Artificial intelligence in automated classification of rat vaginal smear cells.

Microscopic examination of vaginal smears has been used routinely to determine the stage of the estrous cycle of female rats in reproductive research. The stage of the estrous cycle is based on relative counts of nucleated epithelial cells, cornified epithelial cells and leukocytes. The purpose of this project was to explore automation of vaginal smear analysis using image processing and artificial intelligence techniques. A fully connected back-propagation neural network was used to locate all potential objects in a digitized scene. A unique algorithm was then employed to center a subsequent sampling box to collect pixel intensity values from the red and green components of each image. A final neural network was used in the classification of cell type. Neural networks were used because of their ability to generalize among input patterns and to tolerate extraneous noise due to variations in staining artifacts and aberrant illumination of the microscope field. This preliminary cell diagnosing system not only provides the basis for the fully automated system but also provides a method by which many other cytologic image processing problems can be automated.     

Clonogen number and radiosensitivity in rat thyroid follicles

A technique has been developed for transplanting whole thyroid follicles into the fat pads of recipient thyroidectomized rats to assess the ability of the follicular cells to proliferate and form colonies, i.e., clusters of new follicles. Of the transplanted follicles, 80-90% formed follicle clusters, indicative of the presence of transplanted follicles not containing colony-forming cells (clonogens) or some reproducible degree of transplantation trauma. The initial number of clonogens per regenerative follicle was calculated from data from split-dose experiments to be 3.3 +/- 1.5, and their sensitivity was characterized by a D0 value of 350 +/- 95 cGy. Three clonogens among about 80 epithelial cells per regenerative follicle and the 10-20% of nonregenerative follicles represent an overall colony-forming efficiency of about 3%. This is similar to the value of 2-3% reported by others using single-cell transplantation techniques.     

Histochemical characterization of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, Amitraz and Oxytetracycline

A number of techniques were employed to assess cell death induced in honeybee larvae midgut after per os inoculation of bacterium Paenibacillus larvae var. larvae, the causative agent of American foulbrood disease, and separately with acaricide Amitraz and antibiotic Oxytetracycline. In honeybee larvae exposed to Amitraz, which demonstrates both necrosis and apoptosis, cell death was found in 82% of midgut columnar and in 50% of regenerative epithelial cells, 24 h after treatment. Cell death reduced to 36% in the epithelial cells, 48 h after treatment. In Oxytetracycline-treated larvae, cell death was identified in 40% of midgut epithelial cells, 24 h after inoculation and increased to 55% over the next 24 h. In Paenibacillus -infected larvae, all midgut epithelial cells died. Using ApopTag (Oncor) to label the multiple DNA ends generated by DNA fragmentation showed programmed cell death in 49% of columnar midgut cells 24 h after Amitraz application. Cell death was reduced to 9% over the next 24 h. Our data indicate that cell death could be identified and quantified in situ, using TUNEL techniques. This study also shows that the acaricide Amitraz is a trigger for programmed cell death in the midgut epithelial cells of honeybee larvae, unlike Paenibacillus which induces necrosis only. The data show that immunohistochemical methods are useful for studying in situ tissue pathology, and indicate possibilities for monitoring the effects of infective and chemical environmental stressors on cell death in honeybee larvae tissue.     

CD40 on adult human airway epithelial cells: expression and proinflammatory effects

CD40/CD40 ligand interaction is an important pathway for B and T cell cooperation and function; functional CD40 molecules have recently been found on nonhematopoietic cells. We detected CD40 in vivo on normal human respiratory epithelial cells and showed that its expression is increased on inflamed airway epithelium. Subsequently, we analyzed its expression and function on primary cultures of human airway epithelial cells. Our data show that CD40 is up-regulated by IFN-beta and IFN-gamma, its ligation increases the surface expression of CD54 and CD106 and it may stimulate the release of IL-6 and IL-8. The use of Janus kinase 3 (JAK3) and NF-kappaB inhibitors suggests that both basal and CD40-induced release of the two cytokines is JAK3-dependent. Using colocalization techniques, we revealed the existence of CD40/JAK3 and CD40/TNFR-associated factor 2 interplay. The extent of these interactions may be partial (2-40% of the cells) or massive (80-90% of the cells) in cultured cells. Stimulation via CD40 causes a significant increase in the number of cells expressing colocalization only in the cultures displaying low frequency of initial colocalization. Thus, airway epithelial cells, activated by CD40, may behave as effector cells of the inflammation process and should be considered priority targets for anti-inflammatory therapy. This work identifies CD40 and the correlated JAK3 signaling molecule as potential molecular targets to block the inflammatory functions of epithelial cells.     

