Dose-Dependent Effects of Zoledronic Acid on Human Periodontal Ligament Stem Cells: An In Vitro Pilot Study.

Bisphosphonates (BPs) are widely used to treat several metabolic and oncological diseases affecting the skeletal system. Despite BPs' well-known therapeutic potential, they also displayed important side effects, among which is BPs-related osteonecrosis of the jaw, by targeting osteoclast activities, osteoblast, and osteocyte behavior. The aim of this study is to evaluate the biological effects of zoledronic acid (ZOL) in an in vitro model of periodontal ligament stem cells (PDLSCs) by using an experimental setting that resembles the in vivo conditions. PDLSCs were treated with different concentrations of ZOL ranging from 0.1 to 5 µM. The effects of ZOL exposure were evaluated on cell viability via 3-[4,5-Dimethylthiaoly]-2,5-diphenyltetrazolium bromide (MTT), cell cycle analysis, apoptosis detection, and immunofluorescence. Quantitative real-time polymerase chain reaction (PCR), colorimetric detection of alkaline phosphatase activity, and Alizarin Red S staining were performed to investigate the osteogenic potential of PDLSCs exposed to ZOL. MTT analysis showed that the viability of PDLSCs exposed to ZOL concentration ≥1.5 µM for 3 and 6 days was significantly lower (P < 0.001) than that of untreated cells. The percentage of apoptotic cells was significantly higher in PDLSCs exposed for 4 days to ZOL at 2 µM (P < 0.01) and 5 µM (P < 0.001) when compared to the control. Moreover, ZOL treatment (3 days) accounted for alterations in cell cycle distribution, with an increase in the proportion of cells in G0/G1 phase and a reduction in the proportion of cells in S phase. Chronic exposure (longer than 7 days) of PDLSCs to ZOL accounted for the downregulation of ALP, RUNX2, and COL1 genes at all tested concentrations, which fit well with the reduced alkaline phosphatase activity reported after 7 and 14 days of treatment. Reduced Col1 deposition in the extracellular matrix was reported after 14 days of treatment. Increased calcium deposits were observed in treated cells when compared to the control cultures. In conclusion, chronic exposure to 1 µM ZOL induced significant reduction of osteogenic differentiation, while ZOL concentrations ≥1.5 µM are required to impair PDLSCs viability and induce apoptosis.

Innate immunity in cutaneous melanoma.

The skin immune system is composed of a vast network of immune cells, including lymphocytes, macrophages, neutrophils, dendritic cells and Langerhans cells, which not only are involved in inflammatory responses but also contribute to homeostatic function and may participate in the various steps of carcinogenesis. Many studies support the notion that innate immunity has a key role in the development, growth and prognosis of cutaneous malignant melanoma (MM), through the release of pro- and/or anti-inflammatory cytokines and tumour growth factors. The tumour environment in a major subset of cutaneous MM shows evidence of a T cell-infiltrated phenotype, but there is less known about the presence and the phenotype of other immune system cells. Response to immunotherapy is largely correlated with the presence of T cells in the tumour microenvironment, while the regulation exerted by stromal components such as macrophages and mast cells has been less investigated. In the current report, we review the recent literature, focusing our attention on the role of macrophages, dendritic cells, mast cells and natural killer cells in orchestrating MM progression, to better understand tumour immunobiology. The identification of new therapeutic targets and the application of approaches aimed at modulating crosstalk between immune and tumour cells, could have a crucial impact on immunotherapy and result in better clinical outcome. We hope this review will be helpful in cutaneous MM research.

Elastofibroma dorsi: a histochemical and immunohistochemical study.

