Matrix-driven translocation of cells and nonliving particles.

Cells of metazoan organisms produce and react to complex macromolecular microenvironments known as extracellular matrices. Assembly in vitro of native, compositionally nonuniform collagen-fibronectin matrices caused translocation of certain types of cells or polystyrene-latex beads from regions lacking fibronectin into regions containing it. The translocation process was not due to diffusion, convection, or electrostatic distribution effects, but may depend on nonequilibrium phenomena at the interface of contiguous collagen matrices formed in the presence and absence of fibronectin or particles. Extracellular matrix formation alone was sufficient to drive translocation by a biophysical process that may play a role in cellular migration during embryogenesis, as well as in other types of tissue reorganization such as inflammation, wound healing, and tumor invasion.

A radical demise. Toxins and trauma share common pathways in hair cell death.

The pathologic similarities noted after ototoxic and/or traumatic injury to the cochlea as well as the key features of the cochlea that make it susceptible to reactive oxygen species (ROS) damage are reviewed. Recent evidence linking ROS to cochlear damage associated with both ototoxins and/or trauma are presented. Mechanisms of generation of ROS in the cochlea and how these metabolites damage the cochlea and impair function are also reviewed. Finally, examples of novel therapeutic strategies to prevent and reverse hearing loss due to noise and/or ototoxins are presented to illustrate the clinical relevance of these new findings.

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.

L- and D- methionine provide equivalent long term protection against CDDP-induced ototoxicity in vivo, with partial in vitro and in vivo retention of antineoplastic activity.

Treatment of metastatic tumors with ionic platinum compounds is hampered by the potent nephrotoxic, ototoxic and neurotoxic properties of these drugs. Recent studies have shown that sulfur-containing antioxidants relieve the dose limiting side effects of cis-diamminedichloroplatinum (CDDP), the most commonly used ionic platinum therapy. Here we report that both isomers of the sulfur-containing antioxidant methionine (MET) completely block the in vivo ototoxic and nephrotoxic effects of CDDP, and the duration of MET otoprotection is longer than has been previously reported. Rats treated with either L- or D-MET in addition to CDDP exhibited no signs of auditory system damage after 7 days, as evaluated by the auditory brainstem response and scanning electron microscopic examination of the organ of Corti, while CDDP-treated rats exhibited pronounced evidence of ototoxic damage after only 3 days. Microscopic examination of kidney tissue revealed moderate to severe nephrotoxic damage to CDDP-treated rats after 5 days, while rats co-treated with either MET isomer showed no evidence of kidney damage. Mortality among CDDP-treated subjects increased steadily over the period of the study, while all of the MET-protected rats survived. Finally, the efficacy of CDDP in the presence of L- or D-MET was evaluated in vitro using cultures of MTLN-3 breast tumor cell lines, and in vivo using implanted MTLN-3 tumors. Both L- and D-MET reduced the ability of CDDP to kill tumor cells in vitro and in vivo, however, our data suggest that a higher proportion of the antineoplastic activity of CDDP is retained in the presence of L- MET.

Fine structure of extracellular matrix and basal laminae in two types of abnormal collagen production: L-proline analog-treated otocyst cultures and disproportionate micromelia (Dmm/Dmm) mutants.

