Evaluation of HPV Infection and Smoking Status Impacts on Cell Proliferation in Epithelial Layers of Cervical Neoplasia

Accurate cervical intra-epithelial neoplasia (CIN) lesion grading is needed for effective patient management. We applied computer-assisted scanning and analytic approaches to immuno-stained CIN lesion sections to more accurately delineate disease states and decipher cell proliferation impacts from HPV and smoking within individual epithelial layers. A patient cohort undergoing cervical screening was identified (n = 196) and biopsies of varying disease grades and with intact basement membranes and epithelial layers were obtained (n = 261). Specimens were sectioned, stained (Mib1), and scanned using a high-resolution imaging system. We achieved semi-automated delineation of proliferation status and epithelial cell layers using Otsu segmentation, manual image review, Voronoi tessellation, and immuno-staining. Data were interrogated against known status for HPV infection, smoking, and disease grade. We observed increased cell proliferation and decreased epithelial thickness with increased disease grade (when analyzing the epithelium at full thickness). Analysis within individual cell layers showed a ≥50% increase in cell proliferation for CIN2 vs. CIN1 lesions in higher epithelial layers (with minimal differences seen in basal/parabasal layers). Higher rates of proliferation for HPV-positive vs. -negative cases were seen in epithelial layers beyond the basal/parabasal layers in normal and CIN1 tissues. Comparing smokers vs. non-smokers, we observed increased cell proliferation in parabasal (low and high grade lesions) and basal layers (high grade only). In sum, we report CIN grade-specific differences in cell proliferation within individual epithelial layers. We also show HPV and smoking impacts on cell layer-specific proliferation. Our findings yield insight into CIN progression biology and demonstrate that rigorous, semi-automated imaging of histopathological specimens may be applied to improve disease grading accuracy.     

Cytometric evidence that cervical intraepithelial neoplasia I and II are dysplasias rather than true neoplasias. An image analysis study of factors involved in the progression of cervical lesions.

Image analysis was performed on 40 Feulgen-stained histologic samples and 48 Feulgen-stained cytologic preparations representing normal squamous epithelium and all grades of cervical lesions (from mild dysplasia to invasive carcinoma) in order to characterize the evolutionary progressive changes in cervical epithelial proliferative disease toward malignancy. Quantitative studies included the analysis of proliferative features, differentiation features, nuclear morphology and DNA content. The data obtained on the histologic sections showed that the various features, to a different extent, detected a gradual increase in phenotypic cellular disarrangements related to the progression of the cervical lesions toward malignancy--that is, the modifications to nuclear area, perimeter, DNA content, percentage of nuclei with nucleoli, nuclear/cytoplasmic ratio and percentage of cells with no membrane positivity for soybean agglutinin lectin were progressively greater, moving from normal epithelium and mild dysplasia toward infiltrating carcinoma. In particular, all the morphologic and histochemical features appeared to parallel a diploid reduction and the appearance of aneuploidy. The simultaneous evaluation of proliferation- and differentiation-related features, together with those of nuclear DNA content, showed two main successive preneoplastic lesions: one characterized by an increase in cell turnover without alterations in its organization and another by a true neoplastic disorder. The data obtained on sequential cytologic examinations showed that individual cell changes are detectable and seem basically to be characterized by the appearance of clusters of cells with somatic characteristics not observed in previous cytologic checks. From the results of our study, the cervical intraepithelial neoplasia (CIN) concept appears to be inaccurate. In fact, only CIN III (severe dysplasia/carcinoma in situ) lesions have the morphologic and proliferative alterations of true neoplasia. In contrast, CIN I and some cases of CIN II lesions lack these characteristics and seem to be properly classified as dysplasia, thus avoiding the term neoplasia, implicit in CIN. Moreover, the multivariate study of data sets of features related to the progressive somatic changes, both in histologically and cytologically studied cases, allows us to detect the steps of progression; they are marked by the appearance of cell clusters with qualitatively different phenotypic characters when compared to the cell populations from which they presumably arise. These results seem to provide a further argument against the CIN theory, which stresses the concept that progression is related only to a gradual numerical increase in an initially established phenotype with the characteristics of malignancy.     