A new culture medium for human skin epithelial cells

Summary A new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed, by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1∶1 mixture of these two media with 10% horse serum supplement was found to promote epithelial cell outgrowth from human skin explants. The buffer system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10% horse serum is capable of initiating and sustaining larger epithelial cell outgrowths. Explants in serum-supplemented NCTC 168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of fibroblasts after only 19 to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial growth factor, hydrocortisone and insulin at 5 ng per ml, 4 μg per ml and 5 μg per ml, respectively did not appreciably enhance the growth of the epithelial cells.     

Rapid isolation of viable circulating tumor cells from patient blood samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of >50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.     

The determination of the cytodifferentiation of the adenohypophysis in embryonic development

This is a review of the literature and author's own data on determination of various cell types of adenohypophysis during embryonic development. Recent studies using techniques of organ culture and immunohistochemistry have established the time of determination of glandular cells of adenohypophysis. It has been shown in rat embryos that the direction of differentiation of all major cell types of adenohypophysis is programmed late during the development of the epithelial anlage of this organ. Similar data as concerns somatotropic and prolactin cells have been obtained on chick embryos. Chick embryos possess regional type of determination of prolactin and somatotropin-containing cells in the anlage in correspondence with their location in definitive adenohypophysis.     

Quantification of collagen synthesis by cultured human glomerular cells

This study examines the amount of total collagen and its different fractions synthesized by cultured human glomerular epithelial and mesangial cells. Two quantitative techniques were used, namely estimation of proline (Pro) plus hydroxyproline (Hyp) present in the collagenase-sensitive proteins and ELISA or RIA of the different types of collagen. In addition, the pattern of collagen synthesis for both cell types was further examined using immunofluorescence methods and polyacrylamide gel electrophoresis. Glomerular epithelial cells synthesized mainly type IV collagen and it was, for the better part, cell-associated. Mesangial cells synthesized approx. 4-times more collagen than epithelial cells. Type I collagen was predominant, but there were also type IV and III collagens. Secreted and cell-associated collagens were present in roughly equivalent amounts. In both cell lines 10-14% of the newly synthesized collagen had been degraded within the cells. These results provide quantitative data on collagen synthesis by human glomerular cells in vitro and represent the first necessary stage before studying which factors mediate the development of glomerular sclerosis.     

14-3-3sigma is down-regulated in human prostate cancer

The 14-3-3sigma is a negative regulator of the cell cycle, which is induced by p53 in response to DNA damage. It has been characterized as an epithelium-specific marker and down-regulation of the protein has been shown in breast cancers, suggesting its tumor-suppressive activity in epithelial cells. Here we demonstrate that 14-3-3sigma protein is down-regulated in human prostate cancer cell lines, LNCaP, PC3, and DU145 compared with normal prostate epithelial cells. Immunohistochemical analysis of primary prostate cells shows that the expression of 14-3-3sigma protein is epithelial cell-specific. Among prostate pathological specimens, > 95% of benign hyperplasia samples show significant and diffuse immunostaining of 14-3-3sigma in the cytoplasm whereas < 20% of carcinoma samples show positive staining. In terms of mechanisms for the down-regulation of 14-3-3sigma in prostate cancer cells, hypermethylation of the gene promoter plays a causal role in LNCaP cells as 14-3-3sigma mRNA level was elevated by 5-aza-2'-deoxycytidine demethylating treatment. Intriguingly, the proteasome-mediated proteolysis is responsible for 14-3-3sigma reduction in DU145 and PC3 cells, as 14-3-3sigma protein expression was increased by treatment with a proteasome inhibitor MG132. Furthermore, tumor necrosis factor-related apoptosis-inducing ligand enhances 14-3-3sigma gene and protein expression in DU145 and PC3 cells. These data suggest that 14-3-3sigma expression is down-regulated during the neoplastic transition of prostate epithelial cells.     

Intrinsic epithelial cells repair the kidney after injury

Understanding the mechanisms of nephron repair is critical for the design of new therapeutic approaches to treat kidney disease. The kidney can repair after even a severe insult, but whether adult stem or progenitor cells contribute to epithelial renewal after injury and the cellular origin of regenerating cells remain controversial. Using genetic fate-mapping techniques, we generated transgenic mice in which 94%-95% of tubular epithelial cells, but no interstitial cells, were labeled with either beta-galactosidase (lacZ) or red fluorescent protein (RFP). Two days after ischemia-reperfusion injury (IRI), 50.5% of outer medullary epithelial cells coexpress Ki67 and RFP, indicating that differentiated epithelial cells that survived injury undergo proliferative expansion. After repair was complete, 66.9% of epithelial cells had incorporated BrdU, compared to only 3.5% of cells in the uninjured kidney. Despite this extensive cell proliferation, no dilution of either cell-fate marker was observed after repair. These results indicate that regeneration by surviving tubular epithelial cells is the predominant mechanism of repair after ischemic tubular injury in the adult mammalian kidney.     

