Basal body replication and cilogenesis in a suctorian, Tokophrya infusionum.

Basal body replication and ciliogenesis in Tokophrya infusionum were studied in synchronized cultures. Basal body replication occurs during the 1st hr of reproduction, which in Tokophrya is by internal budding. The number of basal bodies increases from about 20 to over 300 within this period. New basal bodies develop in association with mature basal bodies; they are formed at right angles to the mature basal body as short "probasal" bodies, which elongate, slant upward, become parallel to the mature basal body, and elongate to the mature size. Ciliogenesis occurs only during reproduction; the nonreproducing adult is not ciliated, and has only 18-25 barren basal bodies. Cilia first appear as short bulges above the basal body. The axonemal structure is incomplete at first, with one or both central microtubules absent, and occasionally the B fibers of the outer doublets are missing. Several accessory fibers are associated with the basal bodies, both in the adult and during reproduction. One of the fibers appears only after the cilia have sprouted. The scheme of basal body replication and ciliogenesis in Tokophrya is compared to that reported in other organisms, and the role of the accessory fibers is discussed.

Immunolocalization of the retinoblastoma protein in the human eye and in retinoblastoma.

PURPOSE: To determine whether, by employing recent advances in immunocytochemical technique, it is possible to identify reliably the product of the retinoblastoma (RB) susceptibility gene, p110RB1, in formalin-fixed, paraffin-embedded eyes with commercially available primary antibodies. If so, the authors sought to determine the distribution of p110RB1 in normal human eyes and retinoblastomas in hopes of better understanding its function. METHODS: Four antibodies to p110RB1 were tested on normal human and monkey eyes, as well as on six human retinoblastomas. The human tissue was formalin-fixed and paraffin-embedded. Free antigen was used for an absorbed control. The monkey eye had been injected with tritiated (H3) thymidine 24 hours before enucleation. RESULTS: Three of the four antibodies had acceptable reactivity (a polyclonal against the carboxyl-terminal epitope and two monoclonals against epitopes near the amino-terminus). Staining was confined to nucleated cells of the normal eyes and was strongest in the cycling cells of the lenticular and corneal epithelia. Somewhat weaker reactivity was seen in those corneal epithelial cells in S phase as determined by autoradiography for H3-thymidine. Of the six retinoblastomas, three had strong nuclear and cytoplasmic staining and one showed weaker staining in the tumor cells than in the adjacent vascular endothelial cells. Two of the tumors had positive cytoplasmic and negative nuclear staining with an amino-terminal antibody but were completely negative for carboxyl-terminal p110RB1 reactivity. CONCLUSIONS: Using appropriate immunocytochemical techniques, p110RB1 can be identified in paraffin-embedded tissues with commercially available antibodies. The observed staining pattern in retinoblastoma suggests that RB1 transcripts are commonly produced in the tumor cells and that they are sometimes, but not always, capable of nuclear binding. Thus, nuclear binding by the RB1 gene product per se is not sufficient to prevent tumor growth, nor does it indicate the presence of a normal transcript.

Rods and cones contain antigenically distinctive S-antigens.

PURPOSE: S-antigen (48 kDa protein or arrestin) is known to be present in rod photoreceptors. Its localization in cones is less clear with several conflicting reports among various species examined. METHODS: This study employed three different anti-S-antigen antibodies (a48K, a polyclonal antiserum and two monoclonal antibodies, MAb A9-C6 and MAb 5c6.47) and examined their localization in rods and cones of human and cat retinas. To identify the respective cone types, an enzyme histochemical technique for carbonic anhydrase (CA) was employed to distinguish blue cones (CA-negative) from red or green cones (CA-positive). S-antigen localization was then examined by immunocytochemical staining of adjacent sections. RESULTS: In human retinas, a similar labeling pattern was seen with both a48K and MAb A9-C6, i.e., the rods and blue-sensitive cones were strongly positive, whereas the red- or green-sensitive cones showed little immunoreactivity. All human photoreceptors showed reactivity to MAb 5c6.47. In the cat retina, only CA-positive cones could be found. As in the human retina, both rods and cones of the cat were positive for MAb 5c6.47. A difference from the labeling pattern in human retina was noted for the other S-antigen antibodies; a48K labeled rods and all of the cones, whereas MAb A9-C6 reacted strongly with the rods but showed no cone staining. CONCLUSIONS: These results suggest that both rods and cones contain S-antigen but that they are antigenically distinctive.

