Effects of transdifferentiation and EGF on claudin isoform expression in alveolar epithelial cells.

Rat alveolar epithelial type II cells grown on polycarbonate filters form high-resistance monolayers and concurrently acquire many phenotypic properties of type I cells. Treatment with EGF has previously been shown to increase transepithelial resistance across alveolar epithelial cell (AEC) monolayers. We investigated changes in claudin expression in primary cultured AEC during transdifferentiation to the type I cell-like phenotype (days 0, 1, and 8), and on day 5 in culture +/- EGF (10 ng/ml) from day 0 or day 4. Claudins 4 and 7 were increased, whereas claudins 3 and 5 were decreased, on later compared with earlier days in culture. Exposure to EGF led to increases in claudins 4 and 7 and decreases in claudins 3 and 5. Claudin 1 was only faintly detectable in freshly isolated type II cells and remained unchanged over time in culture and after exposure to EGF. These results suggest that increases in transepithelial resistance accompanying AEC transdifferentiation and/or EGF exposure are mediated, at least in part, by changes in the pattern of expression of specific claudin isoforms.     

Claudin 7 is reduced in uterine epithelial cells during early pregnancy in the rat

The non-receptive uterine luminal epithelium forms an intact polarised epithelial barrier that is refractory to blastocyst invasion. During implantation, organised dismantling of this barrier leads to a receptive state promoting blastocyst attachment. Claudins are tight junction proteins that increase in the uterine epithelium at the time of implantation. Claudin 7 is a member of this family but demonstrates a basolateral localisation pattern that is distinct from other claudins. The present study investigated the localisation, abundance and hormonal regulation of claudin 7 to elucidate a role for the protein during implantation. The results showed that claudin 7 demonstrates a distinct basal and lateral localisation in the uterine luminal and glandular epithelium throughout early pregnancy. On day 1, claudin 7 is abundantly present in response to ovarian estrogen. At the time of implantation, claudin 7 decreases in abundance. This decrease is not dependent on blastocyst presence, as shown by results in pseudopregnant animals. We propose that claudin 7 mediates intercellular adhesions in the uterine epithelium and also may be responsible for stabilising adhesion proteins at the basolateral cell surface. Thus, claudin 7 may function under the maintenance of the uterine luminal epithelial barrier, in the non-receptive state preventing implantation from occurring.     

Anterior and intermediate pituitary tissues express claudin 4 in follicle stellate cells and claudins 2 and 5 in endothelial cells

Follicle-stellate cells are pituitary non-granular cells that are arranged between secretory cells or organized in follicles with small lumens. Cells from the follicles exhibit the typical phenotype of a transporting epithelium, including apical microvilli with a cilium and tight junctions. Freeze-fracture electron microscopy images show that the tight junctions consist of 5–7 anastomosing strands and that cultured follicle-stellate cells develop a trans-epithelial electrical resistance characteristic of “tight” epithelia. Here, we investigate the molecular composition of the tight junction from follicle stellate cells. We found that the rat anterior pituitary lobe expresses mRNAs for claudins 2, 4 and 5; the proteins of all these claudins are observed in the anterior lobe, whereas the intermediate lobe expresses claudins 2 and 5 and the posterior lobe contains only claudin 5. Follicle-stellate cells, identified by their protein marker S100β, expresses claudin 4 in the apical membrane, in co-localization with dipeptidyl-peptidase and near acetylated β-tubulin. Claudin 4 partially co-localizes with E-cadherin, indicating that a fraction of the protein is located in the basolateral domain. Follicle-stellate-enriched cell cultures develop patches of polygonal cells expressing claudin 4 and E-cadherin, encircled by extensive monolayers of fusiform cells. Claudin 2 stains specifically blood vessels, identified by claudin 5 and VE-cadherin labels. Thus, follicles in the anterior pituitary consist of “tight” epithelia that can carry out intense vectorial transport, together with a high cation movement in blood vessels, possibly related to the ion requirements of excitable secretory cells for hormone secretion.     

