Neisseria gonorrhoeae coordinately uses Pili and Opa to activate HEC-1-B cell microvilli, which causes engulfment of the gonococci.

This study was undertaken to examine concomitant roles of pili and colony opacity-associated proteins (Opa) in promoting Neisseria gonorrhoeae adherence to and invasion of human endometrial HEC-1-B cells. Adherence of N. gonorrhoeae to cultured HEC-1-B cells was saturable, even though organisms adhered to <50% of the cells. During 4 to 6 h of incubation, adherent mono- and diplococci formed microcolonies on the surfaces of the cells. Microvilli of the HEC-1-B cells adhered by their distal ends to individual cocci within the microcolonies. When the microcolonies grew from isogenic pilus-negative (P-) Opa-, P- Opa+, or P+ Opa- gonococci, microvilli did not elongate, and the colonies were not engulfed. In contrast, the microvilli markedly elongated during exposure to P+ Opa+ gonococci. The microvilli adhered to the organisms along their full lengths and appeared to actively participate in the engulfment of the microcolonies. Internalized microcolonies, with P+ Opa+ gonococci, contained dividing cocci and appeared to be surrounded by cell membrane but were not clearly within vacuoles. In contrast, degenerate individual organisms were within vacuoles. Low doses of chloramphenicol, which inhibits protein synthesis by both prokaryotes and eukaryotes, prevented the microvillar response to and internalization of the P+ Opa+ gonococci; higher doses caused internalization without microvillus activation. Cycloheximide and anisomycin, which inhibit only eukaryotic protein synthesis, caused dose-dependent enhancement of uptake. Cytochalasins reduced engulfment; colchicine had no effect. These results show that gonococci must express both pili and Opa to be engulfed efficiently by HEC-1-B cells.

Apoptosis-associated proteins and the development of oral squamous cell carcinoma.

Expression of apoptosis-associated proteins was evaluated in premalignant and malignant oral epithelial lesions, to test the hypothesis that protein regulation of apoptosis may be altered in the development of oral squamous cell carcinoma. Ninety archived paraffin-embedded specimens from 25 patients (two or more sequential biopsies each) and eight control specimens were evaluated in immunohistochemically stained sections for tumor suppressor protein p53, p53 binding protein mdm-2, and apoptosis regulatory proteins Bcl-2, Bcl-X, Bax, and Bak. The initial histologic diagnosis for 17/25 patients was either focal keratosis, mild dysplasia, or moderate dysplasia; the initial diagnosis for the remaining eight patients ranged from severe dysplasia to moderately differentiated squamous cell carcinoma. Thirty of 90 specimens showed positive p53 expression, nine of which were dysplasias. In patients with one or more lesions displaying p53 expression, there was increased intensity of staining with disease progression. Bak was expressed in 57/90 specimens, including 27 dysplasias of various grades. There was also a significantly increased intensity of Bak staining with disease progression, which did not appear to be dependent upon p53 status. Bcl-X was expressed in 73/90 specimens, with staining displayed earlier in premalignant lesions than either p53 or Bak. Ten of 90 specimens were positive for Bcl-2 (all were dysplasias or carcinomas), and only 2/90 specimens were positive for Bax. Eleven of 90 specimens were positive for mdm-2; six of which were also positive for p53. These data show that apoptosis-associated proteins are altered in variable patterns in both premalignant and malignant oral epithelial lesions. p53 and especially Bak and Bcl-X are expressed early; Bax is largely absent; and Bcl-2 and mdm-2 show sporadic expression in the development of oral premalignant and malignant disease.

Cell cycle proteins and the development of oral squamous cell carcinoma.