Selective loss of blue cones and rods in human retinal detachment.

OBJECTIVE: To determine if there are histopathologic changes in the outer retina that could explain the blue-yellow color confusion previously described following rhegmatogenous retinal detachment in humans. METHODS: Ten eyes with traumatic retinal detachments were studied. Eight of the eyes were removed from 2 1/2 to 11 days following trauma. In the remaining two eyes, the retinas were successfully reattached. Enzyme histochemical studies for carbonic anhydrase and immunochemical studies for S antigen were performed to distinguish blue cones from red/green cones. RESULTS: With the 2 1/2- to 4-day-old detachments, nearly all of the carbonic anhydrase-negative (blue-sensitive) cones and many of the rods were seen to have signs of irreversible necrosis, including extreme swelling of the inner segments and mitochondria, loss of the outer segments, and pyknotic and displaced nuclei. In the 6- and 11-day-old detachments, almost all of the carbonic anhydrase-negative cones and many rods were missing. Blue cones were essentially absent from the reattached retinas, and there were only about half the normal number of rods. CONCLUSIONS: Rhegmatogenous retinal detachment results in rapid and almost total loss of the blue cones. Significant rod loss also occurs in this type of detachment but the red/green cones are comparatively resistant to damage. These findings could explain the observed blue-yellow color confusion in such patients. We discuss other clinical implications.

Retinoblastoma. Cell of origin.

OBJECTIVES: To apply modern techniques of molecular cell biology and to revisit the old question of the cell of origin for retinoblastoma in hopes of gaining a better understanding of the retinoblastoma gene's antioncogenic mechanisms. METHODS: Twenty-two consecutively accessed retinoblastomas were examined with immunocytochemical techniques for numerous retinal proteins. Both single and double labeling were used. Enzyme histochemistry for carbonic anhydrase was used as well. RESULTS: Differentiated areas of the tumors contained abundant Müllerlike cells. Fleurettes stained mostly for red and green cone-specific antibodies while features of blue cones and rods predominated in areas with high cytoplasmic-to-nuclear ratios but no fleurettes. All of the differentiated neoplastic cells were either photoreceptors or Müller's cells. No other retinal cell types were found. CONCLUSIONS: The cells of retinoblastoma are capable only of bipotential differentiation, ie, Müller's cells and photoreceptors. Given this and recent findings concerning retinal embryogenesis, we argue for the rod photoreceptor as the cell of origin. A possible role for the retinoblastoma gene product is discussed.

p53 regulates apoptosis in human retinoblastoma.

OBJECTIVES: To determine whether apoptosis is a significant mode of cell death in human retinoblastoma (RB) and if it is regulated by the expression of p53. METHODS: Apoptosis was analyzed using the criterion of internucleosomal DNA degradation as determined by agarose gel electrophoresis of DNA isolated from tumor specimens. Individual cells undergoing apoptosis were identified using terminal transferase-mediated biotin-dUTP nick end labeling (TUNEL) of fragmented DNA. The expression of p53 and WAF1 (a protein involved in p53-mediated cell cycle arrest) in human RB was determined by immunocytochemical analysis. The function of p53 in human RB cell lines was tested by transfecting them with a complementary DNA encoding a temperature-sensitive isoform of murine p53 under the control of a strong viral promoter. RESULTS: DNA from RB tumor specimens showed a strong nucleosomal ladder of DNA fragments typical of apoptosis. The TUNEL staining indicated that poorly and moderately differentiated cells in tumors were undergoing DNA fragmentation. Immunoreactivity for p53 was variable. Cells expressing low levels of p53 seemed viable and expressed WAF1. Cells expressing high levels of p53 were found immediately adjacent to cells undergoing apoptosis. Human RB cells in culture that were expressing a murine temperature-sensitive isoform of p53 died at temperatures that allow this protein to assume a wild-type conformation. CONCLUSIONS: Apoptotic cell death is prevalent in RB. The close association of p53-immunoreactive cells and cells undergoing apoptosis in human tumors, and the ability of exogenous p53 to stimulate cell death in cultured human RB cells, suggests that p53 plays a role in regulating cell death in RB.

