Sonic hedgehog regulates otic capsule chondrogenesis and inner ear development in the mouse embryo.

Development of the cartilaginous capsule of the inner ear is dependent on interactions between otic epithelium and its surrounding periotic mesenchyme. During these tissue interactions, factors endogenous to the otic epithelium influence the differentiation of the underlying periotic mesenchyme to form a chondrified otic capsule. We report the localization of Sonic hedgehog (Shh) protein and expression of the Shh gene in the tissues of the developing mouse inner ear. We demonstrate in cultures of periotic mesenchyme that Shh alone cannot initiate otic capsule chondrogenesis. However, when Shh is added to cultured periotic mesenchyme either in combination with otic epithelium or otic epithelial-derived fibroblast growth factor (FGF2), a significant enhancement of chondrogenesis occurs. Addition of Shh antisense oligonucleotide (AS) to cultured periotic mesenchyme with added otic epithelium decreases levels of endogenous Shh and suppresses the chondrogenic response of the mesenchyme cells, while supplementation of Shh AS-treated cultures with Shh rescues cultures from chondrogenic inhibition. We demonstrate that inactivation of Shh by targeted mutation produces anomalies in the developing inner ear and its surrounding capsule. Our results support a role for Shh as a regulator of otic capsule formation and inner ear development during mammalian embryogenesis.

A rapid, reproducible method for coating Rotorod Sampler collector rods with silicone grease.

BACKGROUND: The Rotorod Sampler (Sampling Technologies, St. Louis Park, MN) is a rotating-arm impactor that recovers airborne particles on two rapidly moving plastic collector rods. For decades, the standard method for applying silicone grease to collector rods has been with one's finger. Although this method can yield excellent results when performed by practiced investigators, a relatively high skill level is required, and significant intrapreparer variability has been reported in the medical and technical literature. OBJECTIVE: The purpose of this investigation was to develop and evaluate a new method for coating Rotorod collector rods with silicone grease. METHODS: Collector rods were coated with silicone grease by dipping them into a solution consisting of silicone grease and hexane. Pollen recovery by these dipped collector rods was compared with pollen counts obtained with hand-greased collector rods. RESULTS: Twenty-three paired samples were obtained during five sampling periods. Pollen recovery by the hand-greased and dipped collector rods was similar (P = 0.410). Dipped collector rods generally offered a lower standard deviation than hand-greased collector rods, however, differences were not statistically significant (F = 1.782, P = 0.087). Dipped collector rods were also superior to hand-greased collector rods in several qualitative categories such as grease uniformity, time required for microscopic analysis, and visual quality. CONCLUSIONS: Dipped collector rods offered a time-efficient means to obtain atmospheric samples with excellent visual quality. The resulting pollen counts were similar to data obtained via the standard, manual method. Allergists are encouraged to consider using this new method in their office practices and for drug studies.

Growth factor expression in cartilage wound healing: temporal and spatial immunolocalization in a rabbit auricular cartilage wound model.

OBJECTIVE: The ability of cartilage to regenerate following injury is limited, potentially leading to osteoarthritis. Integrative cartilage repair, necessary for durable restoration of cartilage lesions, can be regarded as a wound healing process. Little is known about the effects of growth factors regulating acute cartilage wound healing in vivo. In this study the temporal expression patterns of growth factors and proteoglycan content in cartilage wound edges in vivo were studied. DESIGN: Cartilage wounds were created in rabbit ear cartilage using a 6 mm biopsy punch. Specimens were subsequently harvested 1, 3, 7, 14 and 28 days after surgery. Paraffin sections were thionin stained to visualize proteoglycan loss and replacement. Immunohistochemical staining of TGFbeta1, TGFbeta3, IGF-1, IGF-II and FGF-2 was used to define growth factor expression at the cartilage wound sites. RESULTS: Almost no effect of cartilage wounding was observed one day after surgery. A decrease of proteoglycan content, with a maximal loss at day 7, and a subsequent restoration was observed at the wound edges. Growth factor expression increased simultaneously. Maximal immunostaining for IGF1, IGFII, FGF2 and TGF-beta3 was observed at day 7, followed by a gradual decrease. Increased expression of TGFbeta1 lasted from day 3 until day 14. CONCLUSION: We have demonstrated the ability of chondrocytes to increase growth factor expression and to restore the rapid decrease in proteoglycan content in the initial phase following acute wounding. A temporal increase in intracellular growth factor expression suggests an autocrine and/or paracrine metabolic stimulation, which can be regarded a sign of chondrocytes repair capacity.