Elastofibroma dorsi (ED) is considered a member of a heterogeneous group of benign fibrous (fibroblastic or myofibroblastic) soft-tissue tumors, frequently localized in the periscapular region in middle aged or older individuals. However, the pathogenesis of ED is still unclear and many authors believe that ED results from a reactive hyperproliferation of fibroblastic tissue, while others suggest that it may be a consequence of a mechanical friction. In our study, we examined 11 cases of ED using histochemical and immunohistochemical methods, in order to extend the knowledge about extracellular matrix composition and histopathogenesis of ED. From the results it appeared that stroma and interspersed spindle cells of ED were positive for both periostin and tenascin-C. Mast cells tryptase-positive were also abundant throughout the lesion. The perivascular distribution of periostin and tenascin-C, associated with the CD34 positivity, suggest that endothelial-mesenchymal transition events can account for neovascularization and production of fibroelastic tissue characteristic of elastofibroma. Our data obtained in endothelial cells cultures demonstrated that elastin production is higher when the status of confluence of the cells is low. So, we can assume that such a phenomenon is a characteristic of mesenchymal/endothelial cells CD34 positive, in which elastin production results to be inversely proportional to the vascular differentiation of cellular elements. In the light of these considerations, we think that a cancerous nature of ED is unlikely. Overall, our study report, for the first time, a detailed description of extracellular matrix composition in ED, suggesting that a mechanical strain-dependent reactivation of periostin and tenascin-C expression, as well as of elastin deposition, could be responsible for development of ED.

Negative transcriptional regulation of the human periostin gene by YingYang-1 transcription factor.

Periostin (POSTN), an osteoblast-specific secreted protein known to be associated with cell adhesion activity for bone formation and development by the epithelial cell-derived tumors, leads to a significant enhancement in angiogenesis and tumorigenesis. At present, little is known about the mechanisms underlying its transcriptional control either in physiological or neoplastic conditions. In this study we demonstrate that the ability of the human POSTN promoter to drive transcription mostly depends on the activity of YingYang-1 (YY1) zinc finger transcription factor. YY1, whose regulatory role in biology includes, besides transcriptional control, also chromatin remodeling, DNA damage repair and tumorigenesis, acts as a strong negative modulator of the POSTN expression. We retain that the identification of the functional role of YY1 in the transcriptional control of the human POSTN gene adds new insights in the studies focused on gene expression in normal and transformed cells.

Effect of a vitamin D3 analogue on keratinocyte growth factor-induced cell proliferation in benign prostate hyperplasia.

Prostate enlargement and function is under the dual control of androgens and intraprostatic growth factors. They regulate, in concert, prostate cell proliferation and apoptosis. An increased signaling of both growth factors and androgens are supposed to underlie benign prostate hyperplasia (BPH), one of the more common disorders of the aging male. Since, in clinical practice, androgen ablation resulted in a rather limited decrease in prostate volume, therapeutic strategies targeting intraprostatic growth factors are emerging. The activated form of vitamin D, vitamin D3, and some of its analogues have been described as potent regulators of cell growth and differentiation. In this study, we report the effects of one of these vitamin D3 analogues, 1,25-dihydroxy-16ene-23yne D3, or analogue (V), on the fate of isolated epithelial cells derived from patients with BPH. We essentially found that analogue (V), as well as vitamin D3, inhibited BPH cell proliferation and counteracted the mitogenic activity of a potent growth factor for BPH cells, such as keratinocyte growth factor (KGF). Moreover, analogue (V) induced bcl-2 protein expression, intracellular calcium mobilization, and apoptosis in both unstimulated and KGF-stimulated BPH cells. Since a short-term (5-min) incubation with analogue (V) reduced the KGF-induced tyrosine phosphorylation of a 120-kDA protein, corresponding to the KGF receptor, a rapid and direct cross-talk between these two molecules is suggested. Such a rapid effect of analogue (V), together with the transient induction of intracellular calcium waves, seems to indicate the partial involvement of a membrane, nongenomic receptor for vitamin D3. In conclusion, we demonstrated the antiproliferative and proapoptotic effect of analogue (V) in BPH cells and speculated on its possible use in the therapy of BPH.

