Thomas R. Pasic

Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C

A recently recognized human rhinovirus species C (HRV-C) is associated with up to half of HRV infections in young children. Here we propagated two HRV-C isolates ex vivo in organ culture of nasal epithelial cells, sequenced a new C15 isolate and developed the first, to our knowledge, reverse genetics system for HRV-C. Using contact points for the known HRV receptors, intercellular adhesion molecule-1 (ICAM-1) and low-density lipoprotein receptor (LDLR), inter- and intraspecies footprint analyses predicted a unique cell attachment site for HRV-Cs. Antibodies directed to binding sites for HRV-A and -B failed to inhibit HRV-C attachment, consistent with the alternative receptor footprint. HRV-A and HRV-B infected HeLa and WisL cells but HRV-C did not. However, HRV-C RNA synthesized in vitro and transfected into both cell types resulted in cytopathic effect and recovery of functional virus, indicating that the viral attachment mechanism is a primary distinguishing feature of HRV-C.

The efficacy of hypertonic saline nasal irrigation for chronic sinonasal symptoms.

OBJECTIVE: To assess quality of life (QOL) in patients with sinonasal symptoms in response to hypertonic saline nasal irrigation (HSNI), and to assess HSNI use patterns. STUDY DESIGN AND SETTING: The study was an uncontrolled 12-month follow-up to a randomized controlled trial (RCT) and used HSNI in a community setting. We included 54 participants with recurrent or chronic sinonasal symptoms. Forty participants had been in the intervention group of a previous study; 14 had been control participants. Primary outcome measures were the Rhinosinusitis Disability Index (RSDI), a sinus-symptom severity assessment (SIA), and the Sino-Nasal Outcomes Test (SNOT-20). Secondary outcome measures were frequency and pattern of HSNI use, side effects and satisfaction. RESULTS: Among participants using HSNI in the prior RCT, RSDI scores continued to improve, from 73.2 ± 2.6 points to 80.6 ± 2.4 points (P < 0.001). SIA and SNOT-20 scores remained stable. Former control participants reported QOL improvement similar to that of HSNI users in the prior RCT. RSDI scores improved from 62.0 ± 3.9 points to 79.7 ± 3.7 points (P < 0.05), SNOT-20 scores improved from 43.5 ± 5.7 points to 28.4 ± 4.8 points, and SIA scores improved from 4.2 ± 0.3 points to 2.6 ± 0.3 points (P < 0.01). Mean HSNI use for all participants was 2.4 irrigations per week; 33% of participants used HSNI regularly, 55% when symptomatic. Side effects were minor; satisfaction was high. CONCLUSIONS: Participants with chronic sinonasal symptoms reported improved QOL and frequent, satisfying use of HSNI. SIGNIFICANCE: HSNI is an effective adjunctive treatment of chronic sinonasal symptoms.

Report of Successful Prolonged Antifungal Therapy for Refractory Allergic Fungal Sinusitis

Allergic fungal sinusitis (AFS) is an increasingly recognized cause of refractory chronic sinusitis in the young immunocompetent host, analogous to allergic bronchopulmonary aspergillosis (ABPA), a related process in the lower respiratory tract. Most patients experience remittent disease despite corticosteroid therapy and aggressive sinus surgery. Because controlled trials have shown adjunctive antifungal therapy to be of benefit in treating ABPA, long-term oral itraconazole was used in a young man with remittent AFS, which was able to break the cycle of relapsing disease.

Cis‐platinum ototoxicity in children

Cis‐platinum is an ototoxic antineoplastic drug. Evaluation of auditory thresholds in 33 children receiving cis‐platinum shows that a threshold shift at 6 and 8 kHz is first measurable after a cumulative dose of 201 to 300 mg/m2. A 35‐ to 40‐dB high‐frequency threshold shift is evident after a cumulative cis‐platinum dose of 301 to 400 mg/m2. Increasing cumulative doses of cis‐platinum are associated with a greater degree of hearing loss. Receiver‐operator characteristic curves were used to find a criterion value that effectively identified threshold shifts that were due to cis‐platinum ototoxicity. A 15‐dB or greater shift in the 6‐ and 8‐kHz threshold average identifies a high true‐positive (50%) and low false‐positive (0%) rate of cis‐platinum‐induced hearing loss. Using this criterion, cis‐platinum ototoxicity affected 77% of children who received cis‐platinum (median cumulative dose 360 mg/m2).

Biological characteristics and propagation of human rhinovirus-C in differentiated sinus epithelial cells

Abstract Information about the basic biological properties of human rhinovirus-C (HRV-C) viruses is lacking due to difficulties with culturing these viruses. Our objective was to develop a cell culture system to grow HRV-C. Epithelial cells from human sinuses (HSEC) were differentiated at air–liquid interface (ALI). Differentiated cultures supported 1–2 logs growth of HRV-C15 as detected by quantitative RT-PCR. Two distinguishing features of HRVs are acid lability and optimal growth at 33–34 °C. We used this system to show that HRV-C15 is neutralized by low pH (4.5). In contrast to most HRV types, replication of HRV-C15 and HRV-C41 was similar at 34 and 37°C. The HSEC ALI provides a useful tool for quantitative studies of HRV-C replication. The ability of HRV-C to grow equally well at 34°C and 37°C may contribute to the propensity for HRV-C to cause lower airway illnesses in infants and children with asthma.