Round window membrane delivery of L-methionine provides protection from cisplatin ototoxicity without compromising chemotherapeutic efficacy.

Cisplatin (cis-diamminedichloroplatinum(II) (CDDP)) is a widely used, highly effective, oncolytic agent that has serious ototoxic side-effects. To test the effectiveness of local delivery, of L-methionine (L-Met) as an otoprotective agent against CDDP ototoxicity, we used a rat model of a highly metastatic breast cancer tumor, i.e. Fisher 344 rats implanted with MTLn3 breast cancer cells. Four experimental groups were evaluated--I: untreated; II: CDDP-treated (three dosages); III: systemically-delivered L-Met + CDDP-treated; IV: locally delivered L-Met + CDDP-treated. The integrity of the outer hair cells (OHCs) was determined using scanning electron microscopy (SEM); hearing was assessed by recording auditory brainstem responses (ABRs) at multiple frequencies. The chemotherapeutic effectiveness of CDDP was quantified by measuring changes in tumor mass and the presence of tumor metastasis. L-Met provided otoprotection of the OHCs against CDDP toxicity in the cochleae of rats following either systemic (III) or local (IV) administration. The ABRs were unchanged in each of the L-Met protection Groups (III and IV) and in the untreated animals of Group I. Treatment with CDDP only (II) induced significant hearing losses at both 16 and 18 kHz when compared to ABRs of untreated rats(I). CDDP was effective in controlling the MTLn3 initiated breast cancer tumors in the CDDP-treated (II) and the local L-Met protection, CDDP-treated (IV) Groups. In contrast, the tumors in the systemic L-Met protection, CDDP-treated Group (III) were not controlled by the CDDP treatment regime. This study demonstrates that local delivery of L-Met to the scala tympani of the cochlea via the round window membrane (IV) provides effective protection against CDDP ototoxicity without compromising its ability to control a highly metastatic form of cancer.

Growth factor treatment enhances vestibular hair cell renewal and results in improved vestibular function.

The vestibules of adult guinea pigs were lesioned with gentamicin and then treated with perilymphatic infusion of either of two growth factor mixtures (i.e., GF I or GF II). GF I contained transforming growth factor alpha (TGFalpha), insulin-like growth factor type one (IGF-1), and retinoic acid (RA), whereas GF II contained those three factors and brain-derived neurotrophic factor. Treatment with GF I significantly enhanced vestibular hair cell renewal in ototoxin-damaged utricles and the maturation of stereociliary bundle morphology. The addition of brain-derived neurotrophic factor to the GF II infusion mixture resulted in the return of type 1 vestibular hair cells in ototoxin-damaged cristae, and improved vestibular function. These results suggest that growth factor therapy may be an effective treatment for balance disorders that are the result of hair cell dysfunction and/or loss.

Interpreting atmospheric pollen counts for use in clinical allergy: allergic symptomology.