Metaphase-arrest technique applied to human cervical epithelium. II. Cell production rates in normal and pathological cervical epithelium

An application of the metaphase-arrest technique to human cervical epithelium, in vivo, was utilized to obtain cell birth rate data for seventy-six patients with normal and pathological cervical epithelium. Mean cell production rates for basal and parabasal layers of normal epithelium were 0.91 and 0.92 cells/1000 cells/hr respectively. Histologically normal epithelium adjacent to CIN (cervical intra-epithelial neoplasia) had a significantly higher rate for the parabasal layer compared to the 'normal' group (P less than 0.05). Values for the lower two-thirds of CIN III were 8-10 times higher than for normal epithelium, with microinvasive carcinoma having the highest rates of all. Values for wart-affected cervical epithelium (NCWVI) were intermediate between normal and CIN, but there was activity in the superficial layer, possibly reflecting activity of the papilloma virus. Large variation in birth rates between individuals in the same histological category was noted for each group, this being particularly the case in six patients with early invasive carcinoma. The data is used to attempt to elucidate some of the uncertainties surrounding the aetiology and biological behaviour of cancer of the cervix and its precursors. Sources of inaccuracy are emphasized and practical difficulties discussed.     

Metaphase-Arrest Technique Applied to Human Cervical Epithelium. Ii. Cell Production Rates In Normal and Pathological Cervical Epithelium

An application of the metaphase-arrest technique to human cervical epithelium, in vivo, was utilized to obtain cell birth rate data for seventy-six patients with normal and pathological cervical epithelium. Mean cell production rates for basal and parabasal layers of normal epithelium were 0.91 and 0.92 cells/1000 cells/hr respectively. Histologically normal epithelium adjacent to CIN (cervical intra-epithelial neoplasia) had a significantly higher rate for the parabasal layer compared to the ‘normal’ group (P < 0.05). Values for the lower two-thirds of CIN III were 8–10 times higher than for normal epithelium, with microinvasive carcinoma having the highest rates of all. Values for wart-affected cervical epithelium (NCWVI) were intermediate between normal and CIN, but there was activity in the superficial layer, possibly reflecting activity of the papilloma virus. Large variation in birth rates between individuals in the same histological category was noted for each group, this being particularly the case in six patients with early invasive carcinoma. The data is used to attempt to elucidate some of the uncertainties surrounding the aetiology and biological behaviour of cancer of the cervix and its precursors. Sources of inaccuracy are emphasized and practical difficulties discussed.     

Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2–94.7% high-grade CIN and in 59.3–100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.     

Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2–94.7% high-grade CIN and in 59.3–100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.     

Single modified cilia displayed by cells of human internal stratified epithelia (oral cavity,vagina)

Summary Serial sections of human vaginal and keratinized oral-gingival epithelia were investigated for ciliary structures. Most melanocytes of the gingival epithelium lacked cilia, whereas almost all basal keratinocytes of the deeper portion of the epithelial ridges possessed one cilium each. In the suprabasal layers of the ridges only a few keratinocytes exhibited a single cilium. In the basal layer, at the top of the connective tissue papillae, approximately every second keratinocyte displayed a single cilium. In the suprabasal layers above the ridges no ciliated keratinocytes were observed. The basal cells of the vaginal epithelium were endowed with cilia, while cilia were absent from the suprabasal cells. In the human forearm epidermis most melanocytes and keratinocytes are supplied with a single cilium; it has been suggested that they may play a role in light reception. However, the widespread occurrence of 9 + 0 cilia in epithelial cells of internal epithelia and their coincidence with the sites of renewal of keratinocytes suggests that a relationship may exist between solitary cilia and mitotic activity.     

Immunocytochemical localization of actin and tubulin in the integument of land crab (Gecarcinus lateralis) and lobster (Homarus americanus)