Double gene deletion reveals lack of cooperation between claudin 11 and claudin 14 tight junction proteins

Members of the claudin family of proteins are the main components of tight junctions (TJs), the major selective barrier of the paracellular pathway between epithelial cells. The selectivity and specificity of TJ strands are determined by the type of claudins present. An understanding of the cooperation between different claudins in various tissues is thus important. To study the possible cooperation between claudin 11 and claudin 14, we have generated claudin 11/claudin 14 double-deficient mice, which exhibit a combination of the phenotypes found in each of the singly deficient mutants, including deafness, neurological deficits, and male sterility. These two claudins have distinct and partially overlapping expression patterns in the kidney. Claudin 11 is located in both the proximal and distal convoluted tubules, whereas claudin 14 occurs in both the thin descending and thick ascending limbs of the loop of Henle and in the proximal convoluted tubules. Although daily urinary excretion of Mg(++), and to a lesser extent of Ca(++), tends to be higher in claudin 11/claudin 14 double mutants, these changes do not reach statistical significance compared with wild-type animals. Thus, under normal conditions, co-deletion of claudin 11 and claudin 14 does not affect kidney function or ion balance. Our data demonstrate that, despite the importance of each of these claudins, there is probably no functional cooperation between them. Generation of additional mouse models in which different claudins are abolished should provide further insight into the complex interactions between claudin proteins in various physiological systems.     

Salinity regulates claudin mRNA and protein expression in the teleost gill

The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.     

Clostridium perfringens Enterotoxin Interacts with Claudins via Electrostatic Attraction

Clostridium perfringens enterotoxin (CPE), a causative agent of food poisoning, is a pore-forming toxin disrupting the selective permeability of the plasma membrane of target cells, resulting in cell death. We previously identified claudin as the cell surface receptor for CPE. Claudin, a component of tight junctions, is a tetratransmembrane protein and constitutes a large family of more than 20 members, not all of which serve as the receptor for CPE. The mechanism by which the toxin distinguishes the sensitive claudins is unknown. In this study, we localized the region of claudin responsible for interaction with CPE to the C-terminal part of the second extracellular loop and found that the isoelectric point of this region in sensitive claudins was higher than insensitive claudins. Amino acid substitutions to lower the pI resulted in reduced sensitivity to CPE among sensitive claudins, whereas substitutions to raise the pI endowed CPE-insensitive claudins with sensitivity. The steric structure of the claudin-binding domain of CPE reveals an acidic cleft surrounded by Tyr³⁰⁶, Tyr³¹⁰, Tyr³¹², and Leu³¹⁵, which were reported to be essential for interaction with the sensitive claudins. These results imply that an electrostatic attraction between the basic claudin region and the acidic CPE cleft is involved in their interaction.     

Decrease in claudin‐2 expression enhances cell migration in renal epithelial madin–darby canine kidney cells

Migration of renal epithelial cells increases after renal tubular damage, but its mechanism has not been clarified in detail. Hyperosmotic stress increased a cellular injury concomitant with a decrease in mRNA and protein expression of claudin‐2 in renal tubular epithelial Madin–Darby canine kidney cells. We hypothesized that claudin‐2 is involved in the regulation of cell migration. To knockdown claudin‐2 expression, we made the cells expressing doxycycline‐inducible claudin‐2 shRNA vector. Claudin‐2 knockdown affected neither the endogenous expression levels of claudin‐1, ‐3, ‐4, and ‐7 nor the Triton X‐100 solubility of these claudins. Transepithelial electrical resistance was increased by claudin‐2 knockdown without affecting permeability to FITC‐dextran (4,000 Da). BrdU incorporation assay and cell counting revealed that cell proliferation and viability are unaffected by claudin‐2 knockdown. In the wound‐healing assay, the recovery rate of wound area was increased by claudin‐2 knockdown. The mRNA expression and activity of matrix metalloproteinase‐9 (MMP‐9) were increased by claudin‐2 knockdown. A selective MMP‐9 inhibitor suppressed cell migration in the claudin‐2 knockdown cells. Hyperosmotic stress increased the expression and activity of MMP‐9, which were inhibited by claudin‐2 overexpression. These results suggest that the decrease in claudin‐2 expression enhances cell migration mediated by the increase in the expression and activity of MMP‐9. J. Cell. Physiol. 226: 1471–1478, 2011. © 2010 Wiley‐Liss, Inc.     