BACKGROUND: Allergists generally consider atmospheric pollen counts to be an estimate of the antigenic challenge confronting allergic individuals. The nature of this challenge depends on the particular pollen types found in the atmosphere and also the airborne concentration of these pollen types. Both clinical experience and clinical investigations support these assumptions; however, a coherent system for relating pollen counts and allergic symptomology does not exist. OBJECTIVE: This review article will systematically review the medical and technical literature concerning the clinical significance of atmospheric pollen counts. DATA SOURCES: This review article will consider three independent bodies of literature: 1) data contrasting human exposure patterns with rooftop pollen counts; 2) data concerning dose-response relationships between atmospheric pollen counts and allergic symptomology; and 3) data concerning methods for indexing atmospheric pollen counts based on a pollen type's in vivo allergenicity and terminal velocity. RESULTS: Three principal results emerged. First, rooftop pollen counts imperfectly approximate human exposure to atmospheric pollen. Differences in both the concentration and type of pollen encountered by humans can be expected to differ from samples obtained on rooftops. Second, allergic symptomology is positively correlated with atmospheric pollen counts. Investigations involving Betula (birch) pollen offer quantitative dose-response models. Complex, nonlinear relationships that seem to reflect both the priming effect and late-phase reactions exist. Last, atmospheric pollen counts can be indexed based on a contemporary application of Thommen's postulates. This system provides allergists with a means to estimate the clinical significance of various pollen types by combining data concerning in vivo allergenicity and terminal velocity. CONCLUSIONS: These conclusions should allow allergists to judge the clinical significance of atmospheric pollen counts with greater sophistication than was previously possible.

The effect of windspeed on pollen and spore counts collected with the Rotorod Sampler and Burkard spore trap.

BACKGROUND: A recent review article presented conflicting evidence concerning the relationship between the Rotorod Sampler's particle collection efficiency and windspeed. Conventional wisdom holds that its collection efficiency is not affected by windspeed; experimental data paint a mixed picture. OBJECTIVES: This brief addendum will present additional data concerning the relationship between the Rotorod's collection efficiency and windspeed. This information will be contrasted with similar data for the Burkard spore trap. DATA SOURCE: Data obtained from an obscure technical report published by Ogden et al. in 1969 will be assessed. RESULTS: The Rotorod's collection efficiency appeared to be greatest at moderate windspeeds; lower efficiencies occurred at both lower and higher windspeeds. Maximum changes in collection efficiency ranged from 29% to 39% over the six windspeeds employed. The Burkard exhibited the opposite relationship between collection efficiency and windspeed. Collection efficiency was lowest at moderate windspeeds and higher at both lower and higher windspeeds. Maximum changes in collection efficiency ranged from 38% to 142%. CONCLUSIONS: Four principal conclusions emerged. First, these newly rediscovered data demonstrate that the Rotorod's collection efficiency is affected by windspeed. These results appear to overturn the conventional wisdom which holds that this device is insensitive to wind. Second, the Rotorod is less affected by windspeed than the Burkard. The maximum change in collection efficiency uncovered for the former device was 39%; this represented the minimum maximum change for the latter device. Third, the Rotorod and Burkard are affected by windspeed in fundamentally different ways. The former device's collection efficiency is greatest at moderate windspeeds and lower at both lower and higher windspeeds. The opposite effect occurs with the latter device. Last, the previous two findings may partially explain some of the differences in particle recovery by the two devices noted in the parent publication. Figures and equations presented in this addendum indicate which circumstances are likely to produce these differences.

Interpreting atmospheric pollen counts for use in clinical allergy: spatial variability.

BACKGROUND: Atmospheric pollen counts are ubiquitous data, however, few guidelines exist for interpreting them. A fundamental problem involves appreciating spatial variability in atmospheric pollen concentrations, for example, differences in pollen conditions between neighborhoods within a metropolitan area. The significance of this problem is perhaps most manifest in clinical allergy where pollen counts from a single rooftop pollen sampler are often applied to an entire city. OBJECTIVES: This review will consider experimental data concerning spatial variability in atmospheric pollen concentrations. Several guidelines for interpreting pollen counts with respect to this spatial variability will be presented. DATA SOURCES: This review will consider three independent bodies of literature concerning spatial variability in atmospheric pollen concentrations: (1) controlled-release experiments, (2) the paleoecology literature, and (3) atmospheric surveys. RESULTS: Four principal results emerged. First, pollen concentrations in the atmosphere appear to be inherently heterogeneous. Differences in pollen recovery between adjacent points increase in a predictable manner as a function of distance. Second, spatial variability decreases as the distance between pollen sources and destination regions increases. Pollen recovery across a city is most similar when pollen is transported from distant source regions. Third, the size of the source area represented by a pollen sampler depends on the distance between the sampler and the nearest vegetation. Samplers located near vegetation reflect small source areas; source areas expand considerably when the distance to the nearest vegetation increases. Last, individual pollen types travel characteristic distances from their source. Heavy pollen types collected at a particular point reflect a smaller source areas than lighter types which represent much larger source areas. CONCLUSIONS: These conclusions allow allergists to interpret their atmospheric pollen counts with greater sophistication than was previously possible. Mathematical equations presented in this review permit rough calculations concerning source areas.