Expression of cell cycle regulatory proteins was evaluated in premalignant and malignant oral epithelial lesions, to test the hypothesis that protein regulation of the cell cycle may be altered in the development of oral squamous cell carcinoma. Archived paraffin-embedded specimens (n = 90) from 25 patients with recurrent or persistent lesions were evaluated in immunohistochemically stained sections for cell cycle regulatory proteins p53, Rb, Cyclin D1, p27, and p21. The cell cycle was also evaluated by expression of nuclear protein Ki 67. Sections were graded semiquantitatively using a 0-3 + scale to indicate the percentage of positively stained cells. The initial histologic diagnosis for 17/25 patients was either focal keratosis, mild dysplasia, or moderate dysplasia; the initial diagnosis for the remaining eight patients ranged from severe dysplasia to moderately differentiated squamous cell carcinoma. Thirty-three of 90 specimens showed positive p53 expression, 11 of which were dysplasias. Eighty-nine of 90 specimens, from all stages of disease, showed positive Rb expression. Twenty-three of 90 specimens showed positive Cyclin D1 expression, typically in the later stages (carcinoma) of a patient's disease. Eighty-four of 90 specimens showed positive p21 expression; while 55 of 90 specimens were positive for p27. In control mucosa, p27 was highly expressed, while Rb and p21 proteins were expressed at relatively low levels; p53 and Cyclin D1 proteins were largely absent. Generally, staining of p53, Rb, p21, and Ki 67 increased with time in serial biopsies, while p27 showed decreased staining with disease progression. These data show that cell cycle regulatory proteins are altered in both premalignant and malignant disease, and that protein phenotypes are heterogeneous. P53 expression is seen early, and Cyclin D1 expression is seen late in the development of oral premalignant and malignant disease. Expression of p53, Rb, p21 and Ki67 increased, while p27 decreased, with disease progression.

p53, p21, Rb, and MDM2 proteins in tongue carcinoma from patients < 35 versus > 75 years.

Relatively rare squamous cell carcinomas of the tongue in young patients may be associated with different etiologic factors and pathogenetic mechanisms than carcinomas from the same site in older patients. Alterations in cell cycle proteins likely contribute to the biologic behavior of these neoplasms. The purpose of this investigation was to evaluate cell cycle proteins (p53, p21, Rb, MDM2) in lateral tongue cancers from patients at the two ends of the age spectrum. All available archived lateral tongue carcinomas from patients < 35 years (n = 36, 23 males and 13 females) were sectioned, immunohistochemically stained, and evaluated. Protein expression was scored as percent positive nuclei. An equal number of sequentially accessioned lateral tongue specimens from patients > 75 years (23 males and 13 females) were stained and compared. Positive p53 staining was seen in 18/36 of the < 35-year group versus 24/36 of the > 75-year group (p = 0.149). Increased p21 staining (both percent of positive cells and intensity) was evident in 25/32 of the < 35-year group versus 24/32 of the > 75-year group (p = 1.0). Increased p21 expression was seen in both p53-positive and -negative cases in both age groups. Rb protein was increased in 16/29 of the < 35-year group versus 17/26 of the > 75-year group (p = 0.58). Fourteen cases (4/35 vs 10/36, p = 0.135) showed positive MDM2 staining; MDM2-positive cases were also p53 positive in 4/4 younger and 8/10 older patients. We conclude that p53, p21, Rb, and MDM2 are over-expressed in lateral tongue cancers, and that immunohistochemical profiles are heterogeneous. A p53-independent pathway of p21 induction is supported by the results; p53 suppression may be associated with MDM2 protein expression in a subset of cancers. Significant differences in the expression of p53, p21, Rb, and MDM2 proteins are not evident in lateral tongue carcinomas from patients < 35 years as compared to patients > 75 years.

Benign neural tumors of the oral cavity: a comparative immunohistochemical study.

To determine if immunohistochemistry can be used as adjunct to the diagnosis and classification of oral benign neural tumors, we stained 77 neurally differentiated tumors with a panel of neural-associated antibodies (S-100 protein, CD57, epithelial membrane antigen, factor XIIIa, CD34, CD68, collagen IV). Using standard histologic criteria, we identified 13 schwannomas, 16 neurofibromas, 23 traumatic neuromas, 16 palisaded and encapsulated neuromas, and 9 granular cell tumors from archived oral pathology specimens. Silver stains showed that neurofibromas, traumatic neuromas, and palisaded and encapsulated neuromas consistently contained axon filaments. Although all neural tumors contained S-100-positive cells, schwannomas and palisaded and encapsulated neuromas contained the most. All tumors expressed CD57; traumatic neuromas were stained intensely and the others stained weakly. The consistent epithelial membrane antigen capsular staining of schwannomas and the absence of factor XIIIa-positive dendritic/spindle cells helped distinguish these tumors from others. Many CD34-positive cells were found in schwannomas, and few were found in palisaded and encapsulated neuromas. Variable numbers CD68-positive cells were seen in all neural tumor types; some of these cells appeared to be macrophages and mast cells, but many were thought to be Schwann cells expressing this antigen. Collagen IV staining, apparently representing basement membrane, was generally a feature of all benign neural tumors. The immunophenotype of the granular cells of the GCTs was S-100+, CD57+, and collagen IV+ supporting the putative neural origin of these tumors. We conclude that neural origin/differentiation of a connective tissue tumor can be confirmed with stains for S-100 protein, epithelial membrane antigen, CD57, and collagen IV. Staining patterns and intensities associated with the panel of antibodies tested can be useful in tumor classification.

