Two canine Merkel cell tumours: immunoexpression of c‐KIT,E‐cadherin, β‐catenin and S100 protein

Canine Merkel cell tumours are rare neuroendocrine neoplasms that show a relatively benign biological behaviour when compared with their human counterparts. To date, little information is available on their immunohistochemical properties. This report describes the histopathological and immunohistochemical features of two such tumours. The tumours’ immunoreactivity profile was studied with respect to different cellular molecules including chromogranin A (CGA), neurone‐specific enolase (NSE), S100 protein, c‐KIT, the cytokeratins (CKs) detected by pancytokeratin (AE1/AE3) antibodies (i.e. high molecular weight CKs 1, 2, 3, 4, 5, 6, 10, 14, 15 and 16, and low molecular weight CKs 7, 8 and 19) and three markers proposed to correlate with increased malignancy in human tumours: E‐cadherin, β‐catenin and p63 protein. In both lesions, tumour cells were positive for cytokeratins, CGA, NSE, S100 and c‐KIT. No immunostaining was observed for p63 protein, and there was no loss or change in E‐cadherin or β‐catenin immunoexpression. These results suggest that the generally benign behaviour of canine Merkel cell tumours, when compared with their human counterparts, may be partly explained by the conservation of important intercellular adhesion molecules such as E‐cadherin and β‐catenin. Additionally, expression of S100 but not of the p63 protein suggests that these canine tumours present a trend towards neural, rather than basal, epithelial differentiation and do not readily compare with human Merkel cell tumours.     

Endometrial cytology of the normal bitch throughout the reproductive cycle

Samples for endometrial cytology were collected both from live bitches using transcervical uterine cannulation (n=48) and postmortem (n=10). The cells identified were endometrial epithelial cells, leucocytes, erythrocytes, spermatozoa, bacteria and cervical or vaginal cells. The endometrial epithelial cells varied morphologically throughout the reproductive cycle and had signs of degeneration during late dioestrus and during early and mid-anoestrus following dioestrus and postpartum. Neutrophils were the most common leucocytes observed during pro-oestrus, oestrus, dioestrus and early pregnancy, and lymphocytes during anoestrus. Macrophages were frequently seen during anoestrus. Erythrocytes were found in variable numbers at all stages of the reproductive cycle. Spermatozoa were detected in samples collected during oestrus and early pregnancy in bitches which had their last mating one to three days previously. Bacteria were commonly observed during pro-oestrus and oestrus. Cornified cervical or vaginal cells were present during pro-oestrus and oestrus. This study demonstrated that the numbers, types, proportions and morphology of ceils in endometrial cytological samples from normal bitches varied throughout the reproductive cycle.     

Pseudo-placentational Endometrial Cysts in a Bitch

Cystic alterations of the canine endometrium compromise reproduction and fertility of the bitch and may lead to life-threatening diseases, such as pyometra. Even without clinical evidence, reduction of the uterine lumen by cysts implicates disturbances during migration, nidation and development of the embryo. Several studies point to the high variability of morphology of uterine endometrial cysts but they lack detailed analyses of alterations. In the present study, immunohistochemistry was used to investigate the expression of steroid hormone receptors (oestrogen, progesterone), proliferation activity, inflammation and infection in the cystic affected tissue regions in contrast to the normal endometrium. Oestrogen receptor expression showed a high density of receptors throughout the surface epithelial cells, crypt epithelial cells, glandular epithelial cells and stromal cells of the normal endometrium as well as the cystic affected regions. Proliferation in the cysts was verified in the middle and basal cells of the crypts. Neither in the endometrium nor in the cysts inflammatory processes or evidence of infection could be detected. Furthermore, lectin histochemistry and electron microscopic methods showed that lectin binding patterns and cell morphology of internal epithelial lining and surface epithelium of the cysts can be used to characterize and distinguish different types of cystic alterations. Analogies between epithelial cells of the glandular chambers of the canine placenta and the cystic cellular morphology, steroid hormone receptor distribution as well as lectin binding patterns of the endometrial cysts, as observed in this study, suggest to introduce the term 'pseudo-placentational endometrial cysts'.     