Immunocytochemical features of retinoblastoma in an adult.

OBJECTIVE: To describe the clinical course and immunocytochemical characteristics of an unusual intraocular tumor. METHODS: Immunocytochemical analysis of the enucleated eye with an intraocular mass that markedly waxed and waned in size during 1 year of close observation of a 29-year-old woman. RESULTS: Most of the tumor was composed of either dying or rapidly proliferating cells. One area located near the retina consisted mostly of well-differentiated cells in uniform sheets (bacillettes) with lacelike glial processes between the tumor cells. Almost all of the differentiated tumor cells were positive for S antigen. In particular, the dominant cell type stained positively for both antibodies known to be specific for those isoforms of S antigen found only in blue cones and rods but not in red or green cones. Only a few of these cells labeled positively with an anti-rhodopsin antibody. CONCLUSIONS: This is the first case of adult retinoblastoma to be confirmed immunocytochemically. The tumor was unusual because the differentiated regions contained bacillettes composed mostly of blue cones. It is possible that this and other adult retinoblastomas may arise from previously existing retinocytomas.

Swelling and loss of photoreceptors in chronic human and experimental glaucomas.

OBJECTIVE: To determine whether outer retinal changes occur in chronic, presumed primary open-angle glaucoma (POAG). METHODS: The outer retinas from 128 human eyes with a diagnosis of chronic glaucoma (presumably POAG in most cases) and 90 control eyes were examined histologically by 3 masked observers for photoreceptor swelling and loss. Retinas from 9 rhesus monkeys with glaucoma induced experimentally by laser trabecular destruction were compared with 7 fellow (control) eyes. The mean pressure elevations in the eyes with laser trabecular destruction ranged from 26.6 to 53.6 mm Hg with durations varying from 7 to 33 weeks. RESULTS: Swelling of the red- and green-sensitive cones was observed in a statistically significantly greater proportion of human eyes with presumed POAG compared with the control eyes. Patchy loss of red/green cones and rods was also found in some of the glaucomatous retinas. In a subset of the human eyes with end-stage disease, cone swelling was a variable finding. Although no photoreceptor loss was found in the 9 monkey eyes with experimental glaucoma, 8 had swelling of their red/green cones that was remarkably similar to that seen in the human eyes. Swelling was not present in any of the control monkey eyes. CONCLUSIONS: The photoreceptors are affected by chronically elevated intraocular pressure. CLINICAL RELEVANCE: These findings may explain some of the abnormalities of color vision and the electrophysiological effects that have been observed in patients with POAG.

Effects of denervation and reserpine on nexuses in the rat vas deferens.

Permanganate-fixed vasa deferentia from rats were examined for the presence of nexal-like contacts by electron microscopy. There was a significantly greater incidence of nexuses (2X) in chronically denervated tissues (5-7 days) but not in tissues from reserpine-pretreated animals (1.0 mg/kg/day for 5-7 days). The results suggest that an increase in nexal regions may not be a general feature of postjunctional supersensitivity but rather may contribute to other denervation-induced changes in contractile response.

Brief exposure of air-filled guinea-pig isolated trachea to low levels of toluene diisocyanate (TDI) vapor in vitro increases reactivity to methacholine.