Human GnRH-secreting cultured neurons express activin betaA subunit mRNA and secrete dimeric activin A.

OBJECTIVE: To evaluate the expression of activin betaA-subunit mRNA and the secretion of activin A in/from cultured GnRH-secreting neuronal cells cloned from human olfactory epithelium (FNC-B4), which showed biochemical and antigenic properties of GnRH-secreting neurons. DESIGN: FNC-B4 cells were cultured in basal and conditioned media. METHODS: Reverse transcription-polymerase chain reaction (RTR-PCR) evaluated the expression of activin betaA-subunit mRNA. By using a specific ELISA, dimeric activin A concentrations were measured in culture media, in the absence or presence of carvone or forskolin and with different doses of progesterone, GnRH, and estradiol. RESULTS: RT-PCR experiments performed on total RNA isolated from FNC-B4 cells, using specific primers for the activin betaA gene, showed a 787bp DNA band corresponding to the betaA gene. FNC-B4 cells secreted activin A, and the highest accumulation in conditioned medium was achieved after 3h culture: the addition of forskolin, but not of carvone, was able to stimulate the release of activin A from cultured neuronal cells (P<0.01). When progesterone or GnRH was added, a significant accumulation of activin A was observed (P<0.01), while estradiol administration did not significantly affect activin A secretion. CONCLUSION: To date, this is the only study, in an in vitro human model reporting, that GnRH-secreting neuronal cells expressed activin betaA-subunit mRNA, and released dimeric activin A in culture medium. The expression and secretion of activin suggests that in these cells activin A might exert its action by autocrine/paracrine mechanisms.

Expression and biological effects of endothelin-1 in human gonadotropin-releasing hormone-secreting neurons.

In a previous report, we demonstrated that in FNC-B4 cells, derived and characterized from a human fetal olfactory epithelium, both sex steroids and odorants regulate GnRH secretion. We now report the presence and biological activity of endothelin (ET)-1 in this GnRH-secreting neuronal cell. By in situ hybridization and immunohistochemistry, we found gene and protein expression of ET-1 and its converting enzyme ECE-1 in both fetal olfactory mucosa and FNC-B4 cells. The presence of authentic ET-1 in the conditioned media of FNC-B4 cells was further supported by combined RIAs and high-performance liquid chromatography studies. Experiments with radiolabeled ET-1 and ET-3 strongly indicated the presence of two classes of binding sites, corresponding to the ETA (16,500 sites/cell) and the ETB receptors (8,700 sites/cell). Functional studies, using selective analogs, indicated that these two classes of receptors subserve distinct functions in human GnRH-secreting cells. The ETA receptor subtype mediated an increase in intracellular calcium and GnRH secretion. Conversely, stimulation of the ETB subtype induced DNA synthesis and mitogen-activated protein kinase p44ERK1 expression. This is the first demonstration, in a human in vitro model, of a neuroendocrine role for ET-1 as regulator of GnRH-secreting neuron activity.

Sex steroids and odorants modulate gonadotropin-releasing hormone secretion in primary cultures of human olfactory cells.

