Paracrine effects of bombesin/gastrin-releasing peptide and other growth factors on pulmonary neuroendocrine cells in vitro

Pulmonary neuroendocrine cells (PNEC) are numerous in the fetus where they have been implicated to have a role in fetal lung development. We assessed the effects of putative growth factors, gastrin releasing peptide (GRP), cholecystokinin (CCK), gastrin (GN), serotonin (5-HT), and epidermal growth factor (EGF), some of which are produced by PNEC, either alone or in combination, on cultured fetal rabbit PNEC from 20, 24, and 28 day fetuses. GRP increased the total protein of the cultures over a 7 day period in an age-dependent manner, with greatest effect in cultures from the 24 day fetus, no effect with the 28 day fetus, and an inhibitory effect on 20 day cultures. This was accompanied by an increase in PNEC, which could be blocked by treatment of the cultures with a monoclonal antibody to GRP (2A11). There was no increase in ³H-thymidine labeling of PNEC in GRP treated cultures but an increase in numbers of cells partially stained for 5-HT, suggesting the induction of a precursor cell. Other growth factors had neither an inhibitory nor a stimulatory effect either alone or in combination with GRP. Preliminary studies with ¹²⁵I-GRP receptor localization suggests that the GRP receptor is mostly expressed on pulmonary fibroblasts, and less on epithelial cells, so that the role for GRP in fetal lung development, at least in the rabbit, is probably indirect, acting via a paracrine mechanism. © 1993 Wiley-Liss, Inc.     

Detection of small cell lung cancer metastases in bone marrow aspirates using monoclonal antibody directed against neuroendocrine differentiation antigen.

To detect metastases in the bone marrow of patients with small cell lung cancer, immunofluorescence with a monoclonal antibody detecting a membrane antigen (MOC-1) associated with small cell lung cancer was performed on 53 bone marrow aspirates from 30 patients. In 19 (63%) patients MOC-1 reactive cells were detected. Simultaneous histopathological examination of the bone marrow biopsy specimens detected tumour cells in only six (20%). The method is more sensitive than conventional histochemical staining of bone marrow aspirate and may eventually be able to show additional subgroups, such as patients with limited disease who might benefit from adjuvant radiotherapy or surgery.     

CD10/neutral endopeptidase inhibition augments pulmonary neuroendocrine cell hyperplasia in hamsters treated with diethylnitrosamine and hyperoxia.

In previous studies, we demonstrated that pulmonary neuroendocrine cell (PNEC) hyperplasia in hamsters treated with diethylnitrosamine (DEN) plus 65% hyperoxia (DEN/O2) reflects predominantly neuroendocrine cell differentiation. Several peptides implicated in non-neoplastic PNEC hyperplasia are hydrolyzed by CD10/neutral endopeptidase 24.11 (CD10/NEP), an enzyme known to downregulate neurogenic inflammation of the lung by modulating locally effective concentrations of multiple bioactive peptides. In fetal mice, we observed that CD10/NEP inhibition by SCH32615 potentiates cell proliferation and type II cell differentiation in the lung in utero. Further, CD10/NEP messenger RNA levels parallelled relative PNEC numbers in DEN/O2-treated hamster lung, suggesting that the enzyme might mediate spontaneous regression of PNEC hyperplasia. The goals of the present study were: (1) to determine whether CD10/NEP inhibition would alter the extent of PNEC hyperplasia occurring in these hamsters, and (2) to analyze cellular mechanisms potentially involved in altering numbers of PNECs in this model. We administered SCH32615 chronically to a subset of DEN/O2-treated hamsters. Immunostaining of lungs from the CD10/ NEP-inhibited subset demonstrated significant acceleration of the development of PNEC hyperplasia, increased PNEC proliferation, and diminished PNEC apoptosis as compared with animals receiving no SCH32615. These observations indicate that PNEC hyperplasia can occur as a result of multiple cellular processes, including increased neuroendocrine cell differentiation, proliferation, and survival. CD10/NEP modulates PNEC numbers primarily by promoting cell differentiation and proliferation during lung injury, probably via increasing the half-life of bioactive peptides in the lung.     