Increased abundance of a normal cell mRNA sequence accompanies the conversion of rat mammary cuboidal epithelial cells to elongated myoepithelial-like cells in culture.

Rat mammary cuboidal epithelial cell lines in culture convert to elongated myoepithelial-like cells. This conversion is accompanied by the appearance of a 9,000 molecular weight acidic polypeptide (p9ka), abundant in the elongated convertants, but which is hardly detectable in the cuboidal epithelial cells. A cDNA library corresponding to a low-molecular-weight fraction of poly(A)- containing RNA from a myoepithelial-like cell line, has been constructed. Recombinant plasmids containing cDNA complementary to p9ka mRNA have been identified by hybrid-selected translation. The mRNA for p9ka has been identified by Northern blotting and is found to be at least five-times more abundant in cultured myoepithelial-like rat mammary cells when compared to the cuboidal epithelial cells. This cytoplasmic mRNA sequence, which is present in increased abundance in cultured mammary myoepithelial-like cells, is also present, at lower levels, in normal rat tissues, including the mammary glands.     

Adrenomedullin and mucosal defence: interaction between host and microorganism

Many surface epithelial cells express adrenomedullin (AM) and it is postulated that it may have an important protective role. This peptide has many properties in common with other cationic antimicrobial peptides including the human beta-defensins. Antimicrobial activity against members of the human skin, oral, respiratory tract and gastric microflora has been demonstrated. Both pathogenic and commensal strains of bacteria are sensitive; Gram-positive and Gram-negative bacteria being equally susceptible. No activity against the yeast Candida albicans was observed. Minimum inhibitory and minimum bacteriocidal concentrations range from 7.75 x 10(-4) to 12.5 and 0.003 to >25.0 microg ml(-1), respectively. On exposure of oral, skin and gastric epithelial cells to whole cells and culture supernatants from bacteria isolated from these sites an increase in AM peptide and gene expression has been observed. No upregulation was detected with C. albicans. In cultured cells and an animal infection model increased AM peptide and gene expression has been demonstrated using immunohistochemical and in situ hybridization techniques. These collective findings suggest that AM represents a new category of antimicrobial peptide, which contributes to the mucosal host defence system.     

Enhanced synthesis of basement membrane proteins during the differentiation of rat mammary tumour epithelial cells into myoepithelial-like cells in vitro

Rama 25, an epithelial cell line obtained from a dimethylbenzanthracene-induced rat mammary tumour differentiates spontaneously in culture forming elongated myoepithelial-like cells. The elongated cells form multilayered ridge structures from which cultures of elongated cells, relatively uncontaminated by epithelial cells, can be obtained. By using immunofluorescence techniques, both the elongated cells and the cells in ridges, but not undifferentiated Rama 25 cells, have been demonstrated to synthesize three basement membrane proteins, laminin, type IV collagen, and fibronectin. The identity of these basement membrane proteins has been confirmed by immunoprecipitation. These proteins appear to be located in a fibrillar extracellular matrix. We suggest that the ability to synthesize basement membrane proteins by mammary epithelial cells in vitro on plastic is a characteristic of myoepithelial-like cells.     

Establishment of primary cultures of rat and human parotid epithelial cells for transfection experiments

Summary The molecular mechanisms that regulate the synthesis of salivary proteins are unknown. The paucity of homogeneous cell populations of parotid acinar cells has become a limiting factor for such a study. Therefore, the establishment of immortalized clones of acinar cells is essential. This study has established primary cultures of rat and human parotid epithelial cells that are suitable for transfection with plasmid vectors, pSV2, pSV3, and pSV5 to generate immortalized cells in vitro. Among various techniques used, the rat and human parotid tissue or cellular clumps when restrained in chicken plasma clot allowed the outgrowth of epithelial cells that maintained epithelial cell morphology for over 4 wk. However, the initial growth requirements for rat and human parotid cells were different. The presence of 10% heat inactivated fetal bovine serum in supplemented MCDB-LB medium was essential for the outgrowth of rat parotid epithelial cells, but this was not needed for the outgrowth of human parotid epithelial cells. The growth of both human and rat parotid epithelial cells can be maintained in serum-free supplemented MCDB-LB. These primary cultures contained amylase-producing cells as demonstrated by immunofluorescent technique, and they were transfected with pSV2, pSV3, and pSV5 using primarily the calcium phosphate-DNA co-precipitation technique. After initial extensive cell death, many cells with epithelial cell morphology survived.     

