Octavio Ramilo

Staphylococcus aureus has emerged as a significant pathogen causing severe invasive disease in otherwise healthy people. Despite considerable advances in understanding the epidemiology, resistance mechanisms, and virulence factors produced by the bacteria, there is limited knowledge of the in vivo host immune response to acute, invasive S. aureus infections. Herein, we report that peripheral blood mononuclear cells from patients with severe S. aureus infections demonstrate a distinctive and robust gene expression profile which is validated in a distinct group of patients and on a different microarray platform. Application of a systems-wide modular analysis framework reveals significant over-expression of innate immunity genes and under-expression of genes related to adaptive immunity. Simultaneous flow cytometry analyses demonstrated marked alterations in immune cell numbers, with decreased central memory CD4 and CD8 T cells and increased numbers of monocytes. CD14+ monocyte numbers significantly correlated with the gene expression levels of genes related to the innate immune response. These results demonstrate the value of applying a systems biology approach that reveals the significant alterations in the components of circulating blood lymphocytes and monocytes in invasive S. aureus infections.

Background: Staphylococcus aureus remains the most common etiologic agent of acute osteomyelitis in children. Recently, methicillin-resistant S. aureus (MRSA) has emerged as a major pathogen. Methods: Records of all children admitted with acute osteomyelitis from January 1999 to December 2003 were reviewed. For the comparative analysis, the study population was evenly distributed in 2 periods: period A, January 1999 to June 2001; n = 113; and period B, July 2001 to December 2003; n = 177. In addition, clinical findings of MRSA osteomyelitis were compared with non-MRSA osteomyelitis, including methicillin-sensitive S. aureus infections. Results: Two hundred ninety children (60% male subjects) with acute osteomyelitis were identified. Median (25th-75th percentile) age at diagnosis was 6 years (range, 2-11 years). Significant clinical findings included the following: localized pain (84%), fever (67%), and swelling (62%). Affected bones included the following: foot (23%), femur (20%), tibia (16%), and pelvis (7%). Thirty-seven percent of blood cultures were positive, and a bacterial isolate was obtained in 55% of cases. Bacteria most frequently isolated included the following: methicillin-sensitive S. aureus (45%) (57% in period Avs 40% in period B), MRSA (23%) (6% in A vs 31% in B; P < 0.001), Streptococcus pyogenes (6%), and Pseudomonas aeruginosa (5%). Children with MRSA compared with those with non-MRSA osteomyelitis had significantly greater erythrocyte sedimentation rate and C-reactive protein values on admission and increased length of hospital stay, antibiotic therapy, and overall rate of complications. We observed significant changes in antibiotic therapy related to increased use of agents with activity against MRSA. Conclusions: Methicillin-resistant S. aureus was isolated more frequently in the second study period and was associated with worse clinical outcomes. Level of Evidence: II. Retrospective study.

Motavizumab (MEDI-524) is a monoclonal antibody with enhanced neutralizing activity against RSV. In mice, motavizumab suppressed RSV replication which resulted in significant reduction of clinical parameters of disease severity. We evaluated the effect of motavizumab on the local and systemic immune response induced by RSV in the mouse model. Balb/c mice were intranasally inoculated with 10^6.5 PFU RSV A2 or medium. Motavizumab was given once intraperitoneally (1.25 mg/mouse) as prophylaxis, 24 h before virus inoculation. Bronchoalveolar lavage (BAL) and serum samples were obtained at days 1, 5 (acute) and 28 (long-term) post inoculation and analyzed with a multiplex assay (Beadlyte Upstate, NY) for simultaneous quantitation of 18 cytokines: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, KC (similar to human IL-8), IL-10, IL-12p40, IL-12p70, IL-13, IL-17, TNF-α, MCP-1, RANTES, IFN-γ and GM-CSF. Overall, cytokine concentrations were lower in serum than in BAL samples. By day 28, only KC was detected in BAL specimens at low concentrations in all groups. Administration of motavizumab significantly reduced (p < 0.05) BAL concentrations of IL-1α, IL-12p70 and TNF-α on day 1, and concentrations of IFN-γ on days 1 and 5 compared with RSV-infected untreated controls. In the systemic compartment, the concentrations of IL-10, IFN-γ and KC were significantly reduced in the motavizumab-treated mice compared with the untreated controls. In summary, prophylactic administration of motavizumab was associated with significant reductions on RSV replication and concentrations of cytokine and chemokines, which are likely related to the improvement observed in clinical markers of disease severity.