Olfactory neurons and GnRH neurons share a common origin during development. In the nasal epithelia, GnRH neurons persist throughout fetal life and adulthood. The fate and function of these neurons in vivo have remained unknown. In a previous in vitro study, we isolated, cloned, and propagated primary long term cell cultures from the olfactory neuroepithelium of 8- to 12-week-old human fetuses. These cells expressed both neural proteins as well as olfactory genes and were responsive to odorant stimuli. We now report that these human olfactory cells also express the GnRH gene and protein. Combined HPLC and RIA studies have indicated that these cells release authentic GnRH in spent media. The release of GnRH was time dependent and was positively affected by sex steroids and odorants. Immunohistochemical data demonstrated the presence of sex steroid receptors in these cells. The presence of the alpha- and beta-subtypes of the estrogen receptor was also demonstrated by RT-PCR and Western blot analysis. When the cells were stimulated with increasing concentrations of 17beta-estradiol in the presence of a fixed concentration of progesterone (10(-7) mol/L), the combination of the two steroids induced a 3- to 4-fold increase in GnRH secretion. This stimulatory effect was completely blunted by tamoxifen. Neither 17beta-estradiol nor progesterone was effective when tested separately. Treatment with increasing concentrations of the odorant, l-carvone, induced a time- and dose-dependent dramatic increase in GnRH protein release (1000-fold increase) and gene expression. Repeated application of the stimulus resulted in a progressive lower responsiveness of the cells. To our knowledge, this is the first time that primary cell cultures from human fetal olfactory neuroepithelium have been shown to express and release GnRH. Our results also demonstrate that these cultures, which are sensitive to sex steroids and odorants, can be useful models in the study of the complex array of regulatory factors that finely tune GnRH secretion in humans.

Effect of 0.2 T static magnetic field on human neurons: remodeling and inhibition of signal transduction without genome instability.

We describe the effect of the static magnetic field generated by a 0.2 T magnetic resonance tomograph on a normal human neuronal cell culture (FNC-B4). After 15 min exposure cells showed dramatic changes of morphology: they formed vortexes of cells and exposed branched neurites featuring synaptic buttons. At the same time, thymidine incorporation and inositol lipid signaling were significantly reduced. Control (sham exposed) or non-neuronal cells (mouse leukemia, and human breast carcinoma cells) did not show any alteration following exposure. Endothelin-1 release from FNC-B4 cells was also dramatically reduced after 5 min exposure. However, PCR analysis of 12 DNA microsatellites selected as gauges of genome instability, did not reveal any alteration following exposure, thus ruling out a direct effect of the magnetic field on DNA stability. These data can be interpreted as a specific effect of the static magnetic field on human neuronal cells and are consistent with the induction of remodeling and differentiation; they demonstrate that fields below 0.5 T have significant biological effects on human neurons.

Basic fibroblast growth factor supports human olfactory neurogenesis by autocrine/paracrine mechanisms.

Throughout life, olfactory sensory neurons are renewed from a population of dividing stem cells. Little is known about the molecular mechanisms that regulate the activation, self-renewal and differentiation of olfactory neuronal precursors; however, evidence indicates that soluble mediators may play a central role in olfactory neurogenesis. To identify molecules that regulate olfactory self-renewal and differentiation, we have recently established, cloned and propagated in vitro primary long-term cell cultures from the human fetal olfactory neuroepithelium. Here we show that primary human olfactory neuroblasts synthesize and release biologically active basic fibroblast growth factor which, in turn, supports neuroblast growth by autocrine/paracrine mechanisms. The growth-promoting activity of basic fibroblast growth factor is dose dependent and is accompanied by morphological changes of the cells and by an increase in the expression of neuronal-related genes. These observations indicate that endogenous basic fibroblast growth factor participates in controlling olfactory self-renewal and suggest that this cytokine represents a key regulatory element of olfactory neurogenesis.

Presence and location of endothelin and endothelin-binding sites in human nasal mucosa under normal and metaplastic conditions.

The presence and site of production of endothelin-1 (ET-1) was investigated in biopsies obtained from the nasal mucosa of 10 healthy human subjects and 10 patients affected by chronic rhinitis. The presence and localization of receptors for ET-1 was also investigated. Bioptic fragments were examined by scanning electron microscopy. ET-1 was present in the vessels and in the respiratory epithelium of normal subjects, whereas in patients affected by epithelial metaplasia induced by chronic rhinitis, it was absent in the metaplastic epithelium and present in the endothelium and vascular wall. Receptors for ET (A- and B-receptor subtypes) were localized in the vessels of the nasal mucosa, both in normal and in pathological subjects. In particular, A-receptors were identified in the vascular wall, whereas B-receptors were mainly distributed in the endothelium. We suggest that ET-1 is involved in the homeostasis of nasal blood flow (shunting the blood toward the deep cavernous plexus and inducing mucosal swelling) by an autocrine and/or paracrine mechanism. Normal epithelium seems to be important in this mechanism, since it is able to produce ET. However, when pathologic conditions induce squamous or cuboidal metaplasia, the epithelium is no longer able to play this role.