Apoptosis-associated markers in oral lichen planus.

Hypothesizing that loss of basal cells in oral lichen planus is due to apoptosis, we evaluated LP specimens for apoptosis-regulating proteins [positive regulators Bcl-xS, Bax, Fas/Fas-ligand, p53, and negative regulators (anti-apoptotic) Bcl-2, Bcl-xL and compared results with reactions in normal mucosa and chronically inflamed gingiva. Also, sections were evaluated with an in situ TUNEL assay that identifies apoptotic DNA fragments. Basal keratinocytes in normal buccal mucosa, nonspecific gingivitis, and LP were negative for Bcl-2 protein, but melanocytes and lymphoid cells were positive. Keratinocyte staining for Bcl-x was negative to weak in normal buccal mucosa and gingivitis, and moderate in LP. Keratinocytes (especially upper prickle cells) in all tissues stained similarly for Bax at weak to moderate levels. Also, no differences in Fas and Fas-ligand staining were evident. Prominent p53-positive staining was seen in all LP biopsies (10-100% of basal keratinocytes) but not in normal buccal mucosa and gingivitis. Few basal keratinocytes in 5/10 LP cases exhibited a positive in situ signal for DNA fragment-associated apoptosis. That the Bcl-2 family of proteins and Fas/Fas-ligand were detected in normal and diseased tissues, and were occasionally expressed differently in oral LP, supports the notion that apoptosis is a potential mechanism of keratinocyte loss, especially in LP. The pattern of p53 staining in oral LP suggests over-expression of wild-type protein; a phenomenon that would arrest the cell cycle to allow repair of damaged DNA, or trigger apoptosis. While immunohistochemical evidence for apoptosis-associated basal keratinocyte death in LP was slight, it appeared that it may be p53 protein, and possibly Bcl-x associated.

Apoptosis in oral erythema multiforme.

OBJECTIVE: Cell death was evaluated in oral erythema multiforme to test the hypothesis that apoptosis may be a mechanism by which keratinocytes die in this condition. STUDY DESIGN: Ten erythema multiforme and five control oral mucosa biopsy specimens were evaluated in immunohistochemically stained sections for apoptosis-regulating proteins Bcl-2, Bcl-x, Bax, p53, Fas, and Fas-ligand. Apoptotic keratinocytes, determined by a detection method for DNA fragmentation (TUNEL) and by conventional morphologic criteria were counted per high power field. RESULTS: Keratinocyte staining for Bcl-2 protein was comparable in erythema multiforme and controls. Bcl-x expression was reduced in five erythema multiforme cases. Staining for Bax protein differed in six erythema multiforme cases and showed variable intensity in layers under the parakeratin. Only slight differences in staining patterns of Fas and Fas-ligand proteins were noted between erythema multiforme and controls. The number of apoptotic keratinocytes evaluated by morphologic examination was significantly higher in erythema multiforme (mean per high power field, 0.90 +/- 0.2; controls, 0.06 +/- 0.04; p < 0.05, Mann-Whitney test) and was limited in significance by the TUNEL method (erythema multiforme, 0.43 +/- 0.1; controls, 0.02 +/- 0.02). Overexpression of p53 protein was seen in basal keratinocytes in five erythema multiforme specimens (mean, 17.5 +/- 4.03 per high power field; controls 1.2 +/- 0.3). CONCLUSIONS: There is evidence that cell death in erythema multiforme is at least in part due to apoptosis. The apoptotic mechanism may be related to an altered expression of apoptosis-regulating proteins. Although measurable alterations in the phenotypic expression of Fas and Fas-ligand proteins were not apparent, activation of Fas/Fas-ligand system could still be involved in the induction of apoptosis in erythema multiforme.