Toluene diisocyanate (TDI) causes occupational asthma characterized by inflammation and hyperreactivity of airways to irritants and bronchoconstrictor drugs. We examined the non-immune, direct effect of TDI on airway reactivity in vitro in the absence of an inflammatory response using the guinea-pig isolated, perfused trachea preparation to measure reactivity to methacholine (MCh), and fixed point ion mobility spectrometry to measure moment to moment levels of TDI vapor in air that was delivered to the tracheal mucosa. MCh was added to the mucosal modified Krebs-Henseleit (MKH) perfusing solution to generate control concentration-response curves for contractile responses. The lumen was then emptied and perfused with air or air containing 5, 20 or 70 ppb TDI vapor, after which the trachea was perfused with MKH solution and reactivity to MCh was re-examined. After only 30 min of treatment, TDI vapor concentration-dependently increased reactivity of the trachea to MCh (2.4- and 2.9-fold, respectively, for 20 and 70 ppb TDI; 5 ppb TDI and air alone had no effect). In tracheas treated in vitro with 2 microM capsaicin to deplete tachykinins, TDI caused the same (4-fold) increase in reactivity to MCh that was observed in control tracheas. However, TDI vapor (70 ppb) no longer enhanced reactivity to MCh in tracheas from which the epithelium had been removed. Our results indicate that a direct, non-immune, non-inflammatory action of TDI on respiratory epithelium leads to hyperreactivity of airways in vitro.

Treatment and histopathology of a congenital vitreous cyst.

OBJECTIVE: This study aimed to evaluate the treatment efficacy of a congenital vitreous cyst and to examine the cyst histopathologically to determine its cellular makeup and possible origin. STUDY DESIGN: The study design was a case report, including a clinicopathologic correlation. INTERVENTION: A 35-year-old woman with a known vitreous cyst since childhood became increasingly troubled by its symptoms. The cyst was treated initially with argon laser photocoagulation. Vitrectomy subsequently was performed because the deflated cyst remained near the visual axis. Histopathologic studies included light and electron microscopy; immunocytochemistry for actin and glial fibrillary acidic protein (GFAP); and enzyme histochemistry for carbonic anhydrase (CA). RESULTS: The cyst was composed of a single layer of heavily pigmented cells with a thick basement membrane along the internal borders of the cells. Ultrastructurally, the cells were connected with tight junctions, had microvillous processes at their apices, and contained numerous large melanosomes in various stages of maturity, including premelanosomes. Immunochemistry showed the cells were positive for actin but negative for GFAP. Enzyme histochemical staining for CA also was strongly positive. CONCLUSIONS: The confinement of this cyst to the region of Cloquet's canal, the presence of a Mittendorf's dot, the cyst's existence for many years, and the finding of pigment epithelial-type cells having immature melanosomes (a feature not seen after birth in normal pigment epithelium) lead the authors to believe that this cyst was a congenital remnant of the primary hyaloidal system. Because pigmented cells are not normally present in this part of the eye, the cyst was a choristoma of the primary hyaloidal system.

Fluticasone propionate and pentamidine isethionate reduce airway hyperreactivity, pulmonary eosinophilia and pulmonary dendritic cell response in a guinea pig model of asthma.

In this study, we examined the effects of fluticasone propionate (FP) and pentamidine isethionate (PI) on antigen-induced lung inflammation and airway hyperreactivity in guinea pigs. Male guinea pigs were sensitized on days 0 and 14 with 10 micrograms of ovalbumin (OVA) plus 1 mg of Al(OH)3. On day 21, animals were challenged with a 2% OVA aerosol inhalation until they developed pulmonary obstruction. Animals were treated with aerosol inhalation of FP (2 ml of 0.5 mg/ml, five consecutive doses at 12-hr intervals with the last dose given 6 hr before OVA challenge) or PI (30 mg/ml for 30 min 1 hr before OVA challenge), and control animals received no drug before OVA challenge. Airway reactivity to methacholine (MCh) was assessed before sensitization and 18 hr after OVA challenge. At 18 hr after challenge, histological sections of trachea and lung were examined for eosinophil, dendritic cell (DC) and macrophage cell densities in the airways. In control animals, OVA evoked airway hyperreactivity to MCh in conjunction with pulmonary eosinophilia and increases in DC prevalence in the trachea and bronchi. Treatment with FP or PI abolished the OVA-induced hyperresponsiveness and significantly reduced the OVA-induced increases in eosinophils and DCs in the airways. FP and PI had no effect on saline-treated animals. Our study indicates that both inhaled FP and inhaled PI reduce antigen-induced airway hyperreactivity and pulmonary inflammation in guinea pigs. The results also suggest that the DC is a target of the anti-inflammatory effects of these drugs in the airways.

