Holliday T. Carper

Human metapneumovirus infection in children hospitalized for wheezing

To the Editor: Recently, the prevalence of viral respiratory tract pathogens associated with acute wheezing exacerbations was reported in a study of infants and children admitted to the Children’s Hospital at the University of Virginia.1 Subsequently, we analyzed the nasal washes from these children for human metapneumovirus (hMPV), a paramyxovirus closely related to respiratory syncytial virus (RSV). hMPV has also been shown to be a significant cause of respiratory tract illnesses in young children.2–4 The study we previously reported from the University of Virginia was an observational, case-control study of 133 children (age 2 months to 18 years) hospitalized for wheezing between April 1, 2000, and March 31, 2001. This represented 93% of all admissions for acute wheezing during the study period. Children who had evidence of cardiopulmonary disease or immunosuppression were excluded. The control subjects included 133 age-matched and gender-matched hospitalized children without wheezing. They were enrolled during the same season of each matched wheezing subject. Demographic information was obtained from parent-administered questionnaires and hospital charts. The Human Investigation Committee at the University of Virginia approved the study. Informed consent was obtained from parents, and assent was obtained from older children. Nasal washes were obtained from patients to test for viruses by culture and for RSV and influenza by antigen testing. In addition, nucleic acids extracted from the samples had been tested previously by RT-PCR for rhinovirus, enterovirus, coronavirus, influenza virus, parainfluenza virus, and RSV, and by PCR for adenovirus.1 Metapneumovirus testing was performed by a real-time RT-PCR assay on a Smart Cycler (Cepheid, Sunnyvale, Calif) using primers and probe for the N gene5 with the Quantitect RT-PCR kit (Qiagen, Valencia, Calif). The probe was altered slightly, with BHQ-3 (Invitrogen, Carlsbad, Calif) substituted for 5-carboxytetramethyl-rhodamine as the 3′ fluorescent quencher. These primers and probe have been shown to detect all 4 genetic lineages of hMPV, and the limit of detection in our assay was 50 copies of viral genome per reaction. Patient demographic data and the frequencies for positive tests for virus among wheezing and control patients were analyzed by nonparametric exact methods. Exact 2-sided 95% CIs for the difference of proportion were constructed as described by Agresti and Min.6 Multivariate analyses related to predicting wheezing as a function of the patient’s atopic status and evidence for viral infection were performed by multiple logistic regression. Tests of association were evaluated on the basis of the generalized Wald χ2 statistic, and 95% CI construction for the adjusted odds ratio was based on the Wald approximation. Total serum IgE data were analyzed on the logarithmic scale by way of 1-way ANOVA. Demographic characteristics of the study population were previously described in detail.1 Thirteen children in this study tested positive for hMPV (10 children hospitalized for wheezing and 3 controls; Table I). Seven of the 10 wheezing children were <3 years old, and half were males. The mean age of the children <3 years old who were infected with hMPV was 7 months (range, 2–13 months). The majority of subjects with positive tests for hMPV (85%; 11/13) were hospitalized from January through April. TABLE I Characteristics of subjects infected with hMPV The prevalence of positive tests for hMPV among children <3 years old was 8.9% (7/79) in wheezing children compared with 1.3% (1/77) of controls (P = .035). Overall, the children in this age group who tested positive for any virus (including hMPV) were more likely to be hospitalized for wheezing than the children who tested negative for virus (odds ratio, 6.48; 95% CI, 2.83–14.81; P < .001). More wheezing children than controls who were younger than 3 years had positive tests for 1 or more viruses (32% compared with 16%; P = .02). Among those who tested positive for hMPV, 3 of the wheezing subjects as well as the control tested positive for other viral pathogens (Table I). Only 1 of these 4 children (all admitted during the midwinter) tested positive for RSV, and none tested positive for influenza. Mean serum total IgE levels were not different between hMPV-infected children with wheezing and hMPV-infected controls. Among the children 3 to 18 years old, the children who tested positive for any virus were more likely to be hospitalized for wheezing than the children who tested negative for virus (odds ratio, 6.00; 95% CI, 2.62–13.73; P < .001). As previously reported,1 rhinovirus was the dominant pathogen, whereas hMPV was not significantly associated with wheezing in this age group. Among children who were 3 through 9 years of age, hMPV was detected in 8.8% (3/34) of the wheezing subjects compared with 5.7% (2/35) of the controls (P = .71). None of the children who tested positive for hMPV in this age group were coinfected with another virus. No subject older than 9 years of age had a positive test for hMPV. In this analysis, we found a significant association between hMPV infection and wheezing among children younger than 3 years, especially during the midwinter months. This is consistent with a highly significant association between hMPV and wheezing exacerbations observed in a 25-year prospective study of lower respiratory tract illness in otherwise healthy outpatient children (in Tennessee) who were younger than 5 years.3 In contrast, hMPV was not significantly associated with wheezing requiring hospitalization among children 3 years of age and older in our study. Instead, rhinovirus was the dominant pathogen associated with severe exacerbations, which has also been observed in other studies of children hospitalized for wheezing.7 In addition, the large majority (at least 80%) of the wheezing children age 3 years and older who were hospitalized in Virginia had striking atopic characteristics.1 Combined with test results for other viral pathogens reported previously in the same patients,1 the detection of hMPV in our current analysis increased the overall prevalence of viral infections among the wheezing subjects younger than 3 years to almost 90%. These results confirm and strengthen the observation that viral respiratory tract pathogens are the dominant risk factor for wheezing exacerbations in early childhood. Similar to other viruses that are significantly associated with wheezing during early childhood, more information is needed about the infants who are infected with hMPV and their long-term prognosis with respect to persistent wheezing and their risk for developing asthma.8