Macrophages and vascular adhesion molecules in oral Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a heterogeneous tumor where spindle cells are predominant and macrophages and factor XIIIa positive dendrocytes are abundant. The origin of the macrophages and dendrocytes is unclear, although their numbers suggest a critical role in KS pathogenesis. To determine if KS macrophages are recruited from the blood stream or proliferate on-site, we examined biopsy specimens 1) for expression and distribution of vascular adhesion molecules (PECAM-1, ELAM-1, ICAM-1, VCAM-1, P-selectin, L-selectin) and the macrophage-associated adhesion-molecule ligand, VLA-4; 2) for dual expression of proliferation (Ki-67) and lineage-associated markers (KP-1, CD34, factor XIIIa, LCA); and 3) for dual expression of macrophage (KP-1) and endothelial cell (CD34) associated markers. Avidinbiotin peroxidase techniques were used. Resident vessels were found to strongly express PECAM-1, ELAM-1, ICAM-1, P-selectin, and moderately express VCAM-1 and VLA-4. Tumor spindle cells showed less intense expression of ELAM-1, ICAM-1 and P-selectin. The most frequent double-stain combination was Ki-67 + CD34+. In contrast, the combinations of Ki-67 + KP-1+, Ki-67 + XIIIa+ and Ki-67 + LCA+ were rarely seen. The enhanced expression of adhesion molecules on resident vessels and the lack of evidence of macrophage proliferation suggest that the abundant macrophages in KS are recruited from the blood stream.

Altered expression of epithelial integrins and extracellular matrix receptors in oral erythema multiforme.

Inflammation and ulceration at the epithelium-connective tissue interface, a characteristic of erythema multiforme (EM), may be associated with altered molecular attachment of basal keratinocytes. To determine the expression of basal keratinocyte-associated integrins and their basement membrane ligands in oral EM, specimens of clinically and microscopically confirmed EM (n = 12) and mucosal controls (n = 7) were stained immunohistochemically for the integrins alpha 3, beta 6, beta 1, and beta 4 and for extracellular matrix proteins laminin 1, laminin 5, collagen IV, and collagen VII using a standard avidin-biotin-peroxidase technique. In EM, results showed increased staining intensity for all integrins studied in basal and suprabasal keratinocytes. Basement membrane-associated staining of a6 and b4 was intense, but disrupted and fragmented. In EM, integrin staining was most marked at the summit of the connective tissue papillae. Laminin 5 staining was more intense than in controls, was frequently fragmented, and extended into the lamina propria. Laminin 1 staining was discontinuous and was frequently less intense than in controls. Collagen IV staining in EM was interrupted along the basement membrane. Collagen VII staining was fragmented but unchanged in intensity. These alterations in interface adhesion molecules suggest that hemidesmosome-associated molecules are important in the pathogenesis of EM. The staining intensities and patterns of expression of these adhesion molecules suggest that oral EM is initially focused in the connective tissue papillae.

Altered interface adhesion molecules in oral lichen planus.

OBJECTIVE: To evaluate expression of key epithelial-connective tissue interface adhesion molecules (basal keratinocyte integrins and extracellular matrix receptors) in oral lichen planus (LP). DESIGN: Integrins alpha 3, alpha 6, beta 1, beta 4 and basement membrane proteins laminin 1, laminin 5, collagen IV, and collagen VII were immunohistochemically identified in frozen biopsy specimens (14 oral LP and II matched controls) using a standard avidin-biotin-peroxidase technique. RESULTS: An increased staining intensity of all antigens in LP was shown, as compared to controls. Integrin expression by LP keratinocytes was generally more intense and appeared on more upper level cells. Staining for basement membrane-associated extracellular matrix proteins was also generally more intense, although fragmentation and gaps were typically seen. Reactions for alpha 6, beta 4, laminin 5, and collagen VII stains were particularly intense along the basement membrane. In LP, strands of laminin 5, collagen IV, and collagen VII appeared in the submucosa approximating or duplicating the basement membrane. CONCLUSIONS: The apparent increased expression of the interface-associated adhesion molecules may be reflective of a keratinocyte compensatory response (due to lymphocyte-mediated damage) that would functionally help resist epithelial separation (ulceration). Expression of alpha 3 beta 1 and alpha 6 beta 4 would also assist in epithelial migration associated with wound repair. We interpret the submucosal extensions and deposits of basement membrane proteins as representing remnants of basement membrane, indicating recent remodeling or atrophy of epithelial rete ridges.