Extracellular signal-regulated kinases modulate capacitation of human spermatozoa.

Recent evidence indicates the presence of p21 Ras and of a protein with characteristics similar to mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERKs), in mammalian spermatozoa, suggesting the occurrence of the Ras/ERK cascade in these cells. In the present study we investigated the subcellular localization of ERKs and their biological functions in human spermatozoa. Immunohistochemistry, immunofluorescence, confocal microscopy, and immunoelectron microscopy demonstrated localization of ERKs in the postacrosomal region of spermatozoa. After stimulation of acrosome reaction with the calcium ionophore A23187 and progesterone, ERKs were mostly localized at the level of the equatorial region, indicating redistribution of these proteins in acrosome-reacted spermatozoa. Two proteins of 42 and 44 kDa that are tyrosine phosphorylated in a time-dependent manner during in vitro capacitation were identified as p42 (ERK-2) and p44 (ERK-1) by means of specific antibodies. The increase in tyrosine phosphorylation of these proteins during capacitation was accompanied by increased kinase activity, as determined by the ability of ERK-1 and ERK-2 to phosphorylate the substrate myelin basic protein. The role of this activity in the occurrence of sperm capacitation was also investigated by using PD098059, an inhibitor of the MAPK cascade. The presence of this compound during in vitro capacitation inhibits ERK activation and significantly reduces the ability of spermatozoa to undergo the acrosome reaction in response to progesterone. Since only capacitated spermatozoa are able to respond to progesterone, these data strongly indicate that ERKs are involved in the regulation of capacitation. In summary, our data demonstrate the presence of functional ERKs in human spermatozoa and indicate that these enzymes are involved in activation of these cells during capacitation, providing new insight in clarifying the molecular mechanisms and the signal transduction pathways of this process.

Endothelin-1 is synthesized and biologically active in human epididymis via a paracrine mode of action.

In a previous study, we reported the presence of endothelin-1 and endothelin receptors in the human testis. We have now extended our investigations to the human epididymis. Since sperm appear to be immotile during their transit through the epididymis, it is conceivable that specific local factors promote smooth muscle contraction, enabling sperm transport. In this paper, we show that endothelin-1 mRNA and protein are readily detectable in the epithelial compartment of the human epididymis, and that endothelin converting enzyme- 1, which converts the precursor pro-endothelin-1 into active endothelin-1, is expressed in the epididymis, consistent with active processing of the prohormone. In addition, two classes of endothelin receptors were characterized and located in the muscle cells of the epididymis. These receptors correspond, in terms of affinity constants and capacity, to the previously characterized endothelinA and endothelinB receptor. These receptors appear to be biologically active and mediate the contractile activity of the epididymis in vitro. Our data suggest that endothelin-1, via a paracrine mode of action, may be responsible for sperm progression through this organ.

Progesterone stimulates p42 extracellular signal-regulated kinase (p42erk) in human spermatozoa.

Mitogen-activated protein kinases (MAPK), also known as extracellular signal-regulated kinases (ERKs) are cytoplasmic and nuclear serine/threonine kinases involved in signal transduction of several extracellular effectors. Recently, we have demonstrated that ERKs are present in spermatozoa and are involved in the regulation of the process of capacitation. We report here the effect of progesterone, a well-known inducer of the acrosome reaction in mammalian spermatozoa, on the immunolocalization, phosphorylation and activity of ERKs in capacitated human spermatozoa. We demonstrated that short-term incubation of spermatozoa with progesterone induces phosphorylation and activation of ERKs, resulting in redistribution of the proteins from the post-acrosomal region to the equatorial segment within the sperm head. To investigate the role of ERKs on the biological effects of progesterone, we used the MAPK cascade inhibitor PD098059, which strongly inhibited progesterone-induced activation of ERK-2. This compound did not inhibit progesterone-induced acrosome reaction, although it prevented redistribution of the enzyme to the equatorial region of the sperm head. These results suggest that the two processes, although temporally related, are independent. In conclusion, we provide new insight into the signal transduction pathways involved in the non-genomic action of progesterone in spermatozoa and suggest a possible involvement of ERKs in the process of fertilization.