The crustacean integument consists of the exoskeleton and underlying epithelium and associated tissues. The epithelium, which is composed of a single layer of cells, is responsible for the cyclical breakdown and synthesis of the exoskeleton associated with molting (ecdysis). During premolt (proecdysis) the epithelial cells lengthen and secrete the two outermost layers (epicuticle and exocuticle) of the new exoskeleton while partially degrading the two innermost layers (endocuticle and membranous layer) of the overlying old exoskeleton. This increased cellular activity is associated with increased protein synthesis and a change in cell shape from cuboidal to columnar. The cytoskeleton, composed of microfilaments (actin) and microtubules (tubulin), plays important roles in the intracellular organization and motility of eukaryotic cells. Immunoblot analysis shows that the land crab exoskeleton contains actin, tubulin, and actin-related proteins (Varadaraj et al. 1996. Gene 171:177-184). In the present study, immunocytochemistry of land crab and lobster integument showed that both proteins were localized in various cell types, including epithelia, connective tissue, tendinal cells, and blood vessels. Muscle immunostained for actin and myosin, but not for tubulin. The membranous layer of land crab (the other layers of the exoskeleton were not examined) and membranous layer and endocuticle of lobster also reacted specifically with anti-beta-actin and anti-alpha-tubulin monoclonal antibodies, but not with an anti-myosin heavy chain antibody. During proecdysis immunolabeling of the membranous layer decreased probably due to protein degradation. The staining intensity for actin and tubulin in the proecdysial epithelium was similar to that in the intermolt (anecdysial) epithelium, suggesting that there was a net accumulation of both proteins proportional to the increase in cellular volume. These results support the previous biochemical analyses and, more specifically, localize actin and tubulin in exoskeletal structures, suggesting that they may serve both intracellular and extracellular functions in crustaceans. J. Exp. Zool. 286:329-342, 2000.     

Simultaneous localization of six antigens in single sections of transgenic mouse intestine using a combination of light and fluorescence microscopy.

To study the geographic differentiation of the intestinal epithelium and to understand the complex lineage relationships of its cell populations, it is often necessary to visualize the protein products of multiple genes in sections prepared from different positions along the duodenal-to-colonic and/or crypt-to-villus axes. Multilabel fluorescence or brightfield immunohistochemical techniques have previously been used for this purpose. However, the number of antigens that can be identified on single sections is limited in fluorescence microscopy by the number of fluorophores with non-overlapping absorption and emission characteristics, in brightfield microscopy by the number of visually distinguishable chromogens, and in both methods by the availability of primary antisera raised in multiple species. We have now used a combination of light and fluorescence microscopic techniques to increase the number of antigens that can be detected in a single section to six. Sections were sequentially stained using immunogold with silver intensification, peroxidase-antiperoxidase with diaminobenzidine chromogen, and peroxidase-anti-peroxidase with alpha-naphthol/basic dye as chromogen, followed by simultaneous fluorescent detection with fluorescein, 7-amino-4-methylcoumarin-3-acetic acid, and beta-phycoerythrin. This method enables up to four separate antigens to be visualized within a single cell and two additional antigens to be detected in unrelated cells. The technique is illustrated by examining the cellular patterns of expression of liver fatty acid binding protein/human growth hormone fusion genes in the intestinal epithelium of adult transgenic mice. It should be generally applicable to other experimental systems that require localization of multiple antigens in single tissue sections.     

Expression of p53, bcl-2 and Ki-67 in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the uterine cervix

OBJECTIVE: To assess the expression of p53, bcl-2 and Ki-67 in the progression of cervical neoplasia. STUDY DESIGN: A total of 131 cervical specimens, consisting of normal cervical epithelium (n = 43), cervical intraepithelial neoplasia (CIN) lesions (n =40) and cervical squamous cell carcinomas (SCCs) (n = 48) were examined immunohistochemically in paraffin sections for expression of p53, bcl-2 and Ki-67. RESULTS: Immunoreactivity of p53 was found in 27% of SCC cases, but it had no significant relationship with SCC staging (p = 0.791). Immunoreactivity of bcl-2 was observed in 33% of CIN 3 cases. We found a significant relationship (chi2 test: p = 0.009) between the expression of bcl-2 and CIN grading. Ki-67 index was higher in high grade CIN (HGCIN: CIN 2 and 3) and SCC lesions compared to normal cervices. Ki-67 index showed a correlation with bcl-2 protein expression (p = 0.030), but not with p53 protein expression (p = 0.239). CONCLUSION: HGCIN is an early stage to demonstrate the alteration of bcl-2 and Ki-67 expressions. Progression of neoplasia in the uterine cervix is accompanied by an increase of antiapoptotic protein, bcl-2 as well as cellular proliferation.     