Effects of Wnt10b on dermal papilla cells via the canonical Wnt/β‐catenin signalling pathway in the Angora rabbit

Wnt10b is a member of Wnt family that plays a variety of roles in biological functions, including those in the development of hair follicles. To investigate the effect of Wnt10b on hair growth in the Angora rabbit and to determine the underlying molecular mechanism, we cultured dermal papilla (DP) cells with exogenous Wnt10b in vitro. We observed the expressions of downstream critical gene β‐catenin and lymphoid enhancer‐binding factor 1 (LEF1) in Wnt/β‐catenin pathway. The levels of β‐catenin mRNA and protein were higher in the Wnt10b group of DP cells than in the Control group, and the mRNA level of LEF1 in the Wnt10b group was higher than in the Control group. Moreover, translocation of β‐catenin from cytoplasm to nucleus was activated in the Wnt10b group. Furthermore, the mRNA levels of the hair follicle‐regulatory genes, insulin‐like growth factor‐1 (IGF‐1) and alkaline phosphatase (ALP), and the protein activity of ALP was also upregulated in the Wnt10b group compared to their corresponding levels in the Control group. These data suggest that Wnt10b could activate the canonical Wnt/β‐catenin signalling pathway to induce DP cells in the Angora rabbit. In addition, the proliferation of DP cells was significantly promoted when cultured with Wnt10b for 48 and 72 hr, suggesting that Wnt10b plays a pivotal role in the proliferation and maintenance of DP cells in vitro. In conclusion, this study demonstrates that Wnt10b may promote hair follicle growth in Angora rabbit through the canonical Wnt/β‐catenin signalling pathway that promotes the proliferation of DP cells.     

Is Mechanically Induced Cystic Endometrial Hyperplasia (CEH) a Suitable Model for Study of Spontaneously Occurring CEH in the Uterus of the Bitch?

Cystic endometrial hyperplasia (CEH) was experimentally reproduced by the intraluminal insertion of a silk wire in the uterus of metestral bitches to obtain a model for the study of spontaneously occurring CEH in the bitch. This mechanically reproduced CEH corresponded histologically very well to spontaneously occurring CEH. With respect to sex hormone receptor expressions, however, there was no similarity. In the mechanically induced CEH, progesterone receptor expression was reduced in the epithelial cells (surface epithelium and endometrial glands) and slightly increased in the stromal fibroblasts and myometrium as compared with the normal metestrus uterus. The oestrogen receptor expression in mechanically induced CEH was reduced in the epithelial cells (surface epithelium and endometrial glands) and more or less unchanged in the stromal fibroblast and myometrium as compared with the normal metestrus uterus. This is in contrast to the increased sex hormone receptor expressions in all uterine cell types, observed in spontaneously occurring CEH. The mechanically induced CEH model corresponds immunohistochemically more to early placentation and to pyometra in the bitch. Thus this experimental CEH model is not suitable for pathogenic studies of spontaneously occurring CEH in the bitch.     

Seasonal or pathological findings? Morphofunctional characteristics of the equine endometrium during the autumn and spring transition

The deep anoestrous phase in winter is part of the anovulatory season in mares and is bordered by the autumn and spring transitional periods (ATP/STP). To define an annual time span for effective prognostic biopsy sampling, the aim of this study was to provide a morphofunctional characterization of the endometrium during ATP and STP. To outline both transitional periods, endometrial specimens were taken in September, October and November (n = 76) as well as February, March and April (n = 184) with the requirement of a detailed clinical documentation. Tissue samples were examined histologically with special emphasis on the functional endometrial morphology. Additionally, an immunohistochemical evaluation was performed on selected specimens regarding the expression of oestrogen receptor α, progesterone receptor and Ki67‐antigen. An absent to low endometrial activity was ascertained in more than 60% of all specimens from late October onwards, whereas a comparably lacking or low activity in STP was observed until early April. Approximately 30% (ATP) to 22% (STP) of all samples exhibited a predominantly “irregular” endometrial differentiation. During the transitional periods, the clinically evaluated ovarian status (transrectal palpation, transrectal ultrasonography and/or serum progesterone and oestrogen analyses) and the endometrial functional morphology were in accordance with approximately 70% of all cases. The expression of steroid hormone receptors and Ki67‐antigen was generally low. Given that endometrial maldifferentiations were frequently found during ATP and STP, its occurence might display a characteristic and physiological feature of the transitional periods. Regarding the functional endometrial morphology, a diagnostic biopsy sampling should therefore be performed between late April and before September.     