Role of endothelin in the human craniofacial morphogenesis.

Human craniofacial morphogenesis is a complex biological event: it is mediated by several factors and different types tissue interaction. Recent studies on animal models have led to an improved understanding of human craniofacial malformations. In particular, the endothelins, peptides that are involved in various biological functions in many tissues and organs, have been shown to play a crucial role in the development of the first branchial-arch-derived structures in mice [Kurihara et al., Nature 368:703-710, 1994]. We previously reported the identification and localization of endothelin-1 (ET-1) and its receptors in human fetal jaw [Barni et al., Dev Biol 168:373-377, 1995]. In the present study, the gene expression of ET-1 and its receptors were demonstrated in human jaw from 11-12-week-old fetuses. By using in situ hybridization, mRNA for ET-1 was localized in the epithelial cells of the oral mucosa: mRNA for ET receptors (ETA and ETB subtypes) was expressed in the mesenchyme. In situ binding experiments confirmed the presence of ETA and ETB receptors in the cells involved in the osteogenesis of the mandible. Furthermore, ET-1 was able to stimulate thymidine uptake and the expression of the oncoprotein c-fos in the same cell types. Our results indicate that ET-1 may play a putative role in epithelium-mesenchyme interaction during human craniofacial morphogenesis. Our findings are in complete accord with those of the most recent works by Yanagisawa [Yanagisawa H et al., 1998] and Clouthier [Clouthier et al., Development 125:813-824, 1998]. They most probably confirm the primary role of ET-1 in the development of the pharyngeal arches.

Identification, characterization, and biological activity of endothelin receptors in human ovary.

We previously reported the expression of endothelin-1 (ET-1) in granulosa cells (GCs) of the human ovary and the presence of ET-1-like immunoreactivity in human follicular fluid obtained from women in an in vitro fertilization program. In follicular fluid, but not in plasma, the levels of ET-1-like immunoreactivity were higher in gonadotropin-stimulated vs. spontaneous cycles, suggesting hormonal regulation of follicular ET-1. To identify and characterize ET receptors in human ovary, we performed autoradiography, radioligand binding, and functional studies. Mathematical analysis of families of self- and cross-competition curves among [125I]ET-1, [125I]ET-3, and selective analogs indicates that human ovary expresses both subtypes of ET receptors, i.e. ETA and ETB receptors. However, the concentration of the ETB site was 100-fold lower than that of the ETA one. By using [125I]ET-1, we demonstrated that the density of binding sites in human ovary is not affected by the hormonal milieu (similar concentrations in normal cycling, postmenopausal, and combined oral contraceptive-treated women). In situ binding studies indicate that the majority of ETA and ETB receptors are expressed in the blood vessels of the ovary. In particular, ETA receptors are abundant in the ovulatory follicles and localized in the theca interna, in close proximity to the granulosa layer. Few GCs of the ovulatory follicle were specifically labeled. Conversely, in the rat ovary, used as a control, ETB receptors were predominantly expressed and localized in GCs. Accordingly, ETB receptors negatively regulated estrogen accumulation in rat GCs. In human granulosa-luteal cells, neither ET-1 (unselective ligand) nor ET-3 or sarafotoxin 6c (ETB ligands) affected estrogen or progesterone secretion. ET-1 was 2.5-fold more potent than noradrenaline in eliciting contraction of ovarian artery, acting through the ETA receptor. Our results indicate that in human ovary, at variance with rat ovary, the endothelin system is primarily involved in the regulation of ovarian blood flow and not steroidogenesis.