Each infectious agent represents a unique combination of pathogen-associated molecular patterns that interact with specific pattern-recognition receptors expressed on immune cells. Therefore, we surmised that the blood immune cells of individuals with different infections might bear discriminative transcriptional signatures. Gene expression profiles were obtained for 131 peripheral blood samples from pediatric patients with acute infections caused by influenza A virus, Gram-negative (Escherichia coli) or Gram-positive (Staphylococcus aureus and Streptococcus pneumoniae) bacteria. Thirty-five genes were identified that best discriminate patients with influenza A virus infection from patients with either E coli or S pneumoniae infection. These genes classified with 95% accuracy (35 of 37 samples) an independent set of patients with either influenza A, E coli, or S pneumoniae infection. A different signature discriminated patients with E coli versus S aureus infections with 85% accuracy (34 of 40). Furthermore, distinctive gene expression patterns were observed in patients presenting with respiratory infections of different etiologies. Thus, microarray analyses of patient peripheral blood leukocytes might assist in the differential diagnosis of infectious diseases.

Respiratory syncytial virus (RSV) is the leading viral pathogen responsible for bronchiolitis and pneumonia in infants and young children worldwide. We have previously shown in the mouse model that treatment with an anti-RSV neutralizing monoclonal antibody (MAb) against the F glycoprotein of RSV, palivizumab, decreased lung inflammation, airway obstruction, and postmethacholine airway hyperresponsiveness. MEDI-524, or Numax, is a new MAb derived from palivizumab with enhanced neutralizing activity against RSV. We compared the effects of these two MAbs on different markers of disease severity using the murine model of RSV infection. BALB/c mice were intranasally inoculated with RSV A2. Palivizumab or MEDI-524 was administered once at either 24 h before or 48 h after RSV inoculation. Regardless of the time of administration, all treated mice showed significantly decreased RSV loads in bronchoalveolar lavage samples measured by plaque assay. Only MEDI-524 given at −24 h significantly decreased lung RSV RNA loads on days 5 and 28 after RSV inoculation. Pulmonary histopathologic scores, airway obstruction, and postmethacholine airway hyperresponsiveness were significantly reduced in mice treated with MEDI-524 at 24 h before inoculation, compared with untreated controls and the other regimens evaluated. MEDI-524 was superior to palivizumab on several outcome variables of RSV disease assessed in the mouse model: viral replication, inflammatory and clinical markers of acute disease severity, and long-term pulmonary abnormalities.

Background: Respiratory syncytial virus (RSV) is the leading respiratory viral pathogen in young children worldwide. RSV disease is associated with acute airway obstruction (AO), long-term airway hyperresponsiveness (AHR), and chronic lung inflammation. Using two different mouse strains, this study was designed to determine whether RSV disease patterns are host-dependent. C57BL/6 and BALB/c mice were inoculated with RSV and followed for 77 days. RSV loads were measured by plaque assay and polymerase chain reaction (PCR) in bronchoalveolar lavage (BAL) and whole lung samples; cytokines were measured in BAL samples. Lung inflammation was evaluated with a histopathologic score (HPS), and AO and AHR were determined by plethysmography.Results: Viral load dynamics, histopathologic score (HPS), cytokine concentrations, AO and long-term AHR were similar in both strains of RSV-infected mice, although RSV-infected C57BL/6 mice developed significantly greater AO compared with RSV-infected BALB/c mice on day 5. PCR detected RSV RNA in BAL samples of RSV infected mice until day 42, and in whole lung samples through day 77. BAL concentrations of cytokines TNF-α, IFN-γ, and chemokines MIG, RANTES and MIP-1α were significantly elevated in both strains of RSV-infected mice compared with their respective controls. Viral load measured by PCR significantly correlated with disease severity on days 14 and 21.Conclusion: RSV-induced acute and chronic airway disease is independent of genetic background.