Vascular adhesion molecules in oral lichen planus.

OBJECTIVE: Because recruitment and retention of lymphoid cells appear to be critical components of the pathogenesis of lichen planus, we have compared the expression and distribution of a panel of vascular adhesion molecules (ELAM-1, P-selectin, ICAM-1, VCAM-1, PECAM-1, CD34) and leukocyte adhesion molecule ligands (LFA-1, Mac-1, VLA4, L-selectin) in biopsies of this disease. STUDY-DESIGN: Frozen sections of 12 clinically and histologically confirmed cases of lichen planus and 9 normal control tissues were evaluated immunohistochemically with a standard 1-day avidin-biotin peroxidase technique. Staining intensity of vascular endothelium was evaluated semiquantitatively. Three microvascular zones or compartments were defined and evaluated separately. RESULTS: Generally, different staining patterns were observed in association with the various endothelium-associated adhesion molecules. In normal controls, PECAM was intensely expressed and VCAM-1 was weakly expressed. Intermediate staining was associated with ELAM-1, P-selectin, ICAM-1, and CD34. Staining within the three microvascular compartments frequently showed variations in intensity. In lichen planus, increased staining for ELAM-1, P-selectin, ICAM-1, and VCAM-1 was evident in one or more of the microvascular compartments. In the subepithelial vascular compartment where the infiltrate was the most dense, VCAM-1 appeared to show the greatest positive change. Almost all cells in the lichen planus infiltrates stained positive for ICAM-1, L-selectin, LFA-1, and VLA4, and large numbers of cells also exhibited VCAM-1, PECAM-1, and Mac-1 immunoreactivity. CONCLUSIONS: It appears that upregulation of ELAM-1, ICAM-1, and VCAM-1 (especially by endothelial cells in the subepithelial vascular plexus) could play a role in the pathogenesis of lichen planus. The expression of leukocyte receptors L-selectin, LFA-1, and VLA4 by most of the cells in the lichen planus infiltrate suggest that these molecules may be responsible for recruitment as well as retention in the active lichen planus lesion.

Heat shock (stress) proteins and gamma delta T lymphocytes in oral lichen planus.

OBJECTIVE: Heat shock proteins (Hsps), a highly conserved class of protective cellular proteins that are produced under various conditions of environmental challenge, have been implicated as the antigenic stimulus in autoimmune diseases. Because lichen planus (LP) appears to be an autoimmune or hyperimmune condition (mediated by T cells), Hsps may have a role in the pathogenesis of this disease. We believe that if keratinocyte Hsps are antigenic targets of a cellular immune response, upregulation of these proteins should be demonstrable in tissue sections. STUDY DESIGN: Immunohistochemistry was used to evaluate expression of several families of Hsps in oral lichen planus tissues. The number and distribution of gamma delta T cells, a subset of T lymphocytes with an immune surveillance function that may contribute to autoimmunity, were also evaluated. Monoclonal antibodies to Hsps 27, 60, 70, 90, gamma delta receptor, and CD3 (pan-T lymphocyte marker) were incubated with frozen sections of LP (n = 22) and normal oral mucosa (n = 17) followed by an avidin-biotin-peroxidase labeling method. Antibodies to bacterial Hsps (GroEL and DnaK) were used as negative controls, and antibody to constitutive eukaryotic Hsp (Hsc70) was used as a positive control. RESULTS: In six cases of LP, basal keratinocytes stained intensely for Hsp27, whereas controls showed only slight staining. Otherwise LP and normal tissues showed comparable positive staining of upper level keratinocytes with anti-Hsp27. Subjective increases in antibody staining were noted for Hsp60 in LP, which was due in part to staining of infiltrating lymphocytes and in part to keratinocyte expression. Normal tissues showed weak basal cell antibody staining for Hsp60. Hsp70 staining was observed at a less intense level in LP than in controls. Except for more intense basement membrane staining with anti-Hsp90 antibody in gingiva and palate, no differences in the occurrence of this protein were found. Absolute numbers of gamma delta T cells were increased in LP when compared with those in control specimens (n = 10 vs n = 1, respectively, per high-power field). However, gamma delta T cells represented less than 1% of the CD3+ lymphocytes. CONCLUSIONS: It was concluded that normal oral mucosa expresses Hsps 27, 60, 70, and 90 and contains few gamma delta T cells. Although the expression of Hsps was altered in LP, the differences demonstrated were slight and were therefore inconclusive. The Hsps expressed in LP could have contributed to the persistence or chronicity of the disease, or they could have simply reflected cellular injury.