Claudins蛋白在胃癌组织中的表达

紧密连接（tightjunctions,TJs）对于维持上皮细胞或内皮细胞的正常结构和发挥生理功能起着重要作用。Claudins（CLDNs）蛋白是构成细胞膜紧密连接的主要成分之一,其异常表达导致上皮细胞或内皮细胞结构破坏、功能受损,与多种疾病的发生、发展关系密切。近年来,越来越多的文献报道了claudins蛋白在胃癌组织中的异常表达。不同claudin蛋白亚型在胃癌组织中的表达降低或增高所起作用亦不相同。Claudins蛋白与肿瘤形成、癌细胞的增殖、转移、以及预后关系密切。因此,深入研究claudins蛋白与胃癌发生、发展的关系具有重要意义。     

Epithelial tight junction proteins as potential antibody targets for pancarcinoma therapy

Recombinant monoclonal antibodies are beginning to revolutionize cancer therapy. In combination with standard chemotherapy, high response rates have been reported with antibodies of the human IgG1 isotype for treatment of non-Hodgkins lymphoma and breast cancer. It is becoming apparent that targets for antibody-based therapies do not necessarily need to be absent from normal tissues but can be present there either in low copy numbers or with binding epitopes shielded from the therapeutic antibody. Here, we studied whether claudin proteins that form tight junctions in normal epithelia are still expressed on carcinoma cells and whether their extracellular domains can be recognized by antibodies. We show that mRNAs of claudins 1, 3, 4, and 7 are all expressed in different human carcinoma cell lines, while claudin 8 was selectively expressed in breast and pancreas cancer lines. Chicken polyclonal antibodies were raised against peptides contained within predicted extracellular domains of claudins 1, 3, and 4. Affinity-purified IgG fractions for claudins 3 and 4 were monospecific and bound to human breast and colon carcinoma lines, but not to a line of monocytic origin. Claudin 3 antibodies also homogeneously stained human renal cell carcinoma tissue and micrometastatic tumor cells as identified by cytokeratin staining in bone marrow biopsies of breast cancer patients. Fluorescence-activated cell sorting and immunocytochemistry indicated that claudin antibodies bound to the surface of tumor cells. By analogy to other tumor-associated antigens that are differentially accessible to antibodies on tumor vs normal tissue, we propose that certain claudin proteins have potential as targets for novel antibody-based therapies of carcinomas.     

Protein Kinase C Activation Has Distinct Effects on the Localization, Phosphorylation and Detergent Solubility of the Claudin Protein Family in Tight and Leaky Epithelial Cells

We have previously shown that protein kinase C (PKC) activation has distinct effects on the structure and barrier properties of cultured epithelial cells (HT29 and MDCK I). Since the claudin family of tight junction (TJ)-associated proteins is considered to be crucial for the function of mature TJ, we assessed their expression patterns and cellular destination, detergent solubility and phosphorylation upon PKC stimulation for 2 or 18 h with phorbol myristate acetate (PMA). In HT29 cells, claudins 1, 3, 4 and 5 and possibly claudin 2 were redistributed to apical cell–cell contacts after PKC activation and the amounts of claudins 1, 3 and 5, but not of claudin 2, were increased in cell lysates. By contrast, in MDCK I cells, PMA treatment resulted in redistribution of claudins 1, 3, 4 and 5 from the TJ and in reorganization of the proteins into more insoluble complexes. Claudins 1 and 4 were phosphorylated in both MDCK I and HT29 cells, but PKC-induced changes in claudin phosphorylation state were detected only in MDCK I cells. A major difference between HT29 and MDCK I cells, which have low and high basal transepithelial electrical resistance, respectively, was the absence of claudin 2 in the latter. Our findings show that PKC activation targets in characteristic ways the expression patterns, destination, detergent solubility and phosphorylation state of claudins in epithelial cells with different capacities to form an epithelial barrier.     