Genetic patterning of embryonic inner ear development.

The commitment of the otocyst to form balance (pars superior) and hearing (pars inferior) receptors is under the control of patterning genes. Intersecting patterns of gene expression has been proposed to explain the regionalization of inner ear sensory receptor development. Insights into the roles of patterning genes is being acquired from the analysis of inner ear development of null mutation mice. An example of the consequences of the loss of expression of a single patterning gene is the effect of a null mutation of paired-box gene 2 (Pax2) on formation of the cochlea. Pax2 transcripts are expressed in the pars inferior of the otocyst and null mutant inner ears show agenesis of the cochlea. This is an example of the profound effect that loss of a single patterning gene can have on inner ear development. However, more typically there is redundancy of gene action during inner ear development as exemplified by the overlapping pattern of expression of two closely related homeobox-containing genes (i.e. Hmx2 and Hmx3) in the pars superior of the otocyst. Hmx2 and Hmx3 genes are both expressed early in otic development with Hmx3 transcripts present in the placode. However, null mutation of the Hmx3 patterning gene produced only a limited vestibular defect, and did not result in the agenesis of the vestibule. This result suggests that there is redundancy in genes that have similar patterns of regional expression in the otic anlagen.

Treatment with all-trans-retinoic acid decreases levels of endogenous TGF-beta(1) in the mesenchyme of the developing mouse inner ear.

BACKGROUND: Previous studies have shown that in utero exposure of the mouse embryo to high doses of all-trans-retinoic acid (atRA) produces defects of the developing inner ear and its surrounding cartilaginous capsule, while exposure of cultured periotic mesenchyme plus otic epithelium to high doses of exogenous atRA results in an inhibition of otic capsule chondrogenesis. METHODS: In this study, we examine the effects of atRA exposure on the endogenous expression of transforming growth factor-beta(1) (TGF-beta(1)), a signaling molecule that mediates the epithelial-mesenchymal interactions that guide the development of the capsule of the inner ear. RESULTS: Our results demonstrate a marked reduction in immunostaining for TGF-beta(1) in the periotic mesenchyme of atRA-exposed embryos of age E10.5 and E12 days in comparison with control specimens. Consistent with these in vivo findings, high-density cultures of E10.5 periotic mesenchyme plus otic epithelium, treated with doses of atRA that suppress chondrogenesis, showed significantly decreased levels of TGF-beta(1), as compared with TGF-beta(1) levels in untreated control cultures. Furthermore, we demonstrate a rescue of cultured periotic mesenchyme plus otic epithelium from atRA-induced chondrogenic suppression by supplementation of cultures with excess TGF-beta(1). CONCLUSIONS: Our results support the hypothesis that TGF-beta(1) plays a role in mechanisms of atRA teratogenicity during inner ear development.

Foundational readings concerning pollen for allergists and aerobiologists.

BACKGROUND: In a medical culture being revolutionized by MEDLINE, MD-Consult, and full-text electronic journals, books are frequently dismissed as antiquated and hopelessly out of date. For those involved in the study of atmospheric pollen, however, books--many several decades old--still represent foundational reading. OBJECTIVES: This paper will present and briefly review 17 books dealing with the ecology of plants that cause pollinosis, pollen morphology, pollen movement in the atmosphere, and air sampling techniques. CONCLUSIONS: This small collection of books provides a foundation with which to approach the study of atmospheric pollen. Allergists and aerobiologists are encouraged to become familiar with these titles, particularly if they intend to publish pollen data in medical journals.