Comparison of Viral Load in Individuals with and without Asthma during Infections with Rhinovirus

RATIONALE Most virus-induced attacks of asthma are caused by rhinoviruses (RVs). OBJECTIVES To determine whether people with asthma are susceptible to an increased viral load during RV infection. METHODS Seventy-four children (4-18 yr old) were enrolled; 28 with wheezing, 32 with acute rhinitis, and 14 without respiratory tract symptoms. Nasal washes were evaluated using quantitative polymerase chain reaction for RV to judge viral load along with gene sequencing to identify strains of RV. Soluble intercellular adhesion molecule-1, IFN-λ1, and eosinophil cationic protein in nasal washes, along with blood eosinophil counts and total and allergen-specific IgE in sera, were also evaluated. Similar assessments were done in 24 young adults (16 with asthma, 8 without) who participated in an experimental challenge with RV (serotype 16). MEASUREMENTS AND MAIN RESULTS Fifty-seven percent of wheezing children and 56% with acute rhinitis had nasal washes testing positive for RV. The geometric mean of viral loads by quantitative polymerase chain reaction in washes from wheezing children was 2.8-fold lower, but did not differ significantly from children with rhinitis (7,718 and 21,612 copies of viral RNA per microliter nasal wash, respectively; P = 0.48). The odds for wheezing were increased if children who tested positive for RV were sensitized to one or more allergens (odds ratio, 3.9; P = 0.02). Similarly, neither peak nor cumulative viral loads differed significantly in washes from adults with asthma compared with those without asthma during the experimental RV challenge. CONCLUSIONS During acute symptoms, children infected with RV enrolled for wheezing or acute rhinitis had similar viral loads in their nasal washes, as did adults with and without asthma infected with RV-16 experimentally.

Both recombinant interleukin-1 (beta) and purified human monocyte interleukin-1 prime human neutrophils for increased oxidative activity and promote neutrophil spreading.

Both purified human monocyte interleukin‐1 and recombinant interleukin‐1 (beta) primed neutrophils for increased superoxide production and chemiluminescence in response to f‐met‐leu‐phe. In addition, purified human monocyte interleukin‐1 and recombinant interleukin‐1 (beta) altered neutrophil shape. Recombinant interleukin‐1 (alpha) used at the same concentration of interleukin‐1 (beta) did not prime neutrophils for increased superoxide production after stimulation with f‐met‐leu‐phe. interleukin‐1 expressed by monocytes in response to endotoxin stimulation could act as a modulator of neutrophil function.

In vitro demonstration of transport and delivery of antibiotics by polymorphonuclear leukocytes.