Risk of significant gynaecological pathology in women with ?glandular neoplasia on cervical cytology

A. Talaat, D. Brinkmann, J. Dhundee, Y. Hana, J. Bevan, R. Irvine, S. Bailey and R. Woolas
Risk of significant gynaecological pathology in women with ?glandular neoplasia on cervical cytology Objective: To review the risk of pre‐invasive and invasive gynaecological pathology in women referred with cervical cytology reporting ?glandular neoplasia. Methods: Review of the case notes of all women referred with cervical cytology reported as ?glandular neoplasia between January 1999 and December 2008 at two UK hospitals: Portsmouth Hospitals NHS Trust and Queen Mary’s Hospital Sidcup. The category of ‘borderline nuclear change in endocervical cells’, result code 8 according to the national health service cancer screening programme (NHSCSP), was excluded from the study. Results: A total of 200 women were identified using the hospitals’ pathology computer systems. Invasive carcinoma was found in 48 women (24%): 28 endocervical adenocarcinomas, eight squamous cell carcinomas (SCC), ten endometrial and two ovarian adenocarcinomas. Pre‐invasive neoplasia was found in 115 (57.5%), including 14 cervical glandular intraepithelial neoplasia (CGIN), 31 cervical intraepithelial neoplasia (CIN) grade 2/3 and 70 concomitant CGIN and CIN2/3. CIN1/HPV was found in 25, simple endometrial hyperplasia in three and no histological abnormality in three. Thirty‐four (70.8%) of 48 invasive carcinomas (of which 23 were endocervical adenocarcinomas) were in asymptomatic women investigated for abnormal cytology. Fourteen of 34 (41.4%) of those with ?glandular neoplasia thought to be endometrial were CGIN or CIN2/3. Colposcopic appearances were normal in 47.6% of women with pure cervical glandular neoplasia (adenocarcinoma or CGIN) compared with 12.8% with squamous cell lesions (CIN2/3 or SCC): P = 0.0001. Thus, colposcopy was more sensitive for detecting squamous cell abnormalities than their glandular counterparts. Although cervical adenocarcinomas are less amenable to prevention by screening than cervical SCC, in our study cervical cytology predominantly detected these abnormalities at their early asymptomatic stages. Conclusion: At least CIN2 was found in 81.5% in women referred with cervical cytology reporting ?glandular neoplasia. A thorough evaluation of the whole genital tract is needed if colposcopy is negative.     

Diverse glandular pathologies coexist with high-grade squamous intraepithelial lesion in cyto-histological review of atypical glandular cells on ThinPrep specimens

Objective: To identify in cytology, high‐grade squamous intraepithelial lesions with endocervical glandular extension in cases previously diagnosed as atypical glandular cells (AGC), analyse possible reasons for the diagnostic pitfall and document the frequency of glandular pathology coexisting with high‐grade cervical intraepithelial lesion in histology. Methods: Thirty‐nine ThinPrep® cervical smear (Pap) tests reported as AGC of undetermined significance and showing high‐grade lesions on histology [cervical intraepithelial neoplasia (CIN) 2 or 3, endometrial or extrauterine adenocarcinoma] were reviewed retrospectively to identify the cases of high‐grade squamous intraepithelial lesion with endocervical glandular extension, using the Bethesda 2001 system. Cyto‐histological correlation was performed. Results: A high frequency of diverse glandular pathologies coexisted with high‐grade cervical intraepithelial lesions on histology. This included endocervical glandular extension in 63%, benign glandular pathology in 33% and pre‐neoplastic or malignant glandular pathology (endocervical glandular dysplasia, adenocarcinoma in situ and metastatic breast carcinoma) in 17% cases. On cytology, the sensitivity was 40%, specificity was 80% and positive predictive value was 86% for endocervical gland extension in high‐grade squamous intraepithelial lesions. Conclusions: Special efforts to recognize endocervical glandular extension in high‐grade squamous intraepithelial lesions and glandular neoplasia coexisting with squamous intraepithelial lesions from the heterogeneous category of AGC can contribute to increasing the diagnostic accuracy. The identification of endocervical glandular extension on cervical cytology would alert the gynaecologist to perform a thorough assessment of the endocervix during colposcopy. This could also help to decide on the need to perform deeper conization rather than loop electrosurgical excision procedure to ensure negative margins when colposcopic biopsy shows CIN 2 or 3.     