Comparing pollen and spore counts collected with the Rotorod Sampler and Burkard spore trap.

BACKGROUND: The Rotorod Sampler and Burkard spore trap are air-sampling instruments commonly used by allergists in the United States. Although both devices are volumetric, their principles of operation and particle recoveries differ. OBJECTIVE: This review will develop some guidelines for interpreting and comparing pollen counts obtained with these instruments. DATA SOURCES: Investigations examining particle recovery by each device will be reviewed. Five studies where the Rotorod and Burkard were operated in parallel will also be assessed. RESULTS: The Rotorod's theoretical and empirical collection efficiencies are low for particles <10 microm but typically exceed 80% for particles above this threshold. This instrument has traditionally been considered insensitive to wind; experimental data present a mixed picture. The Burkard offers high collection efficiencies, particularly for small particles, when an aerosol's velocity is low. Bi-directional errors in collection efficiency occur as a function of increasing wind speed and particle size. Parallel trapping investigations demonstrated that the Burkard yielded a higher estimate of the atmospheric particle concentration for all particle sizes. Differences were widest for small fungus spores but narrowed for pollen-sized particles. Some recovery differences are readily explained by sampling theory. Other disparities may reflect over-sampling, under-sampling or each device's principles of operation. CONCLUSIONS: Both instruments appear to record the same relative changes in airborne particle concentrations. The Burkard appears to be a superior instrument for sampling particles <10 microm. The Rotorod appears to be equal or superior to the Burkard for collecting particles >10 microm. A rough empirical means for comparing differences in particle recovery is presented.

Pollen recovery in atmospheric samples collected with the Rotorod Sampler over multiple-day periods such as weekends.

BACKGROUND: Conventional wisdom holds that multiple-day samples should not be obtained with the Rotorod Sampler because sampling efficiency declines over long sampling periods such as weekends. OBJECTIVE: The purpose of this present investigation was to test this position under experimental conditions. METHODS: Atmospheric pollen samples were collected over 24-hour, 48-hour, and 72-hour periods by five Rotorod Samplers operated in a compact sampling array. Pollen counts obtained from the latter two sampling periods were compared with corresponding values computed from 24-hour samples. Differences in pollen recovery were assessed using Pearson's correlation coefficient and paired comparisons t-tests. RESULTS: Two hundred seventeen atmospheric samples were obtained between March 30 and October 8, 1998. Differences between the 48-hour and 72-hour samples and their computed counterparts were not statistically significant when the atmospheric pollen concentration was less than 100 pollen grains per cubic meter of air (p/m3). At higher pollen concentrations large quantitative and statistically significant differences were noted. CONCLUSIONS: These data suggest that particle loading on Rotorod collector rods caused sample quality to deteriorate when atmospheric pollen concentrations exceed 100 p/m3. Multiple-day sampling periods appear to be justified when the atmospheric pollen concentration is less than this threshold. Adjusting the Rotorod Sampler's duty cycle may offer a means to collect samples over periods longer than 24 hours during times of the year when pollen shed is heavy.

A summary of the atmospheric surveys published in the United States allergy literature, 1966-1996.

OBJECTIVE: In this investigation we sought to summarize the atmospheric surveys that were published in two United States allergy journals (and subsequent series) during a 31-year period beginning in 1966. DATA SOURCES: All original articles published in the Annals of Allergy (and its subsequent series) and the Journal of Allergy (and its subsequent series) were cataloged in a computer database beginning with the first issues of 1966. Publications were classified in a manner reflecting their aerobiologic content. STUDY SELECTION: From this database of articles (n = 7,403), atmospheric surveys for pollen, spores, and other aeroflora were identified and summarized according to standard criteria. For each study we documented the sampling instruments used, the height of these instruments off of the ground, the study period, and the sampling schedule. RESULTS: Sixty-one atmospheric surveys were summarized: 30 from locations in the United States and 31 from outside of the country. Various volumetric and nonvolumetric samplers were used; the height of instruments above the ground and the sampling protocols varied widely. CONCLUSIONS: This investigation can serve as a companion to earlier compilations. These data should prove more useful than information available via electronic and paper indexes.