阴道镜及宫颈活组织检查对早期宫颈上皮内瘤变诊断价值分析

目的:探究阴道镜及宫颈活组织检查对早期宫颈上皮内瘤变(cervicalintraepithelialneoplasia,CIN)的诊断价值。方法:选择2015年3月至2018年5月于我院接受诊治的543例疑似宫颈上皮瘤变患者,分别对其实施阴道镜及宫颈活组织检查,以病理学检测结果为金标准,分别评估两种方式单独检测及联合检测对早期CIN的诊断一致性、灵敏度和特异度,并进行组间对比。结果:(1)543例疑似CIN患者病理诊断早期CIN阳性患者168例,阴性患者375例,诊断率为30.94%;阴道镜对早期CIN诊断发现阳性患者有143例,良性患者有400例,诊断率为26.34%;宫颈活组织检测对早期CIN诊断发现阳性患者有159例,良性患者有384例,诊断率为29.28%;阴道镜联合颈活组织检测对早期CIN诊断发现阳性患者有163例,良性患者有380例,诊断率为30.02%。(2)检测发现,阴道镜对早期CIN诊断一致性为81.77%,灵敏度为60.12%,特异度为91.47%。(3)宫颈活组织对早期CIN诊断一致性为91.71%,灵敏度为83.33%,特异度为95.47%。(4)阴道镜联合宫颈活组织对早期CIN诊断一致性为96.50%,灵敏度为92.86%,特异度为98.13%。(5)联合检测对早期CIN诊断的一致性、灵敏度和特异度均明显优于阴道镜及宫颈活组织单独检测。结论:阴道镜及宫颈活组织检测对早期CIN具有较好的诊断效果,但联合检测诊断准确率更高,适用于早期CIN临床筛查中。     

Fluorescence lifetime: Beating the IRF and interpulse window

Fluorescence lifetime imaging captures the spatial distribution of chemical species across cellular environments employing pulsed illumination confocal setups. However, quantitative interpretation of lifetime data continues to face critical challenges. For instance, fluorescent species with known in vitro excited-state lifetimes may split into multiple species with unique lifetimes when introduced into complex living environments. What is more, mixtures of species, which may be both endogenous and introduced into the sample, may exhibit 1) very similar lifetimes as well as 2) wide ranges of lifetimes including lifetimes shorter than the instrumental response function or whose duration may be long enough to be comparable to the interpulse window. By contrast, existing methods of analysis are optimized for well-separated and intermediate lifetimes. Here, we broaden the applicability of fluorescence lifetime analysis by simultaneously treating unknown mixtures of arbitrary lifetimes—outside the intermediate, Goldilocks, zone—for data drawn from a single confocal spot leveraging the tools of Bayesian nonparametrics (BNP). We benchmark our algorithm, termed BNP lifetime analysis, using a range of synthetic and experimental data. Moreover, we show that the BNP lifetime analysis method can distinguish and deduce lifetimes using photon counts as small as 500.     

Exploratory analysis of quantitative histopathology of cervical intraepithelial neoplasia: objectivity, reproducibility, malignancy-associated changes, and human papillomavirus.

BACKGROUND: As part of a project to evaluate emerging optical technologies for cervical neoplasia, our group is performing quantitative histopathological analyses of biopsy specimens from 1,190 patients. Objectives in the interim analysis are (a) quantitatively assessing progression of the neoplastic process of cervical intraepithelial neoplasia (CIN)/squamous intraepithelial lesions (SIL), (b) detecting malignancy-associated changes (MACs), and (c) phenotypically measuring human papillomavirus (HPV) detected by DNA testing. METHODS: The diagnostic region of interest (ROI) from immediately adjacent sections were imaged, and the basal lamina and surface of the superficial layer were delimited. Nonoverlapping quantitatively stained nuclei were selected from 1,190 samples with histopathological characteristics of normal (929), koilocytosis (130), CIN 1 (40), CIN 2 (23), and CIN 3/carcinoma in situ (CIS) (68). A fully automatic procedure located and recorded the center of every nucleus in the region of interest (ROI). We used linear discriminant analysis to assess the changes between normal and CIN 3/CIS. RESULTS: Scores computed from the cell-by cell features and the clinical grade of CIN/SIL were highly correlated, as were those of the architectural features and the clinical grade of CIN/SIL. We found even higher correlations between a combination of cell-by-cell and architectural scores, and clinical grade. Using these scores, we found MACs in the normal biopsy specimens from patients with high-grade CIN/SIL. Furthermore, the same scores correlated with the molecular detection of HPV. CONCLUSIONS: Quantitative histopathology can be used in large clinical trials as an objective and reproducible measure of CIN/SIL. Detectable phenotypic changes correlate well with CIN/SIL neoplastic progression. It can also be used to infer the presence of CIN/SIL (MACs) and molecular changes associated with increased risk of cancer development (high-risk HPV).     