Glucocorticoid regulation of surfactant-associated proteins in rabbit fetal lung in vivo

The effects of a maternally administered synthetic glucocorticoid, betamethasone, on the levels of mRNA for the surfactant proteins SP-A, SP-B, and SP-C and on the levels of SP-A protein were investigated in day 27 gestational age rabbit fetal lung tissue. Betamethasone administration to the pregnant rabbit caused approximately a twofold increase in the fetal lung level of SP-A protein and a threefold increase in fetal lung SP-A mRNA levels when compared to levels in fetuses obtained from saline-treated or uninjected animals. SP-B mRNA was increased fourfold in fetal lung tissue obtained from glucocorticoid-treated pregnant does when compared to levels in fetuses of uninjected pregnant does. However, SP-B mRNA levels in fetal lung tissue from saline-injected controls were also significantly elevated, ～twofold, when compared to fetal lung SP-B mRNA levels in the uninjected control condition. SP-C mRNA levels in lung tissue of fetuses from both saline-injected and betamethasone-injected pregnant does were increased similarly, ～twofold, over SP-C mRNA levels in fetal lung tissue obtained from uninjected control does. These data are suggestive that betamethasone treatment increases fetal lung SP-A and SP-B mRNA levels and that maternal stress alone can increase the expression of SP-B and SP-C mRNA in rabbit fetal lung tissue. Using in situ hybridization, SP-A mRNA was shown to be present primarily in alveolar type II cells in fetuses of control and saline-injected does. However, SP-A mRNA was easily detected in both alveolar type II cells and bronchiolar epithelial cells of rabbit fetal lung tissue following maternal betamethasone treatment. In contrast, SP-B and SP-C mRNA were present only in alveolar type II cells of lung tissue obtained from fetuses of control, saline, or betamethasone-treated does. Thus maternal administration of glucocorticoids increased SP-A protein as well as SP-A and SP-B mRNA levels in rabbit fetal lung tissue. SP-A mRNA was localized to both alveolar type II cells and in smaller amounts in bronchiolar epithelial cells of rabbit fetal lung tissue. However, SP-B and SP-C mRNA were detected only in alveolar type II cells. © 1993 Wiley-Liss, Inc.     

In vitro characteristics of pulmonary neuroendocrine cells isolated from rabbit fetal lung. I. Effects of culture media and nerve growth factor

Pulmonary neuroendocrine (NE) cells, dispersed throughout the airway mucosa as single cells and as innervated clusters (neuroepithelial bodies), were isolated from rabbit fetal lung and studied in short-term culture. The effects of culture media and nerve growth factor (NGF) on in vitro maintenance, differentation, and cell kinetics of isolated NE cells were examined. For demonstration of NE cells in intact lung, during cell separation and after culture, immunostaining for serotonin, formaldehyde-induced fluorescence method, histochemical reaction for acetylcholinesterase, and electron microscopy were used. The isolation procedure consisted of mechanical and enzymatic dissociation of lung tissue followed by separation of isolated cells on a discontinuous gradient of Percoll, resulting in 5- to 10-fold enrichment in NE cells. Cell fractions enriched in NE cells were cultured up to 7 days either in supplemented alpha-minimal essential medium with fetal bovine serum or in defined, hormone-supplemented, serum-free medium. NGF (2.5 S 5 to 50 ng/ml) was added to both serum-supplemented and serum-free media; cultures without NGF served as control. The number of serotonin-immunoreactive NE cells maintained in serum-supplemented medium (0.5% fetal bovine serum) increased significantly (p less than 0.05) on days 4 and 7 compared with cultures grown in serum-free medium. NE cells maintained in serum-supplemented medium incorporated [3H]thymidine and their labeling index was significantly increased (p less than 0.01) on day 7, whereas few or no NE cells were labeled in cultures grown in serum-free medium. NGF had no effect on the maintenance or kinetics of NE cells. Cultured NE cells formed elongated (unipolar or bipolar) neurite-like cytoplasmic processes with a button-like ending, regardless of the presence of NGF. Amine accumulated in perinuclear cytoplasm and in button-like endings. Staining for acetylcholinesterase (strongly positive in intact neuroepithelial bodies) was not detectable after separation into single cells, culture, or exposure to NGF. This study demonstrates that NE cells can be isolated from rabbit fetal lung and maintained in short-term culture. Low concentrations of fetal bovine serum enhanced the in vitro maintenance of NE cells, whereas NGF had no such effect. A feeder layer may be also important, since NE cells were closely associated with lung epithelial or fibroblast-like cells. The formation of neurite-like processes appears to be an expression of paracrine/paraneuron-type cell differentiation not mediated by NGF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pulmonary neuroendocrine cells, airway innervation, and smooth muscle are altered in Cftr null mice