Histochemical Method for Detecting Nitric Oxide Synthase Activity in Cell Cultures

NADPH diaphorase histochemistry has been used extensively for detecting nitric oxide synthase (NOS) activity in various cell types including neuronal cell bodies, vascular endothelium, cells of the immune system and epithelial cells. The use of the diaphorase technique in cell cultures to study the induction of NOS has not been investigated. In this paper we report the use of diaphorase histochemistry as a good marker for the detection of NOS activity in cultured cells. This technique can be used in conjunction with other established techniques to determine the presence and activity of NOS in cultured cells.     

Growth regulation of the mammalian ocular lens by vitreous humor.

Experiments were performed in our laboratory to study the effects of a mammalian 8 kD vitreous humor (VH) factor on the DNA synthesis and mitosis of the epithelial cells of organ cultured rabbit lens. The 8 kD polypeptide factor was purified from mature rabbit vitreous humor by liquid chromatography. Proliferative activities of the epithelial cells of organ cultured lenses were stimulated by 3% rabbit serum. The data from our experiments depicted that the 8 kD VH factor effectively inhibits DNA synthesis and mitosis by the epithelial cells of the organ cultured lens. Our experiments also showed that this 8 kD VH factor can maintain its growth inhibitory activity even when heated for 3 min at 95 degrees C. The growth inhibitory effect of the 8 kD VH factor was dose dependent. Using iodinated vitreal proteins it was demonstrated that the VH proteins are able to enter or bind to lens epithelial cells. The growth inhibitory effect of the 8 kD VH factor was also tested on tissue cultured lens epithelial cells. These experiments showed that the 8 kD VH factor has no growth inhibitory effect on the tissue cultured lens epithelial cells. This experiment has been repeated many times using different concentrations of the factor. These observations suggest that the 8 kD VH factor may have receptors in the lens capsular material (extracellular matrix) and the factor-receptor binding is essential for the growth inhibitory effect.     

Assessment of pollen assemblages on footwear for evidence of pollen deriving from a mock crime scene: a contribution to forensic palynology

To provide evidence of a link between a crime scene and a suspect, pollen analysis is occasionally employed. However, experimental research linking pollen on footwear to a specific crime scene has been infrequently undertaken such that there are limited references to cite in court. In this blind study, the author had to determine, which of 12 pairs of footwear had walked on a mock scene by comparing their assemblages to those of two scene controls. An additional four control pairs of footwear that had not been worn on or near the scene were also analysed. The pollen data from the footwear was assessed and compared with the scene control samples for: number of taxa, key types, Czekanowski Coefficient and general assemblages. Results indicated that six of the pairs of footwear had walked on the scene and these were, indeed, the ones that actually had done so. The four control pairs were dissimilar in most aspects, particularly key types. This research demonstrates, with some limitations, that evidence of a match between footwear sample and location can be found, even when material from other habitats is present on an exhibit.     

Endocytosis in polarized cells.

Recent biochemical and morphological studies using the MDCK cell line have provided insights into the organization of the endocytic pathways in an epithelial cell. The cytoskeletal organization of these cells has been described and evidence for the involvement of microtubules in facilitating endocytic traffic has been obtained. The findings with this model system are compared to results from in vivo studies of the endocytic pathways from the surface domains of specialized epithelial cells and to studies of endocytosis in neurons.     

The Ca2+-binding S100A2 protein is differentially expressed in epithelial tissue of glandular or squamous origin

It has been previously shown that S100A2 is downregulated in tumor cells. The level of immunohistochemical S100A2 expression was therefore characterized in 424 normal and tumoral (benign and malignant) tissues of various origins, but mostly epithelial (with either glandular, squamous, respiratory or urothelial differentiation). We also investigated whether S100A2 could be co-localized with cytokeratin K14, an intermediate filament protein expressed in basal proliferative keratinocytes. Our data show that S100A2 has a low level of expression in non-epithelial tissue. In epithelial tissue S100A2 expression decreases remarkably in the tumors when compared to the normal specimens, and was correlated with the level of keratin K14. This decrease in S100A2 staining from normal to cancer cases is more pronounced in glandular than in squamous epithelial tissue. In addition, the patterns of S100A2 staining also differ between glandular and squamous tissue. These data suggest distinct functional roles for S100A2 in epithelial tissue of squamous or glandular origins.