Outcomes of cervical liquid-based cytology suggesting a glandular abnormality

Objective: To ascertain the positive predictive value of both ?glandular neoplasia (national standard code 6) and borderline change (national standard code 8) in glandular cells in liquid‐based cervical cytology specimens in Cardiff and Vale NHS Trust and to outline the histological outcomes of these cases. Method: Eighty‐nine liquid‐based (Surepath?) cervical cytology cases were retrospectively identified from a 2‐year period (January 2005 to December 2006) and correlated with histopathological diagnoses. Results: Initial punch biopsy histology revealed 18 cases (21%) of cervical glandular intraepithelial neoplasia (CGIN). A further nine cases (10%) of CGIN were identified following local excision or hysterectomy. Ten cases of invasive malignancy were identified: four endocervical adenocarcinomas (all node negative, TNM stage T1b1), five endometrial adenocarcinomas and one squamous cell carcinoma. There were 10 with high‐grade cervical intraepithelial neoplasia (CIN) alone. Women diagnosed with endometrial malignancy presented later with an average age of 64.6 years compared with 34.9 years for endocervical lesions. Taking high‐grade CIN or worse as a positive outcome, the overall positive predictive value (PPV) of glandular abnormalities on cytology (both code 6 and 8) was 58.1% [95% confidence interval (CI) 47.8, 68.4]. PPV for borderline change in glandular cells alone was 24.1% (95% CI 8.5, 39.6) and for ?glandular neoplasia alone 75.4% (95% CI 64.3, 86.5). Conclusion: With our interpretation of the classification, women with cytological diagnoses of glandular neoplasia of the cervix should initially be investigated by local resection rather than punch biopsy, and those with borderline change in glandular cells with repeat cytology.     

Role of claudin species-specific dynamics in reconstitution and remodeling of the zonula occludens

Tight-junction strands, which are organized into the beltlike cell-cell adhesive structure called the zonula occludens (TJ), create the paracellular permselective barrier in epithelial cells. The TJ is constructed on the basis of the zonula adherens (AJ) by polymerized claudins in a process mediated by ZO-1/2, but whether the 24 individual claudin family members play different roles at the TJ is unclear. Here we established a cell system for examining the polymerization of individual claudins in the presence of ZO-1/2 using an epithelial-like cell line, SF7, which lacked endogenous TJs and expressed no claudin but claudin-12 in immunofluorescence and real-time PCR assays. In stable SF7-derived lines, exogenous claudin-7, -14, or -19, but no other claudins, individually reconstituted TJs, each with a distinct TJ-strand pattern, as revealed by freeze-fracture analyses. Fluorescence recovery after photobleaching (FRAP) analyses of the claudin dynamics in these and other epithelial cells suggested that slow FRAP-recovery dynamics of claudins play a critical role in regulating their polymerization around AJs, which are loosely coupled with ZO-1/2, to form TJs. Furthermore, the distinct claudin stabilities in different cell types may help to understand how TJs regulate paracellular permeability by altering the paracellular flux and the paracellular ion permeability.     

