Kristine Grindle

The infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development.

Summary The nasopharynx (NP) is a reservoir for microbes associated with acute respiratory infections (ARIs). Lung inflammation resulting from ARIs during infancy is linked to asthma development. We examined the NP microbiome during the critical first year of life in a prospective cohort of 234 children, capturing both the viral and bacterial communities and documenting all incidents of ARIs. Most infants were initially colonized with Staphylococcus or Corynebacterium before stable colonization with Alloiococcus or Moraxella. Transient incursions of Streptococcus, Moraxella, or Haemophilus marked virus-associated ARIs. Our data identify the NP microbiome as a determinant for infection spread to the lower airways, severity of accompanying inflammatory symptoms, and risk for future asthma development. Early asymptomatic colonization with Streptococcus was a strong asthma predictor, and antibiotic usage disrupted asymptomatic colonization patterns. In the absence of effective anti-viral therapies, targeting pathogenic bacteria within the NP microbiome could represent a prophylactic approach to asthma.

Preseasonal treatment with either omalizumab or an inhaled corticosteroid boost to prevent fall asthma exacerbations

BACKGROUND Short-term targeted treatment can potentially prevent fall asthma exacerbations while limiting therapy exposure. OBJECTIVE We sought to compare (1) omalizumab with placebo and (2) omalizumab with an inhaled corticosteroid (ICS) boost with regard to fall exacerbation rates when initiated 4 to 6 weeks before return to school. METHODS A 3-arm, randomized, double-blind, double placebo-controlled, multicenter clinical trial was conducted among inner-city asthmatic children aged 6 to 17 years with 1 or more recent exacerbations (clincaltrials.gov #NCT01430403). Guidelines-based therapy was continued over a 4- to 9-month run-in phase and a 4-month intervention phase. In a subset the effects of omalizumab on IFN-α responses to rhinovirus in PBMCs were examined. RESULTS Before the falls of 2012 and 2013, 727 children were enrolled, 513 were randomized, and 478 were analyzed. The fall exacerbation rate was significantly lower in the omalizumab versus placebo arms (11.3% vs 21.0%; odds ratio [OR], 0.48; 95% CI, 0.25-0.92), but there was no significant difference between omalizumab and ICS boost (8.4% vs 11.1%; OR, 0.73; 95% CI, 0.33-1.64). In a prespecified subgroup analysis, among participants with an exacerbation during the run-in phase, omalizumab was significantly more efficacious than both placebo (6.4% vs 36.3%; OR, 0.12; 95% CI, 0.02-0.64) and ICS boost (2.0% vs 27.8%; OR, 0.05; 95% CI, 0.002-0.98). Omalizumab improved IFN-α responses to rhinovirus, and within the omalizumab group, greater IFN-α increases were associated with fewer exacerbations (OR, 0.14; 95% CI, 0.01-0.88). Adverse events were rare and similar among arms. CONCLUSIONS Adding omalizumab before return to school to ongoing guidelines-based care among inner-city youth reduces fall asthma exacerbations, particularly among those with a recent exacerbation.

Improved Molecular Typing Assay for Rhinovirus Species A, B, and C

ABSTRACT Human rhinoviruses (RVs), comprising three species (A, B, and C) of the genus Enterovirus, are responsible for the majority of upper respiratory tract infections and are associated with severe lower respiratory tract illnesses such as pneumonia and asthma exacerbations. High genetic diversity and continuous identification of new types necessitate regular updating of the diagnostic assays for the accurate and comprehensive detection of circulating RVs. Methods for molecular typing based on phylogenetic comparisons of a variable fragment in the 5′ untranslated region were improved to increase assay sensitivity and to eliminate nonspecific amplification of human sequences, which are observed occasionally in clinical samples. A modified set of primers based on new sequence information and improved buffers and enzymes for seminested PCR assays provided higher specificity and sensitivity for virus detection. In addition, new diagnostic primers were designed for unequivocal species and type assignments for RV-C isolates, based on phylogenetic analysis of partial VP4/VP2 coding sequences. The improved assay was evaluated by typing RVs in >3,800 clinical samples. RVs were successfully detected and typed in 99% of the samples that were RV positive in multiplex diagnostic assays.