Several antibiotics are concentrated inside polymorphonuclear leukocytes (PMN). To investigate whether PMN could act as vehicles for delivery of antibiotics, we combined an assay measuring PMN chemotaxis under agarose with a bioassay measuring levels of antibiotic in agar. Double-layer plates were made by pouring a layer of chemotaxis agarose into tissue culture plates and then adding a thin layer of Trypticase soy agar. Neutrophils were incubated with antibiotic for 1 h and then were washed and placed in wells made in the plates. After allowing PMN to migrate under the agar toward a chemoattractant well containing formyl-methionine-leucine-phenylalanine for 3 h, Streptococcus pyogenes was streaked on top of the agar and grown overnight. PMN migration and zones of inhibition of bacterial growth were measured. Neutrophils migrated 2.51 +/- 0.16 mm toward the chemoattractant well and 1.48 +/- 0.12 mm toward the medium well; migration was not significantly affected by any of the antibiotics used. Plates with PMN incubated without antibiotic showed insignificant inhibition of bacterial growth toward chemoattractant and medium wells (0.38 +/- 0.18 and 0.14 +/- 0.12 mm, respectively; for both, P > 0.05 for difference from 0). PMN incubated with oxacillin (3 micrograms/ml), a drug not concentrated in PMN, caused a similar lack of inhibition (0.28 +/- 0.09 mm toward chemoattractant; 0.14 +/- 0.03 mm toward medium). Incubation with 30 microns of ciprofloxacin per ml resulted in inhibition that was similar in both directions (1.40 +/- 0.16 versus 1.18 +/- 0.13 mm). However, for PMN incubated with azithromycin (3 micrograms/ml), an agent highly concentrated inside phagocytes, there was a large degree of inhibition which was significantly greater in the direction of chemoattractant than in the direction of medium (3.47 +/- 0.30 versus 1.89 +/- 0.25 mm; P < 0.001), indicating that release of bioactive azithromycin by neutrophils occurred after migration. Likewise, after incubation with rifampin (10 micrograms/ml), which is also concentrated by PMN, inhibition was significantly greater in the direction of chemoattractant than in the direction of medium (1.54 +/- 0.24 versus 0.81 +/- 0.28 mm; P = 0.001). We conclude that for certain antibiotics, PMN may act as vehicles for transport and delivery of active drug to sites of infection. Images

Adenosine modulation of tumor necrosis factor-α-induced neutrophil activation

We hypothesized that adenosine, known to be release from inflammatory sites, could lessen the potentially damaging activity of neutrophils (PMN) primed by tumor necrosis factor-alpha (TNF alpha) at sites of infection. We investigated the effect of adenosine on PMN primed with cell-free medium from mononuclear leukocytes (MNL) that had been treated with lipopolysaccharide (LPS) yielding a conditioned medium rich in TNF alpha and on PMN primed with recombinant human TNF alpha (rhTNF alpha). LPS (10 ng/mL) minimally primed PMN, but LPS-MNL-conditioned medium increased PMN chemiluminescence in response to f-Met-Leu-Phe (fMLP) 1242% compared with unprimed PMN. LPS-MNL-conditioned medium contained adenosine (approximately 30 nM). Converting the adenosine in the LPS-MNL-conditioned medium to inosine with adenosine deaminase (ADA) or blocking adenosine binding to PMN with the adenosine receptor antagonist 1,3-dipropyl-8-(phenyl-p-acrylate)-xanthine (BW A1433U) resulted in a near doubling of chemiluminescence. The LPS-MNL-conditioned medium contained TNF alpha (836 pg/mL; approximately 1 U/mL). Recombinant human TNF alpha (1 U/mL) primed PMN for a 1033% increase in chemiluminescence. Added adenosine decreased rhTNF alpha-primed PMN chemiluminescence (IC50 approximately 100 nM), and adenosine (100 nM) decreased both superoxide and myeloperoxidase release from rhTNF alpha-primed fMLP-stimulated PMN. The activity of adenosine was counteracted by ADA and BW A1433U, and the modulating effect of adenosine was on the primed response rather than on priming per se. Thus, physiological concentrations of adenosine reduce the effects of recombinant human TNF alpha and native human TNF alpha (released from LPS-treated MNL) on PMN activity. Endogenous adenosine may preclude or minimize damage to infected tissue by damping the TNF alpha-primed PMN oxidative response.