Histopathologic and Immunohistochemical Exam in One Case of Canine Endometrial Adenocarcinoma

Canine endometrial carcinomas are rare, and mostly occur in geriatric bitches. In this work, the uterus of a 10‐year‐old female Boxer evidencing an endometrial carcinoma on the body of the uterus was used to describe the histopathological features of the tumour and to study its immunophenotype. In this work, a panel of immunomarkers (cytokeratins AE1/AE3 and 14, vimentin, CD10 and Ki‐67) was applied to the endometrial carcinoma to establish the staining patterns indicative of the tumour agressiveness and cellular differentiation. Additionally DNA ploidy was also performed. In this case, the tumour showed papillar pattern, with large pleomorphic, anaplastic cells and also some aberrant multinucleated and giant cells. In some areas of the tumour, it was also observed cytotrophoblastic‐like cells outlining the papillae. Cytokeratin AE1/AE3 expression was detected in the luminal neoplasic cells. Cytokeratin 14 positivity was sporadic and irregular, and was observed mainly in the luminal epithelium. Only stromal and aberrant cells showed a positive staining to vimentin. Positive membranous staining to CD10 was evidenced by clear epithelial, cytotrophoblastic‐like cells at the tumour surface but not by the stromal cells. The mitotic and Ki‐67 indices were low, suggestive of a weak aggressiveness of the tumour. The multinucleated and giant cells evidenced a positive immunostaining to CK AE1/AE3, and CD 10; its positivity to vimentin was sporadic. This study aims to contribute to the advancement of the knowledge in canine endometrial carcinoma immunophenotype.     

Isolation,proliferation and characterization of endometrial canine stem cells

In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT‐PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue‐derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue‐derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the “Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy” for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue‐derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow‐derived MSC in dog.     

The Use of Epostane in an Attempt to Reproduce Cystic Endometrial Hyperplasia in the Bitch

The aetiology and pathogenesis of spontaneous cystic endometrial hyperplasia (CEH) in the bitch is not yet completely understood. Recent research based on the expression of uterine sex hormone receptors in spontaneous cases of CEH suggested that a temporary progesterone deficiency during late oestrus-early metoestrus may be responsible for the onset of CEH development. In the present study a temporary progesterone deficiency during late oestrus–early metoestrus was experimentally created using an inhibitor of progesterone synthesis, epostane. At day 49 of metoestrus, there was a significant reduction in the size of the uterine wall, mainly due to endometrial atrophy, and there was also a significant increase in the mucus-filled uterine lumen in the bitches that had been treated with epostane compared to the control bitches. No significant differences in the expression of sex hormone receptors was observed between the two groups. As no CEH developed in the epostane-treated bitches, an additional oestrogenic stimulus may be required to stimulate endometrial proliferation. Therefore, it is suggested that deficient luteinization of the corpus luteum may be the trigger in the pathogenesis of CEH, as the secretion of varying amounts of sex steroids depends on the degree of luteinization.     

荷斯坦奶牛子宫内膜上皮细胞的体外培养

为建立和完善奶牛子宫内膜上皮细胞的体外培养技术,采用组织培养和胶原酶消化相结合的方法,对中国荷斯坦奶牛的子宫内膜上皮细胞进行了原代分离培养,经胰酶差时消化进行纯化,并以免疫组化法检测角蛋白表达。结果显示,所采用的方法可高效获得呈铺路石样的上皮细胞,角蛋白表达呈阳性,第2~8代的传代细胞在形态特征和增殖状态上保持着与原代细胞相近的生物学特征。     