L-Azetidine-2-carboxylic acid (LACA), a naturally occurring vegetable imino acid, can be incorporated into mammalian proteins in place of proline, thereby eliciting an inhibitory effect on collagen secretion. Exposure of explants of the embryonic mouse inner ear to LACA reduces the number of collagen fibrils in the otic capsule, gives rise to a dose-dependent derangement of the basal lamina, and ultimately results in dysmorphogenesis and retarded differentiation of the inner ear. Disproportionate micromelia (Dmm) is an incomplete dominant form of dwarfism characterized by a reduced quantity of type II collagen in the cartilaginous extracellular matrix (ECM). Abnormal morphogenesis in homozygotic Dmm mice resembles the abnormal morphogenesis observed in LACA-exposed otic explants, resulting in malformed inner ears with a bulky cartilaginous capsule and a lack or reduction of defined perilymphatic spaces (Van De Water and Galinovic-Schwartz, 1987). In this study, we examined by ultrastructural analysis LACA-exposed otic explants and inner ears of Dmm/Dmm mouse embryos for abnormalities in the collagenous constituents of the basal laminae and capsular ECM. We demonstrate, in comparison to normal embryonic mouse inner ears, a reduction in collagen fibrils and irregular cytodifferentiation of chondrocytes in the ECM of LACA-exposed and Dmm/Dmm inner ears as well as in the basal laminae of LACA-exposed specimens. In addition, we provide evidence of dysmorphogenesis of the otic capsule and perilymphatic spaces in LACA-exposed explants. Moreover, while previous studies demonstrated the anomalous development of sensory structures in otocyst explants following LACA exposure, in this study we provide evidence of the normal morphogenesis of otic epithelial-derived sensory structures in homozygotic Dmm/Dmm mouse embryos.

Transforming growth factor beta: does it direct otic capsule formation?

Interactions between the epithelium of the otocyst and surrounding periotic mesenchyme direct the formation of the capsule of the mammalian inner ear. In the present study, we have characterized the temporal-spatial distribution of transforming growth factor beta 1 (TGF-beta 1) in the epithelial and mesenchymal tissues that compose the inner ear between 10 and 14 days of embryonic development. In addition, using high-density cultures of periotic mesenchyme to model otic capsule formation, we have demonstrated that exogenous TGF-beta 1 can modulate otic chondrogenesis by acting as either an enhancer or a suppressor of this process. Our immunohistochemical and in vitro results suggest a paracrine action for this growth factor in modulation of epithelial-mesenchymal tissue interactions and otic morphogenesis.

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.

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.

Mammalian auditory hair cell regeneration/repair and protection: a review and future directions.

Regeneration/repair and protection of auditory hair cells and auditory neurons is an exciting, rapidly evolving field. Simultaneous developments in the fields of otobiology and surgical otology have led to new and exciting possibilities in inner ear medicine and surgery; specifically, the treatment or prevention of a variety of types of hearing losses in the foreseeable future. Sensorineural hearing loss in humans is commonly associated with a loss of auditory hair cells. It has been generally accepted that hearing loss resulting from hair cell damage is irreversible because the human ear has been considered to be incapable of regenerating or repairing these sensory elements following severe injury. An organ of Corti explant study has shown that it is possible to initiate the regeneration/repair of mammalian hair cells. In this study, ototoxin-damaged organ of Corti explants from juvenile rats were treated with a combination of retinoic acid (10-8M) and fetal calf serum (10%). TGF-alpha has been identified as a growth factor capable of evoking auditory hair cell regeneration/repair in ototoxin-damaged organ of Corti explants. Preliminary in vitro experiments with juvenile rat organ of Corti explants and in vivo studies in the cochleae of adult guinea pigs indicate that pretreatment followed by continuous treatment of the inner ear with a combination of retinoic acid and TGF-alpha can protect the auditory hair cells from the ototoxic effects of aminoglycosides. Because the integrity of spiral ganglion neurons is also essential for normal auditory function, there is a parallel series of in vitro and in vivo studies of the effects of neurotrophic factors on the survival of auditory neurons and the regeneration of injured neuronal processes. Clinical studies have demonstrated that it is now possible to perform surgeries on the inner ear, i.e., partial or total labyrinthectomies, and maintain hearing. The field of cochlear implantation has also provided insights into both the short- and long-term effects of cochlear fenestration on inner ear function. Administration of growth factors to the inner ears of animals is now possible with the use of implanted catheters and miniature infusion pumps. These advances suggest that localized application of drugs to the human inner ear may be feasible. The aim of this paper has been to provide an overview of advances in the study of the biology of auditory hair cells and auditory neurons, as well as recent relevant surgical advances. Taken together, these advances in otobiology and surgery will, in the future, be combined to devise new and innovative treatments for inner ear disorders.

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.

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.

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.

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.

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: 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.