Tumor necrosis factor-alpha decreases neutrophil chemotaxis to N-formyl-1-methionyl-1-leucyl-1-phenylalanine : analysis of single cell movement

Tumor necrosis factor‐alpha (TNF‐α), a cytokine produced by mononuclear cells in response to endotoxin, inhibits neutrophil chemotaxis. We analyzed the effects of TNF‐α on the orientation and movement of individual neutrophils in a chemoattractant gradient. Neutrophils, treated or untreated with TNF‐α, were observed migrating in a gradient of the chemotactic peptide N‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine (fMLP) on a specially constructed chamber (Zigmond bridge). The movement of these cells was videotaped, digitized, and then tracked using a newly designed computer algorithm. The data obtained from this algorithm were then utilized to calculate distance traveled, speed and ability to polarize and migrate in a directed manner for each individual cell. TNF‐a‐treated cells behaved like cells not exposed to fMLP in that they failed to orient in a chemotactic gradient and moved in a manner similar to randomly migrating cells. This study provides unique observations of the effect of TNF‐α on multiple parameters of PMN migration.

Pentoxifylline modulates activation of human neutrophils by amphotericin B in vitro.

The antifungal agent amphotericin B (AmB) alters neutrophil (polymorphonuclear leukocyte [PMN]) function, and this may be the mechanism for some of the adverse effects caused by AmB. AmB is a potent inhibitor of PMN migration, increases PMN adherence and aggregation, and primes PMN for increased oxidative activity in response to a second stimulus. AmB also stimulates mononuclear leukocytes (MNLs) to release inflammatory mediators which augment the effects of AmB on PMN function. In the present study, we observed that the methylxanthine derivative pentoxifylline decreased the effects of AmB on PMN function. AmB (2 micrograms/ml) priming doubled PMN chemiluminescence stimulated by fMet-Leu-Phe. In the presence of MNLs, AmB priming increased fMet-Leu-Phe-stimulated PMN chemiluminescence to 622% of unprimed PMN activity. Pentoxifylline (100 microM) blunted the rise in AmB-augmented PMN chemiluminescence in the presence of MNLs to 282% of unprimed PMN activity, and pentoxifylline metabolites were active at 10 microM. Pentoxifylline (100 microM) also blocked AmB-augmented PMN oxidative activity in whole blood, as measured by nitroblue tetrazolium reduction. In the presence of MNL, AmB (2 micrograms/ml) doubled the expression of the important PMN adherence factor Mac-1. Pentoxifylline (1 mM) decreased AmB-stimulated PMN Mac-1 expression back to unstimulated amounts. In the presence of MNLs, AmB (2 micrograms/ml) decreased PMN nondirected and directed migration to fMet-Leu-Phe to 40 and 38% of control PMN migration, respectively. Pentoxifylline (300 microM) counteracted AmB inhibition of nondirected and directed migration to fMet-Leu-Phe, resulting in migration that was 71 and 87% of control PMN migration, respectively. In contrast, the methylxanthine caffeine (100 muM) increased AmB-enhanced chemiluminescence but did not affect AmB-inhibited PMN migration. Pentoxifylline should be evaluated as adjunctive therapy to lessen the inflammatory damage caused by AmB.

Methylxanthines with adenosine alter TNFα-primed PMN activation

Abstract Methylxanthines are best known as phosphodiesterase inhibitors that cause a rise in intracellular cAMP. One would expect the two methylxanthines, caffeine and pentoxifylline, to have similar actions on neutrophils (PMN). However, caffeine stimulated and pentoxifylline inhibited PMN oxidative activity. Micromolar concentrations of pentoxifylline decreased native and recombinant tumor necrosis factor-α (TNFα)-primed formyl met-leu-phe (fMLP)-stimulated PMN chemiluminescence, superoxide production and myeloperoxidase (MPO) release. In contrast, equal concentrations of caffeine increased chemiluminescence and MPO release with no effect on superoxide production. These activities of the methylxanthines were only observed in the presence of physiological concentrations of adenosine, and were abolished by the treatment of the PMN with adenosine deaminase. The activities of adenosine, pentoxifylline and caffeine on PMN activity could not be readily explained by changes in PMN [cAMP]. Thus for TNFα-primed PMN, pentoxifylline decreases PMN activity by enhancing the effect of adenosine on degranulation and superoxide production; whereas caffeine increases PMN activity by counteracting the effect of adenosine on degranulation.