Global analysis of fluorescence lifetime imaging microscopy data

Global analysis techniques are described for frequency domain fluorescence lifetime imaging microscopy (FLIM) data. These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample. Two approaches to implementing the lifetime invariance constraint are described. In the lifetime invariant fit method, each image in the lifetime image sequence is spatially averaged to obtain an improved signal-to-noise ratio. The lifetime estimations from these averaged data are used to recover the fractional contribution to the steady-state fluorescence on a pixel-by-pixel basis for each species. The second, superior, approach uses a global analysis technique that simultaneously fits the fractional contributions in all pixels and the spatially invariant lifetimes. In frequency domain FLIM the maximum number of lifetimes that can be fit with the global analysis method is twice the number of lifetimes that can be fit with conventional approaches. As a result, it is possible to discern two lifetimes with a single-frequency FLIM setup. The algorithms were tested on simulated data and then applied to separate the cellular distributions of coexpressed green fluorescent proteins in living cells.     

Two-Photon Lifetime Imaging of Voltage Indicating Proteins as a Probe of Absolute Membrane Voltage

Genetically encoded voltage indicators (GEVIs) can report cellular electrophysiology with high resolution in space and time. Two-photon (2P) fluorescence has been explored as a means to image voltage in tissue. Here, we used the 2P electronic excited-state lifetime to probe absolute membrane voltage in a manner that is insensitive to the protein expression level, illumination intensity, or photon detection efficiency. First, we tested several GEVIs for 2P brightness, response speed, and voltage sensitivity. ASAP1 and a previously described citrine-Arch electrochromic Förster resonance energy transfer sensor (dubbed CAESR) showed the best characteristics. We then characterized the voltage-dependent lifetime of ASAP1, CAESR, and ArcLight under voltage-clamp conditions. ASAP1 and CAESR showed voltage-dependent lifetimes, whereas ArcLight did not. These results establish 2P fluorescence lifetime imaging as a viable means of measuring absolute membrane voltage. We discuss the prospects and improvements necessary for applications in tissue.     

Fluorescence lifetime of fluorescent proteins as an intracellular environment probe sensing the cell cycle progression

The fluorescence lifetime of fluorescent proteins is affected by the concentration of solutes in a medium, in inverse correlation with local refractive index. In this paper, we introduce the concept of using this dependence to probe cellular molecular environment and its transformation during cellular processes. We employ the fluorescence lifetime of Green Fluorescent Protein and tdTomato Fluorescent Protein expressed in cultured cells and probe the changes in the local molecular environment during the cell cycle progression. We report that the longest fluorescence lifetimes occurred during mitosis. Following the cell division, the fluorescence lifetimes of these proteins were rapidly shortened. Furthermore the fluorescence lifetime of tdTomato in the nucleoplasm gradually increased throughout the span of S-phase and remained constantly long until the end of interphase. We interpret the observed fluorescence lifetime changes to be derived from changes in concentration of macromolecular solutes in the cell interior throughout cell cycle progression.     

Expression of vascular endothelial growth factor in the progression of cervical neoplasia and its relation to angiogenesis and p53 status

OBJECTIVE: To evaluate vascular endothelial growth factor (VEGF) expression in the successive steps of cervical neoplasia and to determine its correlation with angiogenesis and p53 status. STUDY DESIGN: Immunohistochemical staining with a VEGF monoclonal antibody was performed on a total of 161 cervical specimens representing 12 normal epithelium, 33 cervical intraepithelial neoplasia (CIN) 1, 30 CIN 3 and 86 squamous cell carcinomas. Microvessels were immunohistochemically labeled with an antibody to CD34. Computerized image analysis was used to evaluate microvessel density (MVD). p53 Status was determined by immunohistochemistry and direct sequencing of exons 5-8 of the p53 gene. RESULTS: VEGF expression progressively increased along the continuum from normal epithelium to squamous cell carcinoma (P < .05). MVD increased significantly with cervical neoplasia progression, from normal epithelium, through CIN, to squamous cell carcinoma (P < .001). A strong correlation was observed between VEGF expression and MVD (P < .001). p53 Protein expression was not detected in the normal epithelium or in CIN 1, while 3 (10%) of 30 CIN 3 and 28 (33%) of 86 squamous cell carcinomas were positive for p53. VEGF expression correlated statistically with p53 protein expression (P < .001). In double VEGF- and p53-stained sections, the 2 markers were generally expressed in the same tumor cells. Of the 4 p53 gene mutations, 3 exhibited strong VEGF expression, and 1 exhibited moderate VEGF expression. VEGF expression did not correlate significantly with outcome variables in patients with squamous cell carcinoma. CONCLUSION: Our results suggest that VEGF expression is involved in the promotion of angiogenesis in cervical neoplasia and that p53 is likely to be involved in the regulation of VEGF expression.     