In vitro model of Haemophilus ducreyi adherence to and entry into eukaryotic cells of genital origin.

Electron microscopy was used to examine Haemophilus ducreyi adherence to and entry into eukaryotic cells of genital origin. A clinical H. ducreyi isolate (90-244) adhered in snake-like whorls to the surfaces of cervical carcinoma cells (HeLa 229), endometrial adenocarcinoma cells (HEC-1-B), and human neonatal foreskin fibroblast (HFF) cells. A prototype strain of H. ducreyi (CIP542) adhered in randomly organized clumps on the surfaces of HFF. Strain 90-244 entered HFF and HEC-1-B cells but did not enter HeLa cells. The H. ducreyi in the HFF cells at 2 h were partly surrounded by a membrane consistent with that of a phagocytic vacuole. At 2 h, strain CIP542 was found in interstitial spaces between the HFF cells and also in the cytoplasm of the cells. After 7 and 24 h, both strains of H. ducreyi were found in the large interstitial spaces between the HFF cells, in the cytoplasm, and extracellularly. This model of in vitro H. ducreyi infection of eukaryotic cells will allow for more specific study of factors that determine the virulence of H. ducreyi.

Scanning electron microscopy of piliated Neisseria gonorrhoeae processed with hexamethyldisilazane.

Piliated Neisseria gonorrhoeae are virulent and attach readily to some human mucosal cells. The study of interactions between piliated Neisseria gonorrhoeae and surface structures of eukaryotic cells in tissue culture requires consistent high resolution imaging in scanning electron microscopy (SEM). The combination of the fixatives glutaraldehyde, osmium, tannic acid, and uranyl acetate improves preservation of pili and other delicate structures. Following the critical point drying (CPD) process, pili bundles remained intact, but charging produced image distortion in most of the specimens. The use of hexamethyldisilazane (HMDS) with air drying substantially reduced charging and image distortion. Less contrast and greater resolution of pili bundles and surface structures of bacteria or tissue culture cells were obtained at magnifications of 10,000 or higher. As an alternative to CPD, HMDS processing of cell culture monolayers was simple and was more efficient when a large number of samples was processed.

Opa (protein II) influences gonococcal organization in colonies, surface appearance, size and attachment to human fallopian tube tissues.

Opa-expressing variants of Neisseria gonorrhoeae strain F62-SF and an Opa- variant, all non-piliated, were examined for differences in the interaction of the bacteria within colonies and in attachment to and damage of human fallopian tube mucosa. Expression of certain Opas was associated with the formation of transparent colonies where the bacteria were tightly packed and evenly spaced within the colonies. Expression of other Opas was associated with the formation of opaque colonies where the gonococci were less tightly packed and were unevenly spaced. Distinct differences in the size of the gonococci and in their surface characteristics were dependent upon the Opa being expressed. Certain Opas were associated with gonococci that had significantly larger cross-sectional areas and bigger perimeters. Scanning electron microscopy showed that OpaC- and OpaD-containing variants yielded greater mucosal damage than OpaB-containing and Opa- variants with the least damage caused by the OpaA-containing variant (clumped bacteria from dark opaque friable colonies). The mucosal damage after 60 min incubation included shortening and decreased numbers of microvilli on non-ciliated cells and invagination and sloughing of ciliated cells. Differences in the interactions of gonococci within colonies and in attachment to fallopian tube mucosa and damage to the mucosal cells occurred with different Opa-expressing variants of N. gonorrhoeae strain F62-SF.

Motoneuron loss in the abducens nucleus of wobbler mice.

