)-linked glucosamine substituted with N-acetyl and O-succinyl constituents. The icaADBC locus containing the biosynthetic genes for production of PIA has been identified in both S. epidermidis and S. aureus. Whereas it is clear that PIA is a constituent that contributes to the virulence of S. epidermidis, it is less clear what role PIA plays in infection with S. aureus. Recently, identification of a novel polysaccharide antigen from S. aureus termed poly N-succinyl beta-(1-->6)-glucosamine (PNSG) has been reported. This polymer was composed of the same glycan backbone as PIA but was reported to contain a high proportion of N-succinylation rather than acetylation. We have isolated a glucosamine-containing exopolysaccharide from the constitutive over-producing MN8m strain of S. aureus in order to prepare polysaccharide-protein conjugate vaccines. In this report we demonstrate that MN8m produced a high-molecular-weight (>300,000 Da) polymer of beta-(1-->6)-linked glucosamine containing 45-60% N-acetyl, and a small amount of O-succinyl (approx 10% mole ratio to monosaccharide units). By detailed NMR analyses of polysaccharide preparations, we show that the previous identification of N-succinyl was an analytical artifact. The exopolysaccharide we have isolated is active in in vitro hemagglutination assays and is immunogenic in mice when coupled to a protein carrier. We therefore conclude that S. aureus strain MN8m produces a polymer that is chemically and biologically closely related to the PIA produced by S. epidermidis.


Assuntos
Polissacarídeos Bacterianos/imunologia , Polissacarídeos Bacterianos/isolamento & purificação , Staphylococcus aureus/química , Animais , Configuração de Carboidratos , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática , Testes de Hemaglutinação , Ácidos Levulínicos/análise , Ácidos Levulínicos/química , Espectroscopia de Ressonância Magnética , Camundongos , Peso Molecular , Polissacarídeos Bacterianos/química
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