Angiogenesis,cell proliferation and apoptosis in progression of cervical neoplasia

OBJECTIVE: To investigate changes in angiogenesis, cell proliferation and apoptosis in the successive steps of cervical neoplasia and to analyze their interrelationship. STUDY DESIGN: A total of 182 cervical specimens, representing 12 normal epithelium, 33 cervical intraepithelial neoplasia (CIN) 1, 21 CIN 2, 30 CIN 3 and 86 squamous cell carcinomas, were evaluated. The microvessels were immunohistochemically labeled with CD34 antibodies. Computerized image analysis was used to evaluate microvessel density (MVD). The apoptotic cells were visualized by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique and proliferative cells by staining with Ki-67 antibodies. RESULTS: One-way analysis of variance showed that the MVD, Ki-67 labeling index and apoptotic index increased significantly with the progression of cervical neoplasia from normal epithelium, through CIN, to carcinoma (P <.001 for each index). All the indices, determined in all 182 cervical tissues, were significantly and positively associated with each other (P < .001 in all cases), with correlation coefficients ranging from .649 to .819. MVD in patients with recurrence or death was significantly higher than in disease-free patients (P < .05). CONCLUSION: The results suggest that tumor progression in the cervical epithelium is accompanied by angiogenesis and an increase in both cell proliferation and apoptosis. Angiogenesis may be a prognostic indicator in patients with squamous cell carcinoma of the cervix.     

Immunohistochemical localization and quantitative analysis of cellular glutathione peroxidase in foetal and neonatal rat tissues: Fluorescence microscopy image analysis

Summary To quantitate the developmental changes in selenium-dependent cellular glutathione peroxidase during the perinatal period, tissue sections from foetal (day 12 to day 22) and neonatal (day 6) rats were stained immunohistochemically using specific polyclonal antiserum. The intensity of the staining was quantified by fluorescence microscopy image analysis. There was a general trend of enriched glutathione peroxidase in the epithelial linings and metabolically active sites. Significant fluorescence was detected in cardiomyocytes, hepatocytes, renal tubular epithelium, bronchiolar epithelium and intestinal epithelium at day 15. The intensity increased in a stepwise manner therafter. The overall increase in the intensity of staining in the heart, liver, kidneys, lungs and intestine was 1.5-, 2.3-, 1.6-, 1.7- and 3.0-fold, respectively. The phase of most rapid increase occurred during the foetal period in the liver, intestine and heart. In the kidneys and lungs, glutathione peroxidase increased significantly during foetal life, and to a similar extent postnatally. These results suggest that the intracellular H₂O₂-scavenging system develops during the foetal period as an essential mechanism for living under atmospheric oxygen conditions. The late development observed in the kidneys and lungs is consistent with the relative biological immaturity of these organs in full-term neonates.     

Scanning electron microscopy of the channel catfish olfactory lamellae

The olfactory lamellae of the channel catfish (Ictalurus punctatus) are composed of sensory and indifferent (non-sensory) epithelia organized into two distinct regions on both surfaces of each lamella. The smaller sensory region located adjacent to the midline raphe has fewer cilia per unit surface area than the indifferent epithelium and contains the olfactory neurons whose ciliated dendritic terminals occur at the epithelial surface. The indifferent epithelium, comprising the greater surface area of the olfactory lamella, is covered with a dense mat of non-sensory cilia. Fractured carbon dioxide critical point dried lamellar tissue revealed the underlying cellular structure. The lamellae are composed of two layers of epithelium enclosing a thin stromal layer. Olfactory receptors were observed in the fractured tissue only within the sensory epithelium.