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study

BackgroundCryopreservation of peripheral blood mononuclear cells has been used to preserve and standardize immunologic measurements for multicenter studies, however, effects of cryopreservation on cytokine responses are incompletely understood. In designing immunologic studies for a new multicenter birth cohort study of childhood asthma, we performed a series of experiments to determine the effects of two different methods of cryopreservation on the cytokine responses of cord and peripheral blood mononuclear cells.ResultsPaired samples of PBMC were processed freshly, or after cryopreservation in a Nalgene container (NC) or a controlled-rate freezer (CRF). Although there were some differences between the methods, cryopreservation inhibited PHA-induced IL-10 secretion and Der f 1-induced IL-2 secretion, and augmented PHA-induced IL-2 secretion and spontaneous secretion of TNF-α. In separate experiments, NC cryopreservation inhibited secretion of several cytokines (IL-13, IL-10, IFN-γ, TNF-α) by PHA-stimulated cord blood mononuclear cells. With the exception of PHA-induced IL-13, results from fresh and cryopreserved cord blood samples were not significantly correlated. Finally, in reproducibility studies involving processing of identical cell samples in up to 4 separate laboratories, variances in cytokine responses of fresh cells stimulated at separate sites did not exceed those in cryopreserved cells stimulated at a central site.ConclusionCollectively, these studies indicate that cryopreservation can affect mononuclear cell cytokine response profiles, and that IL-10 secretion and antigen-induced responses may be especially vulnerable. These studies also demonstrate that mononuclear cell responses can be standardized for performance in a small number of laboratories for multicenter studies, and underscore the importance of measuring reproducibility and of testing whether cryopreservation techniques alter specific immunologic outcomes.

Lethal Respiratory Disease Associated with Human Rhinovirus C in Wild Chimpanzees, Uganda, 2013

We describe a lethal respiratory outbreak among wild chimpanzees in Uganda in 2013 for which molecular and epidemiologic analyses implicate human rhinovirus C as the cause. Postmortem samples from an infant chimpanzee yielded near-complete genome sequences throughout the respiratory tract; other pathogens were absent. Epidemiologic modeling estimated the basic reproductive number (R0) for the epidemic as 1.83, consistent with the common cold in humans. Genotyping of 41 chimpanzees and examination of 24 published chimpanzee genomes from subspecies across Africa showed universal homozygosity for the cadherin-related family member 3 CDHR3-Y529 allele, which increases risk for rhinovirus C infection and asthma in human children. These results indicate that chimpanzees exhibit a species-wide genetic susceptibility to rhinovirus C and that this virus, heretofore considered a uniquely human pathogen, can cross primate species barriers and threatens wild apes. We advocate engineering interventions and prevention strategies for rhinovirus infections for both humans and wild apes.

Dynamics of the upper airway microbiome in the pathogenesis of asthma-associated persistent wheeze in preschool children

Repeated cycles of infection-associated lower airway inflammation drives the pathogenesis of persistent wheezing disease in children. Tracking these events across a birth cohort during their first five years, we demonstrate that >80% of infectious events indeed involve viral pathogens, but are accompanied by a shift in the nasopharyngeal microbiome (NPM) towards dominance by a small range of pathogenic bacterial genera. Unexpectedly, this change in NPM frequently precedes the appearance of viral pathogens and acute symptoms. In non-sensitized children these events are associated only with “transient wheeze” that resolves after age three. In contrast, in children developing early allergic sensitization, they are associated with ensuing development of persistent wheeze, which is the hallmark of the asthma phenotype. This suggests underlying pathogenic interactions between allergic sensitization and antibacterial mechanisms.

Cohort-wide characterization of the nasopharyngeal microbiome across year 1 in "high risk" infants by PCR and 16S rRNA gene deep sequencing: elucidation of viral-bacterial-host interactions driving early atopic asthma pathogenesis

Banerji, A; Busse, P; Shennak, M; Lumry, W; Davis-Lorton, M; Wedner, J; Jacobs, J; Baker, J; Bernstein, J; Lockey, R; Li, H; Craig, T; Cicardi, M; Riedl, M; Al-Ghazawi, A; Soo, C; Iarrobino, R; Sexton, D; TenHoor, C; Kenniston, J; Faucette, R; Biedenkapp, J; Chyung, Y; Adelman, B Masschusetts General Hospital, Boston, United States; Mount Sinai School of Medicine, New York, United States; Triumpharma, Amman, Jordan; AARA Research Center, Dallas, United States; Winthrop University Hospital, Mineola, United States; Washington University School of Medicine, St. Louis, United States; Allergy and Asthma Clinical Research, Walnut Creek, United States; Baker Allergy Asthma and Dermatology, Lake Oswego, United States; Univeristy of Cincinnati College of Medicine, Cincinnati, United States; University of South Florida, Tampa, United States; Institute for Asthma and Allergy, Chevy Chase, United States; Penn State Hershey Medical Center, Hersey, United States; University of Milan-Ospedale Luigi Sacco, Milan, Italy; University of California, San Diego, La Jolla, United States; Dyax Corp., Burlington, United States