The amine- and peptide-producing pulmonary neuroendocrine cells (PNEC) are widely distributed within the airway mucosa of mammalian lung as solitary cells and innervated clusters, neuroepithelial bodies (NEB), which function as airway O2 sensors. These cells express Cftr and hence could play a role in the pathophysiology of cystic fibrosis (CF) lung disease. We performed confocal microscopy and morphometric analysis on lung sections from Cftr-/- (null), Cftr+/+, and Cftr+/- (control) mice at developmental stages E20, P5, P9, and P30 to determine the distribution, frequency, and innervation of PNEC/NEB, innervation and cell mass of airway smooth muscle, and neuromuscular junctions using synaptic vesicle protein 2, smooth muscle actin, and synaptophysin markers, respectively. The mean number of PNEC/NEB in Cftr-/- mice was significantly reduced compared with control mice at E20, whereas comparable or increased numbers were observed postnatally. NEB cells in Cftr null mice showed a significant reduction in intracorpuscular nerve endings compared with control mice, which is consistent with an intrinsic abnormality of the PNEC system. The airways of Cftr-/- mice showed reduced density (approximately 20-30%) of smooth muscle innervation, decreased mean airway smooth muscle mass (approximately 35%), and reduced density (approximately 20%) of nerve endings compared with control mice. We conclude that the airways of Cftr-/- mice exhibit heretofore unappreciated structural alterations affecting cellular and neural components of the PNEC system and airway smooth muscle and its innervation resulting in blunted O2 sensing and reduced airway tonus. Cftr could play a role in the development of the PNEC system, lung innervation, and airway smooth muscle.     

The type II epithelial cells of the lung. IV. Adaption and behavior of isolated type II cells in culture

Type II lung epithelial cells were isolated from rabbit lungs using the method of Kikkawa and Yoneda (Kikkawa Y, Yoneda K: Lab Invest 30: 76, 1974). Successful primary cultures were obtained only after utilizing high density cell plating (greater than 3 X 10(5) viable cells per sq. cm.) and allowing an attachment time of 48 hours. Attachment efficiency of the isolated cell preparations was highest when medium was supplemented with 10% fetal calf serum. These conditions enabled us to obtain consistently successful primary type II lung cell cultures. Cultures were monitored for a period of 2 weeks after initiation. Light, phase, and electron microscopy examination demonstrated that these primary cultures were indeed type II cells. The principal morphologic feature was the presence of dense lamellar granules in these cells. These primary cultures retained the characteristic type II features for 3 to 5 days in vitro, after which cultures exhibited a progressive deterioration and loss of their phenotypic properties. This behavioral pattern of type II cells in culture may represent both accelerated proliferation and accelerated transformation of these cells into type I epithelial cells.     