How to write about pollen.

BACKGROUND: Clinicians and palynologists (pollen specialists) often have different ways of expressing the same thing when they write about pollen. OBJECTIVE: This brief paper considers the confusing use of the word pollens that is frequently encountered in medical literature. METHODS: Three common uses for the word pollens are considered citing examples from clinical literature. The problems associated with each case are explored. RESULTS: Words and phrases that can serve as suitable alternatives to the word pollens are presented. The advantages of these alternatives are discussed. CONCLUSION: The word pollens represents a vague usage that obscures the author's intended meaning. Consistent application of the suggested alternatives will enhance the transmission of key concepts in the medical literature.

Volumetric ragweed pollen data for eight cities in the continental United States.

BACKGROUND: A previous report presented volumetric ragweed pollen data for 23 cities in the continental United States. Although helpful, it did not represent all areas of the country. OBJECTIVE: The purpose of this present communication is to furnish ragweed pollen data for eight additional cities. METHODS: Ragweed pollen data were obtained from eight pollen sampling facilities in the continental United States. These data were analyzed to determine the length of the season in each city and its peak date. RESULTS: The ragweed pollen season was typically 2 or 3 months in duration. Ragweed pollen counts reached their annual maximum in late August or early September in cities in Minnesota, Nebraska, and South Dakota. The peak was achieved in late September or early October for two cities in Texas. The annual maximums for three remaining cities in Colorado, Utah and Alabama occurred in late August, early September and mid-September, respectively. CONCLUSIONS: The findings of this investigation were consistent with previous reports about the north-south nature of the ragweed pollen season. These data, when combined with the results of the 23-city investigation, indicate when source avoidance is most critical in many areas.

Basilar papilla explants: a model to study hair cell regeneration-repair and protection.

Explants of basilar papillae from 6-7 days posthatch chicks were cultured in growth medium for a period of 1-8 days. Hair cells were counted following staining of stereocilia bundles with FITC-phalloidin, and the percentage of hair cell survival was determined by comparison to control (i.e. uncultured) specimens. Hair cell integrity was evaluated by scanning electron microscopy. Although previous studies have utilized organotypic culture of the basilar papilla to assess cell proliferation and ototoxicity, viability and integrity of hair cells was documented for periods of up to only 2 3 days. Our results demonstrate substantive auditory hair cell viability for a period of 7 days in vitro. We describe a pattern of natural hair cell loss in organotypic culture that progresses along a proximal-distal, abneural-neural gradient, mimicking the pattern of hair cell loss that occurs following ototoxic insult to the chick basilar papilla in vivo and the pattern we observed during a 48-h period of exposure of basilar papilla explants to an ototoxic dose of neomycin. Our results provide an important quantitative step for the use of organotypic culture of the chick basilar papilla as a purposeful model to investigate the process of hair cell regeneration-repair in the avian auditory system.

A survey of television meteorologists about their sources for and understanding of pollen counts.

BACKGROUND: Pollen counts are widely used by television stations in news programming but little is known about the sources for and quality of this information. OBJECTIVE: The purpose of this investigation was to survey television meteorologists about their sources for pollen counts and to assess their general understanding of these data. METHODS: Sixty-seven meteorologists employed by television stations in the nation's 20 largest Designated Market Areas (DMAs) were contacted by telephone. A brief survey was administered verbally and their responses were recorded. RESULTS: Thirty-seven meteorologists indicated that their station used pollen counts in news programming. Most were aware of their station's source for this information and reported that viewers contacted them about the pollen count. Few meteorologists, however, knew the type of instrument that was used to obtain pollen samples they aired and only 10 selected the correct definition of a pollen count from a list of three choices. CONCLUSIONS: Pollen counts are an important element in television news programming that are poorly understood by the meteorologists who present them to the public. Allergists are encouraged to better educate the personnel at stations to which they supply pollen counts.