Pulmonary dendritic cell distribution and prevalence in guinea pig airways: effect of ovalbumin sensitization and challenge.

We characterized the localization and prevalence of dendritic cells (DC) in guinea pig airways before and after s.c. sensitization and aerosol challenge with ovalbumin (OVA). DC, eosinophils, macrophages, T cells and B cells in lung and trachea were identified and quantified in frozen sections using monoclonal antibodies and computer-assisted image analysis. Airway reactivity of conscious animals to inhaled methacholine was examined. In unsensitized animals, DC were localized primarily within the lamina propria of the trachea and bronchi, in the submucosa of the trachea and in the adventitia of the bronchi. In contrast to reported studies on rats, few DC were noted in the epithelium. After OVA challenge, sensitized animals demonstrated an early obstructive response and a late-phase response that was well developed by 18 hr. Challenge with OVA increased DC prevalence in the lamina propria and submucosa of the trachea and in the lamina propria and adventitia of the bronchi. There was widespread eosinophilia throughout the airways, but no changes in B cells or T cells were evident. Macrophages were increased in the epithelium of both OVA-treated and saline-treated animals. At 18 hr after challenge, sensitized guinea pigs but not saline-treated controls were hyperreactive to inhaled methacholine. Except for macrophages, none of these effects were observed after saline treatment. Our findings indicate that inflammation in the airways of OVA-sensitized guinea pigs involves infiltration of DC, which is seen at the time animals are hyperreactive to inhaled methacholine.

Airway hyperreactivity elicited by toluene diisocyanate (TDI)-albumin conjugate is not accompanied by airway eosinophilic infiltration in guinea pigs.

Nonspecific airway hyperresponsiveness is present in many patients with toluene diisocyanate (TDI)-induced asthma; however, the underlying pathophysiological mechanisms of this hyperresponsiveness remain controversial. In the present study, we used a guinea pig model to investigate the association of TDI-induced airway hyperresponsiveness with eosinophilic airway infiltration, which is widely considered to play a key role in the development of allergen-induced hyperresponsiveness. Guinea pigs were sensitized by i.d. injections of 10 microl TDI on day 1 and day 6. Control animals received saline injections. Two weeks after the second injection, airway reactivity to inhaled methacholine and specific airway resistance (sRaw) was measured before and at several times after inhalation challenge with TDI-GSA (guinea pig serum albumin) conjugates. Eosinophils in the airways were detected using enzyme histochemistry and quantified using computer-assisted image analysis. TDI-specific IgG1 antibodies were found in the blood of TDI-sensitized animals. An immediate increase in sRaw was induced in these animals by TDI-GSA challenge; airway hyperresponsiveness to methacholine was observed at 6 h and 18 h after TDI-GSA challenge. However, TDI-GSA challenge did not result in an elevation of eosinophils in the airways, compared with control animals. The results suggest that the development of TDI-induced airway hyperresponsiveness is not dependent upon eosinophil infiltration in airways.

A comparison of the electrical properties and morphological characteristics of the smooth muscle of the rat and guinea-pig vas deferens.