Identification of leptin receptors in lung and isolated fetal type II cells

Leptin is a cytokine involved in regulation of the satiety response. Receptors for this protein have been identified in brain as well as many other peripheral tissues. Some of the highest levels of receptor concentration occur in the lung. Considering the cellular diversity of lung, neither the localization nor the function of leptin in pulmonary tissues has been delineated. The purpose of the present study was to determine if fetal and adult rabbit lung displayed specific binding for leptin, to identify the binding sites, and to explore a potential functional role for leptin in lung surfactant production. Frozen sections of adult and fetal rabbit (24th gestational day) lung were prepared and incubated with increasing concentrations of [125I]leptin in the presence or absence of 1-microM-unlabeled leptin. Sections were removed and radioactivity measured. Concurrently, sections were coated with nuclear Trac emulsion and incubated in the dark at -30 degrees C. Lung showed specific binding for leptin. Microscopically, [125I]leptin was localized to acinar-lining epithelium of developing fetal lung. Larger cells within the epithelial layer appeared to bind leptin more avidly than adjacent cells. Antibodies to the leptin receptor were used to identify binding sites in adult lung and isolated fetal lung type II cells. In adult lung, both the K20 (against the extracellular amino-terminal) and the M18 antibody (against the intracellular carboxy-terminal) displayed several binding sites. In contrast, the isolated fetal type II cells showed only a single binding site for both antibodies. The apparent molecular mass of the receptor using the K20 antibody appeared to be approximately 125 kD. A protein of similar mass bound the M18 antibody suggesting that functional receptor is present in lung and expressed by fetal type II cells. Incubation of isolated fetal type II cells with leptin (0.01-10 microg/ml) stimulated [3H]choline incorporation in disaturated phosphatidylcholine. These results show that fetal and adult lung bind leptin specifically, and fetal type II cells in particular, may be responsive to leptin stimulation of phospholipid production. Leptin may therefore be important in regulating maturation of cells of the fetal lung.     

Epithelial cell differentiation in organotypic cultures of fetal rat lung

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbonstripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbonstripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that (1) the hormones extracted from bovine fetal calf serum by carbonstripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and (2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.     

MOC-31/Ep-CAM immunoreactivity in Merkel cells and Merkel cell carcinomas

AIMS: To evaluate the monoclonal antibody MOC-31 in Merkel cell carcinomas and normal Merkel cells. Merkel cell carcinoma is a rare and aggressive tumour that occurs mainly in elderly individuals. The histological diagnosis of Merkel cell carcinoma can be difficult because it looks similar to other small blue cell tumours, particularly skin metastases of small-cell lung carcinomas. This antibody recognizes the epithelial cell adhesion molecule (Ep-CAM), that has been assigned to the small cell lung cancer cluster 2 of antibodies. To the best of our knowledge, immunostaining for MOC-31/Ep-CAM has not been previously described in Merkel cells or Merkel cell carcinomas. METHODS AND RESULTS: Thirty-one cases of Merkel cell carcinoma and three samples of normal human fingertip were selected to analyse the expression of MOC-31/Ep-CAM by immunohistochemistry. A high number of Merkel cell carcinomas (21/31, 67.7%) showed intense and readily interpretable positivity. Immunostaining was diffuse or focal and always localized to the plasma membrane. Normal Merkel cells of human fingertip also showed plasma membrane immunoreactivity for MOC-31/Ep-CAM. CONCLUSION: The demonstration of positivity for MOC-31/Ep-CAM in Merkel cell carcinomas precludes the use of this immunohistochemical marker to distinguish between tumours and skin metastases of small-cell lung carcinoma.     

人胎肺神经内分泌细胞的发育及其作用

目的：探讨胎肺生长发育中神经内分泌细胞的表达及其功能意义。方法：检测３０例人胎肺组织,采用组织化学Ｇrimelius嗜银反应、免疫组织化学、透射电镜技术及显微图像分析。结果：神经内分泌细胞最早在胚胎第１２周胎肺出现,主要为分泌ＧＲＰ细胞,其分布与细支气管发育分化密切相关;ＣＴ阳性细胞在胚胎１６周胎肺出现;分泌ＣgＡ细胞在２０周 出现。随胎龄增长,神经内泌细胞数量递增,胚胎２４周达高峰。此后细胞数量递减,出生     

Quantifying proliferation of cultured human and rabbit airway smooth muscle cells in response to serum and platelet-derived growth factor.