Development of Cat‐301 immunoreactivity in auditory brainstem nuclei of the gerbil

The developing brainstem auditory system has been studied in detail by using anatomical and physiological techniques. However, it is not known whether immature auditory neurons exhibit different molecular characteristics than those of physiologically mature neurons. To address this issue, we examined the distribution of Cat‐301 immunoreactivity in the developing auditory brainstem of gerbils. Cat‐301 is a monoclonal antibody that recognizes a 680‐kD chondroitin sulfate proteoglycan similar to aggrecan, a high‐molecular‐weight chondroitin sulfate proteoglycan found in cartilage. In the central nervous system, Cat‐301 immunoreactivity is localized to the extrasynaptic surface of neurons. It has been hypothesized by Hockfield and co‐workers (Hockfield et al. [1990a]Cold Spring Harbor Symp. Quart. Biol. 55:504–514) that the Cat‐301 proteoglycan is a molecular marker indicating that a neuron has acquired mature neuronal properties.

Hearing Loss in Townes-Brocks Syndrome:

Townes-Brocks syndrome is an autosomal dominant syndrome consisting of anomalies affecting the ear, hand, foot, anus, and kidney. Anomalies affecting the ear include lop ear, preauricular skin tags, ossicular abnormalities, and a mixed hearing loss. The hearing loss in Townes-Brocks syndrome is predominantly sensorineural, affects high-frequency thresholds more than low-frequency thresholds, and has a variable (usually small) conductive component. The sensorineural component of the hearing loss is slowly progressive. It is typically in the mild range (20 to 40 dB hearing level) during early childhood and progresses to the moderate hearing loss range (40 to 60 dB hearing level) by early adulthood. We present a description of the otologic manifestations and an analysis of audiologic findings in six members of a family With Townes-Brocks syndrome.

Influence of Computed Tomography on Pretherapeutic Tumor Staging in Head and Neck Cancer Patients

Refinements in radiographic techniques have resulted in increased use of radiographic studies in the evaluation of patients with head and neck cancer over the past 20 years. To assess the impact of such studies, we compared tumor clinical stages based solely on physical-examination findings with those obtained with the addition of CT findings. This study was accomplished through case review of 81 head and neck cancer patients who underwent CT after preliminary TNM-stage assignment as determined on the basis of physical examination alone. In this cohort, 44 patients (54%) had a change in assigned clinical stage. We reviewed individual anatomic sites to determine where CT was found to be most useful in modifying tumor stage. Changes in tumor and nodal stage were found across all major sites of the head and neck. Tumors of the hypopharynx were the most likely to change stage (90%) on the basis of CT findings, whereas tumors of the glottic larynx were least likely to undergo a change in stage (16%). The therapeutic implications of these findings are discussed in the context of the published literature.

Rhinovirus C targets ciliated airway epithelial cells

BackgroundThe Rhinovirus C (RV-C), first identified in 2006, produce high symptom burdens in children and asthmatics, however, their primary target host cell in the airways remains unknown. Our primary hypotheses were that RV-C target ciliated airway epithelial cells (AECs), and that cell specificity is determined by restricted and high expression of the only known RV-C cell-entry factor, cadherin related family member 3 (CDHR3).MethodsRV-C15 (C15) infection in differentiated human bronchial epithelial cell (HBEC) cultures was assessed using immunofluorescent and time-lapse epifluorescent imaging. Morphology of C15-infected differentiated AECs was assessed by immunohistochemistry.ResultsC15 produced a scattered pattern of infection, and infected cells were shed from the epithelium. The percentage of cells infected with C15 varied from 1.4 to 14.7% depending on cell culture conditions. Infected cells had increased staining for markers of ciliated cells (acetylated-alpha-tubulin [aat], p < 0.001) but not markers of goblet cells (wheat germ agglutinin or Muc5AC, p = ns). CDHR3 expression was increased on ciliated epithelial cells, but not other epithelial cells (p < 0.01). C15 infection caused a 27.4% reduction of ciliated cells expressing CDHR3 (p < 0.01). During differentiation of AECs, CDHR3 expression progressively increased and correlated with both RV-C binding and replication.ConclusionsThe RV-C only replicate in ciliated AECs in vitro, leading to infected cell shedding. CDHR3 expression positively correlates with RV-C binding and replication, and is largely confined to ciliated AECs. Our data imply that factors regulating differentiation and CDHR3 production may be important determinants of RV-C illness severity.

Chronic Rhinosinusitis with Nasal Polyps: A Proteomic Analysis

Objectives: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a severe subtype of chronic rhinosinusitis that can affect patients despite medical and surgical interventions. The purpose of this study was to utilize the techniques of proteomics to investigate differences in protein abundance within the sinonasal mucosa of patients with CRSwNP compared to healthy controls. Methods: In a case-control study at a tertiary-care academic medical center, sinonasal mucosa was harvested from 3 patients with CRSwNP and 3 control patients undergoing transsphenoidal excision of pituitary tumors. Two-dimensional gel electrophoresis was used to identify proteins with elevated or reduced abundance in CRSwNP patients compared to controls. The proteins showing the greatest abundance differences were characterized by mass spectrometry. Results: More than 300 differentially abundant proteins (p ≤ 0.05) were identified. Many of these protein species were involved in the host inflammatory response. Proteins up-regulated in CRSwNP patients included eosinophil lysophospholipase by a ratio (R) of 18.13, RHO-GDP dissociation inhibitor 2 (R = 2.80), and apolipoprotein A-1 (R = 1.73). Down-regulated proteins in CRSwNP patients included catalase (R = −5.87), annexin A1 (R = −6.27), and keratin II-8 (R = −6.73). A detailed analysis of additional protein species is outlined. Conclusions: The proteomic approach allows detection of significant differences in protein abundance in CRSwNP and provides unique insight into the pathophysiology of this common disease.