Cyclical endometrial steroid hormone receptor expression and proliferation intensity in the mare

The aims of this study were to investigate the steroid hormone receptor expression and the proliferation intensity during the equine endometrial cycle by immunohistological methods, established for routine examination of formalin-fixed, paraplast-embedded specimens. Endometrial biopsy specimens were obtained during one cycle from 7 mares. In comparison with the blood steroid hormone levels the quantity and distribution of oestrogen (ER) and progesterone receptors (PR) and the proliferation marker Ki-67 antigen expression were investigated. Rising 17beta-oestradiol concentrations in preoestrus induce a synchronous expression of ER, PR and Ki-67 antigen in stromal cells. In the early dioestrus 17beta-oestradiol levels decrease and progesterone levels reach their maxima. This correlates with an intense proliferation activity and the highest hormone receptor expression in epithelial cells. In accordance to the morphological features of asynchronous glandular differentiation in fibrotic areas (endometrosis) their epithelial hormone receptor expression is out of phase.     

Role of progestin-induced mammary-derived growth hormone in the pathogenesis of cystic endometrial hyperplasia in the bitch

Endogenous progesterone and synthetic progestins may induce hypersecretion of growth hormone (GH) of mammary origin, hyperplastic ductular changes in the mammary gland, and the development of cystic endometrial hyperplasia (CEH) in dogs. It was investigated whether progestin-induced mammary GH plays a role in the pathogenesis of CEH in the bitch. During 1 year, bitches with surgically excised mammary glands and healthy control bitches received medroxyprogesterone acetate (MPA). Before and after MPA treatment, uterine and mammary tissues were collected for histological, immunohistochemical, and RT-PCR examination. After MPA administration, the mammary tissue in the control dogs had differentiated into lobulo-alveolar structures and CEH was present in all uteri of both dog groups. In the MPA-exposed mammary tissue of the control dogs, GH could only be demonstrated immunohistochemically in proliferating epithelium. After treatment with MPA the dogs of both groups had immunohistochemically demonstrable GH in the cytoplasm of hyperplastic glandular uterine epithelial cells. RT-PCR analysis of the mammary gland tissue after MPA administration demonstrated a significant higher GH gene, and lower GHR gene expression than before treatment. In the uterus, the expression of the gene encoding for GH was significantly increased in the mastectomized dogs, whereas in the control dogs the expression of the gene encoding for insulin-like growth factor-I had significantly increased with MPA administration. MPA treatment significantly down regulated PR gene expression in the uterus in both dog groups. These results indicate that progestin-induced GH of mammary origin is not an essential component in the development of CEH in the bitch.     

Expression of Steroid Hormone Receptors in the Genital Structures of a True Hermaphrodite Pug Dog

Hermaphroditism is a rare and a not well‐understood disordered sexual development (DSD) in dogs. The objective of the study was to analyse the sex steroid hormone receptor (STHR) expression patterns in the internal genital structures, because the responsiveness of the different tissue types to the steroid hormones may have a key role in pathological alterations based on DSDs. Furthermore, the adhesion molecule β‐catenin was investigated by means of immunohistochemistry because of its important role in development, tissue integrity and disease. Molecular sexing was performed via PCR targeting DBX/DBY genes to identify the pug dog as a true XX hermaphrodite. The portions of uterine tissue revealed comparable expression patterns for STHRs as investigated in normal female reproductive tissue. In the male parts, β‐catenin showed strong expression in the Sertoli cells of the seminiferous tubules; this was in contrast to normal testicular tissue. Likewise, the layers of smooth muscle actin‐positive cells surrounding the seminiferous tubules were reduced in the hermaphrodite. The results of this study deepen the knowledge of tissue characteristics in a hermaphrodite dog and highlight the importance of early diagnosis because the STH responsiveness in maldeveloped reproductive tissue might lead to serious problems for the dog.     