Development of suitable methods for the quantification of the proliferative response of airway smooth muscle (ASM) cells in culture will assist the investigation of the cellular mechanisms underlying the hyperplasia and hypertrophy of ASM as seen in asthmatic airways. In this study, two rapid and simple colorimetric assays have been modified to enable the growth of human bronchial and rabbit tracheal smooth muscle in culture to be assessed. One method depends upon the reduction by living cells of the tetrazolium salt MTT to form a blue formazan product, whereas the other relies on rapid binding of the dye Coomassie brilliant blue to protein at acidic pH. Experiments demonstrated the validity of both assays in quantifying the proliferative response of cultured human and rabbit ASM cells. The increase in optical density observed for each assay correlated directly, throughout the duration of culture, with the increase in cell number determined by hemocytometry in human and rabbit ASM cells proliferating in response to fetal calf serum (1.25 to 10%). This relationship held also for rabbit tracheal ASM cells proliferating in response to the heterodimer of platelet-derived growth factor (1 to 50 ng/ml). Application of these methods to adherent proliferating cultures of human and rabbit ASM cells demonstrated their adaptability to the generation of growth curves in response to serum and to a defined growth factor. These methods allow both total cellular protein and proliferation to be estimated in human and rabbit ASM cells in culture, using assays that are rapid, reproducible, inexpensive, and easy to perform while negating the use of radioisotopes. It is intended that these additional methods should be useful in delineating some of the mechanisms that might contribute to the proliferative response of these cells--particularly since there has been a resurgence in interest in culturing smooth muscle cells derived from the airways.     

Cell biology of pulmonary neuroepithelial bodies—validation of an in vitro model. I. Effects of hypoxia and Ca2+ ionophore on serotonin content and exocytosis of dense core vesicles

Pulmonary neuroendocrine (NE) cells including the innervated clusters of NE cells—neuroepithelial bodies (NEB)—are difficult to study because of their small numbers and diffuse distribution within the airway mucosa of the lung. We have previously reported a method for isolation and culture of NE cells from rabbit fetal lung using a combination of mechanical and enzymatic dissociation followed by gradient centrifugation. This method provides single cell suspension of mixed lung cells enriched in NE cells, particularly those originating from NEB. This study further validates our in vitro model by detailed morphologic characterization of cultured NEB cells using high resolution light microscopy, transmission and scanning electron microscopy, HPLC for detection of serotonin (5-HT), and molecular (Northern blot) analysis of mRNA encoding for 5-HT synthesizing enzymes, tryptophane hydroxylase, and aromatic L-amino acid decarboxylase. In addition to effects of hypoxia on NEB cells in vitro were investigated to define the role of these cells as possible airway chemoreceptors. Exposure of NEB cultures to hypoxia resulted in decreased intracellular content of 5-HT accompanied by increased exocytosis of dense core vesicles (DCV). The amount of 5-HT release correlated with the degree of hypoxia, suggesting modulation by ambient pO₂ levels. The role of Ca²⁺ ions in exocytosis of DCV and 5-HT release from NEB cells was tested in experiments with Ca²⁺ ionophore (A23187). Exposure of cultures to 5 μg/ml of ionophore resulted in up to 40% reduction in 5-HT content of NEB cultures as well as increased exocytosis of DCV. Our overall findings are consistent with a view that NEB cells are chemosensory in nature and that Ca²⁺ signaling pathway is involved in stimulus-secretion coupling. Further refinements in cell separation and culture methodology are required before more detailed investigation of NEB cell membrane properties, signal transduction mechanisms, and intracellular signaling pathways can be carried out. © 1993 Wiley-Liss, Inc.     

Localization of surfactant-associated proteins SP-A and SP-B mRNA in rabbit fetal lung tissue by in situ hybridization.

Pulmonary surfactant is a lipoprotein substance, comprised of approximately 80% phospholipid and approximately 10% protein, that lowers surface tension at the air-alveolar aqueous interface. Surfactant is synthesized and secreted by alveolar type II epithelial cells where it is stored intracellularly in lamellar bodies. In the present study, we used the technique of in situ hybridization to localize the mRNA for two surfactant-associated proteins, SP-A and SP-B, in developing rabbit fetal lung tissue. We found that SP-A mRNA was first localized in rabbit fetal lung alveolar type II cells on day 26 of gestation, the time at which lamellar bodies are first observed within fetal lung type II cells. On day 28 of gestation, a very small amount of SP-A mRNA was also detectable in the epithelial cells of some bronchioles. In neonatal and adult rabbit lung tissue, SP-A mRNA was primarily restricted to alveolar type II cells; however, the epithelial cells of some bronchioles contained small amounts of SP-A mRNA. SP-B mRNA was first detected in cuboidal epithelial cells in the prealveolar region of the rabbit fetal lung tissue on day 24 of gestation, i.e., at least 2 days before the appearance of SP-A mRNA and lamellar bodies within differentiated alveolar type II cells. SP-B mRNA was detected in most bronchiolar epithelial cells of the rabbit fetal lung tissue at day 28 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelial differentiation by multipotent fetal mouse lung mesenchymal cells