Human endometrial cancers contain follicle-stimulating hormone receptors: a preliminary study.

In order to investigate whether human endometrial cancers contain follicle-stimulating hormone (FSH) receptors, cancer fragments were collected at hysterectomy in six post-menopausal women affected by histologically confirmed endometrial malignancy. Cryostat sections were prepared for in situ binding investigation. Positive endometrial glandular cells were registered in all cancers; 125I-FSH binding sites seemed to increase with the increasing tumor grade. Our data demonstrated for the first time that human endometrial cancers contain specific FSH receptors.

Gene expression of endothelin-1, endothelin-converting enzyme-1, and endothelin receptors in human epididymis.

We have previously reported the presence of endothelin-1 (ET-1) and its receptors in the human testis. In the present study we extended our investigations to human epididymis. The rationale of our study originated from the fact that sperm appear to be immotile during their transit through the epididymis. Hence, it is conceivable that specific factors, unknown to date, are present in this organ, capable of inducing smooth muscle contractions, thus forcing sperm transport. In this paper it is shown that ET-1 messenger ribonucleic acid and protein are readily detectable in the epithelial compartment of the human epididymis, and that ET-converting enzyme-1, which converts the precursor pro-ET-1 into the active peptide ET-1, is expressed in the epididymis, thus indicating an active processing of the prohormone. In addition, two classes of ET receptors were characterized and located in the muscle cells of the epididymis. These receptors correspond, in terms of affinity constants and capacity, to the ETA and ETB receptors previously characterized. These receptors mediate the contractile activity of the epididymis in vitro, thus suggesting that ET-1 can be responsible of sperm progression through this organ, acting via a paracrine mode of action.

Characterization and localization of epidermal growth factor receptors in human developing tooth.

In this study the characterization and localization of Epidermal Growth Factor (EGF) receptor in human jaws, from fetuses ranging in age from 9 to 12 weeks is reported for the first time. Binding of [125I]-EGF to membranes obtained from three separate pools of fetal jaws was specific and time- and temperature-dependent. Analysis of the binding data revealed the presence of a single class of binding site with high affinity (Kd, 9.2 x 10(-10) mol/L) and mean binding capacity of 128 fmoles/mg protein. Immunohistochemical study demonstrated the presence of EGF receptors in the early developmental stages of human tooth. In the bud stage, the positivity was localized in the epithelial cells. In the cap stage, EGF receptors was present in the outer and inner enamel cells, in some cells of the stellate reticulum and in the mesenchymal papilla and follicle cells. In the bell stage, positivity for EGF receptors was present in the outer enamel epithelium, in some cells of the stellate reticulum and in the mesenchymal cells of the follicle and papilla. The presence of EGF receptors in the proliferative stages in both epithelial and mesenchymal cells suggests that EGF is involved in the early developmental stages of the human tooth germ.

Neuroblast long-term cell cultures from human fetal olfactory epithelium respond to odors.

Primary cell cultures from human fetal olfactory neuroepithelium have been isolated, cloned, and propagated in continuous in vitro culture for approximately 1 year. The two clones we report here synthesize both neuronal proteins and olfactory-specific markers as well as the putative olfactory neurotransmitter, carnosine. In addition, patchclamp experiments reveal that these cells are electrically excitable. Following exposure to a panel of aromatic chemicals one of the cell cultures shows a specific increase in intracellular cAMP, indicating that some degree of functional maturity is expressed in vitro. The results suggest that these cells originate from the "stem cell" compartment that gives rise to mature olfactory receptor neurons. These long-term cell cultures represent models that will be useful in studying the mechanism(s) of olfaction and the regulation of olfactory neurogenesis and differentiation.