Microelectrodes were used to compare a variety of electrophysiological parameters of the rat and guinea-pig vas deferens. In comparison to the guinea pig, spontaneous junction potentials in the rat tissue were of shorter duration and occurred with greater frequency and amplitude. Action potentials induced by nerve stimulation could be observed in the smooth muscle of both species. However, in the rat tissue the majority of action potentials were generated in the impaled cell while 60% of the action potentials in the guinea-pig vas deferens were propagated. When current was intracellularly applied, spike potentials could be induced in approximately 90% of the cells of the rat vas deferens but in less than 10% of the cells of the guinea-pig vas deferens. The space constant was 1.48 mm for the guinea-pig vas deferens, but less than 0.5 mm for the rat vas deferens. Electromicroscopic examination of the homologous tissues indicates that the differences in electrical properties can be accounted for in part by differences in morphology. The incidence and intimacy of neuromuscular contacts was greater in the rat vas deferens while the incidence of nexuses between smooth muscle cells was greater in the guinea-pig tissue.

Ovalbumin aeroallergen exposure-response in Brown Norway rats.

A major route of exposure to allergens is through the respiratory tract. Comparatively few animal studies have used aerosolized high-molecular-weight allergens for sensitization, and in these studies, proper characterization of the aeroallergen exposure was usually missing. The purpose of this study was to profile the exposure-response relationship in Brown Norway rats (BNR) to well-characterized ovalbumin (OVA) aerosols. Rats were exposed 30 min/wk x 6 wk to respirable OVA aerosols from <1 mg/m(3) to 64 mg/m(3) air. Ovalbumin-specific circulating immunoglobulin (Ig)E, IgG, and IgA were measured throughout the study period. Rats were sacrificed 1 day after the last exposure. Pulmonary tissue was processed for histopathological and histochemical analysis. Tracheas were isolated, perfused, and assessed for in vitro responsiveness to methacholine. Serum concentrations of OVA-specific antibodies increased with both exposure concentration and number of exposures. The number of BNR with measurable titers also increased with both dose and time. Pulmonary inflammatory changes were noted only in BNR exposed to higher OVA concentrations (15 and 64 mg/m(3) air). Increased tracheal reactivity to methacholine was not found in any of the sensitized BNR. In summary, sustained aeroallergen concentration-dependent changes in specific antibody responses and pulmonary inflammation have been demonstrated.

Effect of ozone treatment on airway reactivity and epithelium-derived relaxing factor in guinea pigs.

Ozone (O(3)) is toxic to respiratory epithelium and causes airway inflammation and hyperreactivity. To evaluate the role of the epithelium in the development of hyperreactivity, we examined in guinea pigs the effects of inhaled O(3) (3 ppm for 1 h; 0-24 h after exposure) on 1) reactivity to inhaled methacholine (MCh), 2) reactivity of the isolated, perfused trachea (IPT) to MCh, 3) epithelium-derived relaxing factor (EpDRF)-mediated relaxations of IPT induced by mucosal hyperosmolar solutions, 4) neurogenic contraction and relaxation responses, 5) transepithelial potential difference, and 6) microscopic analysis of nitrotyrosine immunofluorescence, substance P fiber density, and tracheal morphology. At 0 h, O(3) caused hyperreactivity to inhaled MCh and mucosally but not serosally applied MCh in IPT (only in the presence of the epithelium) and a decrease in transepithelial potential difference. Inhibition of EpDRF-induced relaxation responses occurred at 2 h. All of these changes returned to control by 12 to 18 h. O(3) had no effect on neurogenic responses. Nitrotyrosine immunofluorescence appeared in the trachea at 0 h in detached epithelial cell ghosts and in intrapulmonary airways by 6 h. Substance P fiber density was elevated in smooth muscle at 0 and 18 h but not in epithelium or lamina propria of intrapulmonary and extrapulmonary bronchi. Loss of cilia and mucosubstances in the mucosa occurred at 0 h; the epithelium became markedly attenuated over 12 to 24 h. A reversible increase in epithelial permeability and a decrease in EpDRF production may contribute to O(3)-induced hyperreactivity to MCh.