During fetal lung development, cells within the mesenchyme differentiate into vascular endothelia. This process of vasculogenesis gives rise to the cells that will eventually form the alveolar capillary bed. The cellular mechanisms regulating lung vasculogenesis are poorly understood, partly due to the lack of experimental systems that model this process. Here, we have developed and characterized a novel fetal mouse lung cell model of mesenchymal to endothelial differentiation. Using mesenchymal cells from the lungs of embryonal day 15 Immortomice, we show that endothelial growth media containing fibroblast growth factor-2 and vascular endothelial growth factor can stimulate formation of vascular endothelial cells in culture. These newly formed endothelial cells retain plasticity, as removing endothelial growth media causes loss of vascular markers and renewed formation of α-smooth muscle actin positive stress fibers. Cells with the highest Flk-1 expression differentiated into endothelia more efficiently. Individual mesenchymal cell clones had varied ability to acquire an endothelial phenotype. These fetal lung mesenchymal cells were multipotent, capable of differentiating into not only vascular endothelia, but also osteogenic and chondrongenic cell lineages. Our data establish a cell culture model for mesenchymal to endothelial differentiation that could prove useful for future mechanistic studies in the process of vasculogenesis both during normal development and in the pathogenesis of pulmonary vascular disease.     

Fetal mouse alveolar type II cells in culture express several type II cell characteristics found in vivo, together with major histocompatibility antigens.

Alveolar type II cells were isolated from fetal mouse lung by differential adherence and obtained in monolayer culture. Cultures display a high degree of purity as shown by histochemical and immunocytochemical staining procedures. Seventy-five percent of cells stained positive with specific anti-lavage serum mouse (SALS-M), an antiserum specific for (pre)alveolar type II cells of the mouse, and osmiophilic bodies were present in 82% of cells. These and other characteristics of type II cells in culture correspond to those of alveolar type II cells in fetal mouse lung. The pattern of reactivity of these cells with various anti-cytokeratin antibodies is described, and we show that, in contrast to rat type II cells, they do not exhibit alkaline phosphatase activity. Identity of the type II cell cultures was shown by their specific phospholipid composition and surfactant protein A (SP-A) content. The fetal alveolar type II cells in culture were found to synthesize and express class I but not class II major histocompatibility complex (MHC) antigens. The possibility to culture fetal alveolar type II cells of the mouse and the availability of genetically well-defined inbred and transgenic mouse strains opens ways to study the genetics of type II cell differentiation and function. Also, the in vitro availability of alveolar type II cells, the progenitor cells of mouse lung tumors, will enable us to study in vitro several of the processes involved in lung tumorigenesis in the mouse.     

Rapid detection of herpes simplex virus in clinical specimens with human embryonic lung fibroblast and primary rabbit kidney cell cultures.

The performance of a culture system for isolation of herpes simplex virus, consisting of one tube each of human embryonic lung fibroblasts and primary rabbit kidney cells, was evaluated. Cultures were incubated at 37 degrees C on a roller drum and observed daily for characteristic cytopathic effect for 5 days. During 1982, a positive isolation rate of 28.1% was seen among 3,154 specimens submitted. Cultures from genital sources were positive more frequently from males (43.8%) than from females (25.5%). Oral lesion cultures were positive as often from males (34.6%) as from females (38.4%). Although detection of herpes simplex virus occurred significantly earlier in rabbit kidney cells on days 1 and 2 of incubation, by day 3 the number of positive cultures was nearly the same in both cell types. By day 4 of incubation, 99.5% of the positive cultures were detected. These results demonstrate that cell culture can be a rapid and sensitive method for detecting herpes simplex virus.