Lipid complexing decreases amphotericin B inflammatory activation of human neutrophils compared with that of a desoxycholate-suspended preparation of amphotericin B (Fungizone).

Amphotericin B (AmB) has toxic effects and alters neutrophil (polymorphonuclear leukocyte [PMN]) function. A lipid-complexed formulation of AmB (AmB-LC) has been reported (A. S. Janoff, L. T. Boni, M. C. Popescu, S. R. Minchey, P. R. Cullis, T. D. Madden, T. Taraschi, S. M. Gruner, E. Shyamsunder, M. W. Tate, R. Mendelsohn, and D. Bonner, Proc. Natl. Acad. Sci. USA 85:6122-6126, 1988) to be less toxic than a desoxycholate-suspended preparation of AmB (AmB-des; Fungizone). In this study we compared the effects of AmB-des and AmB-LC on in vitro PMN function. Neither form of AmB stimulated PMN chemiluminescence, but AmB-des (2 micrograms/ml) nearly tripled PMN chemiluminescence in response to f-Met-Leu-Phe (fMLP), a phenomenon known as priming. Because AmB stimulates monocytes to release cytokines which can affect PMN function, we studied the effects of AmB on PMNs in mixed leukocyte cultures. AmB-des (1 to 2 micrograms/ml) increased the chemiluminescence of PMNs plus mixed mononuclear leukocytes (MNLs) to fMLP. The activity was about three times that of PMNs plus MNLs and seven times the activity of PMNs stimulated with fMLP in the absence of MNLs. Cell-free AmB-des (2 micrograms/ml)-stimulated, MNL-conditioned medium primed pure PMNs to a level equal to that of whole MNLs treated with AmB-des. AmB-LC was much less potent. AmB-LC (20 micrograms/ml) increased fMLP-stimulated chemiluminescence to two times that of PMNs plus MNLs without AmB-LC. AmB-des (2 micrograms/ml) (but not AmB-LC [2 micrograms/ml]) increased nitroblue tetrazolium reduction by PMNs in whole blood from 31 to 52% of positive cells. Neither form of AmB increased Mac-1 (the CD11b/CD18 integrin) expression of pure PMNs. AmB-des (0.5 to 2 micrograms/ml) (but not AmB-LC [< or = 40 micrograms/ml]) nearly doubled PMN Mac-1 expression in the presence of MNLs, and cell-free AmB-des (2 micrograms/ml)-stimulated, MNL-conditioned medium stimulated PMN Mac-1 to 125% of the control level. AmB-des (0.2 to 2 micrograms/ml) (but not AmB-LC [< or = 40 micrograms/ml]) decreased chemotaxis of pure PMNs to fMLP by as much as 35% and that of PMNs in the presence of MNLs by as much as 50%. Desoxycholate by itself had no effect on PMN function. These differences in activity between AmB-des and AmB-LC may explain the lessened toxicity observed with AmB-LC. Images

Rhinovirus infection results in stronger and more persistent genomic dysregulation: Evidence for altered innate immune response in asthmatics at baseline, early in infection, and during convalescence.

Background Rhinovirus (HRV) is associated with the large majority of virus-induced asthma exacerbations in children and young adults, but the mechanisms remain poorly defined. Methods Asthmatics and non-asthmatic controls were inoculated with HRV-A16, and nasal epithelial samples were obtained 7 days before, 36 hours after, and 7 days after viral inoculation. RNA was extracted and subjected to RNA-seq analysis. Results At baseline, 57 genes were differentially expressed between asthmatics and controls, and the asthmatics had decreased expression of viral replication inhibitors and increased expression of genes involved in inflammation. At 36 hours (before the emergence of peak symptoms), 1329 genes were significantly altered from baseline in the asthmatics compared to 62 genes in the controls. At this time point, asthmatics lacked an increase in IL-10 signaling observed in the controls. At 7 days following HRV inoculation, 222 genes were significantly dysregulated in the asthmatics, whereas only 4 genes were dysregulated among controls. At this time point, the controls but not asthmatics demonstrated upregulation of SPINK5. Conclusions As judged by the magnitude and persistence of dysregulated genes, asthmatics have a substantially different host response to HRV-A16 infection compared with non-asthmatic controls. Gene expression differences illuminate biologically plausible mechanisms that contribute to a better understanding of the pathogenesis of HRV-induced asthma exacerbations.