Immunohistochemical and qPCR determination of the expression and serum level of anti‐Müllerian hormone in pre‐pubertal,intact and ovarian remnant syndrome detected bitches

The aim of this study was to determine the serum concentrations, ovarian presence and expression of anti‐Müllerian hormone (AMH) in pre‐pubertal, bitches with signs of ovarian remnant syndrome (ORS) and intact bitches. In addition, we aimed to verify the suitability of serum AMH concentrations for diagnostic purposes in sterilized bitches and/or in suspected cases of ORS in the field of veterinary medicine. For this purpose, 36 healthy female dogs divided into six groups: proestrus, oestrus, dioestrus, anoestrus, pre‐pubertal and ORS. Serum AMH concentrations were determined by electrochemiluminescence immunoassay, and ovarian presence and distribution of AMH was confirmed by immunohistochemical and qPCR techniques. According to the results of qPCR, while the expression values of AMH were at the highest concentrations in the proestrus and oestrus, there was a statistically significant decrease in these values at the later stages of the cycle (p < 0.05). According to hormone analysis, the serum AMH values of the ORS group had decreased significantly compared with the proestrus and oestrus (p < 0.05). Although serum AMH levels of ORS group were increased compared with anestrus and pre‐pubertal groups, this increase was statistically non‐significant (p > 0.05). Immunohistochemically, AMH expression was first observed in the granulosa cells of primordial follicles in folliculogenesis. Expression values were the highest in the proestrous and oestrus groups, but values from bitches in later stages of the cycle were statistically significant decrease in comparison with these groups (p < 0.05). As a result, AMH concentration and expression were found to be higher in proestrus and oestrus than in other periods (p < 0.05). In addition, the measurable level of AMH concentration in bitches with ORS is an indication that it can be used in the diagnosis of ORS.     

Epithelial‐to‐mesenchymal transition: immunohistochemical investigation of related molecules in canine cutaneous epithelial tumours

Background –  Epithelial‐to‐mesenchymal transition (EMT) is a multistep process, important in tumour invasion and metastasis, characterized by loss of epithelial markers, redistribution of β‐catenin and gain of mesenchymal markers. Hyposthesis/Objectives –  Our aim was to investigate the immunohistochemical aberrant expression of cytokeratin, vimentin, survivin and heat shock protein 72 (Hsp72) in canine cutaneous epithelial tumours, to understand the association of expression of these molecules with features of malignancy and their role in the EMT phenotype. Methods –  Ten canine squamous cell carcinomas (SCCs; one with lymph node metastasis), 30 canine hair follicle tumours (six pilomatricomas, eight infundibular keratinizing acanthomas, six trichoepitheliomas and 10 trichoblastomas) and five normal skin samples were investigated by immunohistochemistry using specific anti‐vimentin, ‐cytokeratin, ‐survivin and ‐Hsp72 antibodies. A semi‐quantitative method was used to analyse the results, as follows: 0 to <5%; ≥5 to <10%; ≥10 to <25%; and ≥25% of positive cells. Immunofluorescence was performed to investigate survivin–vimentin and survivin–Hsp72 colocalization in selected SCCs. Results –  In malignant hair follicle tumours and SCCs, a reduced intensity of cytokeratin and increased survivin and Hsp72 expression were observed. In SCCs, loss of cytokeratin expression and vimentin immunolabelling, suggestive of the EMT phenotype, were evident in <5% of neoplastic cells in the front of tumour invasion. In the same areas, strong nuclear survivin and cytoplasmic Hsp72 staining was evident, often colocalizing. Only a few neoplastic cells in the front of tumour invasion showed vimentin–survivin colocalization. Conclusions and clinical importance –  A possible simultaneous involvement of survivin and Hsp72 in tumour invasion and the multistep process of EMT of cutaneous epithelial tumours of dogs is suggested.     