The 'wobbler' mutant mouse can be recognized at about 4 weeks of age by its tremor and atrophy of forelimb muscles. In addition to degeneration of spinal motoneurons, especially in cervical spinal cord, selected bulbar motoneurons have also been reported to degenerate in the mutant. We examined a cranial motor nucleus and found a 31% loss of abducens motoneurons in 4-5-week-old wobbler mice as compared to age-matched control mice.

Role of nonciliated cells in renewal of the bronchial epithelium of rats exposed to NO2.

The purpose of the present study was to identify the proliferative cell types in the nonciliated cell population of the upper airways and determine the capacity of each to act as progenitor cells. Sprague-Dawley rats (30 days old) were exposed to 20 ppm NO2 for 24 hours to stimulate cell division, given injected tritiated thymidine (3H-TdR), sacrificed 1 hour and 1, 3, 5, and 7 days later, and prepared for light- and electron-microscopic autoradiography. One hour after injection of 3H-TdR, the mean labeling index (LI) was 1.6% in control animals and 5.2% in exposed animals. Mean grain counts per cell decreased from 15.6 at 1 hour after 3H-TdR to 6.9 on the third day, indicating that the labeled cell population had divided. Labeled cells in the control and exposed cell populations were identified with electron microscopy. At 1 hour after injection of 3H-TdR, basal cells and nonciliated columnar cells were labeled. However, only nonciliated columnar cells were stimulated to divide by NO2. The labeled nonciliated columnar cell population was made up of serous, "intermediate" and goblet cells. Each of these cell types was stimulated to divide to the same degree. After cell division (1-7 days) labeled cells of all types were observed with labeled ciliated cells appearing on the third day. It was concluded that the basal cell is not a primary progenitor cell. The primary progenitor cell for epithelium in the upper airway is the total columnar secretory cell population (serous, "intermediate," and goblet cells).

Morphological basis of tolerance to ozone.

The purpose of this research was to study Type 1 epithelial cells in the ozone (O3)-tolerant lung epithelium. Rats were made tolerant by exposure to 0.5 ppm O3 for 2 days and allowed to recover in air. Reexposure to a lethal concentration of O3 (6 ppm) at 3, 7, and 15 days of recovery revealed that tolerance was present at 3 days but almost absent at 7 and 15 days of recovery. Using Type 2 cell proliferation as a means of quantitating Type 1 cell injury, it was observed that when the preexposed rats were reexposed to 0.5 ppm at 3, 7, and 15 days, very little Type 1 cell injury occurred at 3 days. However, at 7 and 15 days the amount of Type 1 cell injury was the same as that associated with the original exposure. To determine whether there was any change in the alveolar epithelial cell populations between the periods of tolerance (3 days) and its decline (7 and 15 days), the percentage of tritiated thymidine [( 3H]TdR-labeled Type 1 and 2 cells at these times were determined. There was a significant decrease in [3H]TdR-labeled Type 1 and 2 cells between the third and fifteenth days of recovery as excess cells were sloughed off and the tissue returned to normal. Using electron microscopic morphometry, Type 1 and 2 cells were then studied during the decline of tolerance. No change was found in the morphology of Type 2 cells; however, the morphology of Type 1 cells revealed a 58% decrease in surface area and a 25% increase in the arithmetic mean thickness when tolerance was present at 3 days. As tolerance declined (7 and 15 days), Type 1 cell morphology returned to normal. It was concluded that tolerance exists when the surface area of a cell exposed to a particular concentration of ozone is small enough so that the existing antioxidant mechanism contained within that cell volume can protect it from damage.

Quantitation of damage to the alveolar epithelium by means of type 2 cell proliferation.

The purpose of this study was to determine whether the amount of alveolar epithelial tissue damaged during exposure of NO2 could be quantified by measuring the proliferative response to Type 2 cells. To accomplishe this, we used tissues from previously published experiments in which rats had been exposed to NO2 and the proliferative response to Type 2 cells had been measured during a 5-day period. The proportion of alveolar epithelium damaged was determined by stereologic examination with electron microscopy of tissue sections from those rats exposed to NO2 for 24 hours. These values were then compared with the total proliferative response to Type 2 cells for the 5 days of exposure. The study demonstrated that increasing tissue damage is assocaited with a greater proliferative response to Type 2 cells. The high degree of correlation (r = 0.93) indicates that the proliferative response of Type 2 cells can be used as an indirect means to quantify acute damage to the alveolar epithelium.