Morphological Changes of the Endometrial Epithelium in the Bitch during Metoestrus and Anoestrus

Although cyclic changes of the endometrium in dogs involving both stromal and glandular compartments have been described, the fate of the surface epithelium after progressive growth and secretion is still unclear. In the present study, uteri of 43 healthy bitches in metoestrus and anoestrus were examined macroscopically and histologically. Tissue biopsies were taken from three different locations (cranial and middle parts of uterine horns and bifurcation). The stage of the oestrous cycle was determined by evaluation of progesterone and oestradiol levels in plasma hormone and was also confirmed clinically. Crypts formed in the luteal phase were covered with a columnar epithelium which gradually underwent fatty degeneration. In addition, the stromal part of the crypts disappeared and finally, in early anoestrus, epithelial sheaths desquamated and shed off into the uterine lumen. The surface epithelium was replaced by new cuboidal cells proliferating and migrating from the glandular openings. These findings were confirmed by oil red O staining and immunohistochemical detection of proliferation with Ki‐67 marker.     

Immunohistochemical Expression of Cyclooxygenase‐2 (COX‐2) in Feline Endometrial Adenocarcinoma and in Normal and Hyperplastic Endometria

Cyclooxygenase‐2 (COX‐2) is overexpressed in several human and animal neoplasms, including the human endometrial carcinoma. It has been suggested as a prognostic marker and a potential therapeutic target. This study aimed to (i) clarify histological aspects of feline endometrial adenocarcinomas (FEA) of the papillary serous type and (ii) characterize COX‐2 immunohistochemical expression in normal, hyperplastic and neoplastic endometrium in this species. Archived paraffin‐embedded tissue samples of 33 FEA, eight cystic endometrial hyperplasias (CEHs) and 21 samples of normal, healthy endometrium in the follicular (FS; n = 10) and luteal (LS; n = 11) stages were evaluated. Histological evaluation of haematoxylin and eosin‐stained sections of the FEA revealed a papillary proliferation of neoplastic cells of serous type, accompanied by clear and multinucleated cells. Other architectural arrangements mainly included solid and tubular growth. Randomly distributed areas of necrosis within the tumours were commonly observed. Invasion of the myometrium, of the serosa and of the vascular and/or lymphatic vessels was not constant features. The mean number of mitoses was higher in FEA compared to non‐neoplastic endometrium. COX‐2 scores were lower in FEA (p = 0.003) and CEH (p = 0.05) when compared to normal epithelium (NE). The loss of the membrane apical reinforcement in epithelial cells was observed in FEA samples, which was accompanied by the dislocation of COX‐2 labelling into the cytoplasm and the perinuclear area; in contrast, in epithelial cells in the healthy and hyperplastic endometria, the immunoreaction showed the characteristic pattern of apical membrane reinforcement, suggestive of the membrane polarization. COX‐2 epithelial scores were higher in the FS than in the LS. No differences were found in stromal COX‐2 expression between normal, CEH and FEA groups, but it was higher in the LS than in the FS. In summary, loss of COX‐2 compartmentalization in neoplastic epithelial cells might be one of the molecular events underlying endometrial carcinogenesis.     

Growth hormone and lactogenic hormones can reduce the leptin mRNA expression in bovine mammary epithelial cells

Leptin mRNA is expressed in not only adipocytes but also mammary epithelial cells and leptin protein is present in milk. Although milk leptin is thought to influence metabolism or the immune system in neonates, there is little information about the regulation of leptin expression in mammary epithelial cells. We examined the effect of growth hormone (GH) and/or lactogenic hormone complex (DIP; dexamethasone, insulin and prolactin) on leptin mRNA expression in mammary epithelial cells. We used a bovine mammary epithelial cell (BMEC) clonal line, which was established from a 26-day pregnant Holstein heifer. We confirmed that the mRNA was expressed in BMECs and the expression was significantly reduced by GH and/or DIP, when the cells were cultured on both plastic plates and cell culture inserts at days 2 and 7 after stimulation with lactogenic hormones. GH and/or DIP significantly increased level of alpha-casein mRNA in BMECs after 7 days on the cell culture inserts, but no mRNA expression was detected at day 2. GH and DIP significantly stimulated the secretion of alpha-casein from BMEC on cell culture inserts at 3.5 and 7 days. However, neither alpha-casein mRNA expression nor secretion was observed in the BMECs cultured on plastic dishes, even in the presence of GH or/and DIP. These results indicate that GH and DIP can directly reduce leptin mRNA expression in both undifferentiated and functionally differentiated bovine mammary epithelial cell.