细胞衰老和凋亡相关蛋白表达在宫颈鳞癌变中的意义

目的：许多细胞周期调控因子和衰老相关标志物如p14ARF、p15INK4b、p16INK4a 和p53 在G1 细胞周期阻滞和癌基因诱导的衰老中意义重大。这些关键的调节蛋白在多种恶性肿瘤中经常发生突变或是缺失。在本研究中将探讨这些因子在宫颈癌发生中的意义。方法：在本研究中在正常宫颈上皮、宫颈上皮内瘤变和宫颈鳞癌中,应用免疫组织化学方法检测p14ARF、p15INK4b、p16INK4a、Bcl-2、p53 表达,并分析它们的表达与宫颈癌变的相关性。结果：p16INK4a 在正常宫颈鳞状上皮10%（2/20）表达阴性,在大部分CIN 和宫颈鳞癌中表达阳性,其中在85%（17/20）CIN 和75%（15/20）鳞癌中呈弥漫性强阳性表达,CIN 和宫颈鳞癌中的阳性表达率显著高于正常上皮（P〈0.01）,CIN 和宫颈鳞癌的间表达率无显著差异。p15INK4b 在正常宫颈鳞状上皮中65%（13/20）表达弱阳性,在100% （20/20）CIN 和95%（19/20）宫颈鳞癌中表达弥漫性阳性,各组之间阳性表达率无显著性差异（P〉0.05）。p14ARF在40%（8/20）正常宫颈上皮细胞中表达呈弱阳性（1＋）,在宫颈鳞癌中表达呈弥漫性强阳性90%（18/20）,在45%（9/20）CIN中表达阳性,各组之间阳性表达率无显著性差异（P〉0.05）。Bcl-2 在20%（4/20）正常宫颈上皮表达呈弱阳性,在18/20CIN中其表达强度和比率均增加,阳性表达率为90%（18/20）,Bcl-2 在鳞癌中700%（14/20）呈强阳性和弥漫阳性,CIN 和宫颈鳞癌中的阳性表达率显著高于正常上皮（P〈0.01）,CIN 和宫颈鳞癌的间表达率无显著差异。P53 免疫组化染色显示在正常宫颈上皮为表达为20%（4/20）,在大多数CIN25%（5/20）和鳞癌中核阳性85%（17/20）,在鳞癌中的阳性表达率显著高于正常宫颈上皮和CIN 病变（P〈0.05）。结论：宫颈鳞癌变涉及包括细胞凋亡和细胞衰老在内的多种信号分子表达异常,这些分子可能在宫颈鳞癌发生发挥重要作用并在宫颈癌早期诊断中有重要意义。     

紧密连接蛋白claudins应用于肿瘤治疗的进展

Claudins蛋白家族是组成紧密连接(Tight junctions,TJs)必不可少的骨架蛋白,在维持上皮和内皮细胞中的细胞极性、细胞间的粘附固定、细胞旁路的离子运输等发挥重要作用。近年来大量的研究结果证明,claudins在许多人类恶性肿瘤中异常表达。因此,claudins也被作为癌症治疗的潜在靶标。文中就claudin蛋白家族在肿瘤中的表达情况及其相关药物的研究进展进行阐述。     

P16INK4A as an adjunct test in liquid-based cytology

OBJECTIVE: To assess the utility of P16INK4A as an adjunct test in liquid-based cytology in cases with equivocal morphologic changes of high grade squamous intraepithelial lesion (HSIL). STUDY DESIGN: P16INK4A immunoreactivity was investigated in residual ThinPrep material (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) from 30 cases with equivocal diagnoses of HSIL that had corresponding follow-up biopsies. Two control ThinPrep cases were included: 1 HSIL with biopsy-confirmed cervical intraepithelial neoplasia (CIN) 3 and a negative specimen with a corresponding biopsy of squamous metaplasia. The expression of P16INK4A in ThinPrep specimens and corresponding biopsies was scored as previously described. A ThinPrep case was scored positive if it contained > 10 abnormal cells with nuclear and cytoplasmic immunocytochemical staining. Corresponding biopsies were scored as having negative, sporadic, focal or diffuse staining. RESULTS: The P16INK4A antibody assay was positive in 19 of 30 ThinPrep cases (63.3%). Seventeen of the 19 (89.4%) biopsies corresponding to the positively stained ThinPreps also were positive, with a score of at least focal positivity in the dysplastic regions (2 CIN 1, 4 CIN 2, 11 CIN 3; 2 lesions lost in the tissue recut). The assay was negative in 11 ThinPreps (36.6%) and 10 biopsies (33.3%) with tissue confirmation of chronic cervicitis (5), squamous metaplasia (2), CIN 1 (3) and 1 lesion lost in the tissue recut. Seventeen of 18 (94.4%) ThinPreps confirmed as high grade lesions upon biopsy showed P16INK4A positivity. The control HSIL case with a CIN 3 biopsy was diffusely positive for P16INK4A, and the control negative case with biopsy diagnosis of squamous metaplasia was negative. Nondysplastic squamous and metaplastic epithelium in 7 biopsies and nondysplastic squamous or metaplastic cells in ThinPrep cases were negative. Sporadic staining of bacteria, inflammatory cells and endocervical cells was noted. CONCLUSION: ThinPrep cases in the equivocal cytologic category with the corresponding tissue biopsy assayed for P16INK4A expression showed that there was utility for this type of testing. A larger series comparing corresponding ThinPrep and tissue biopsies will be undertaken. The role of HPV infection in these cases will also be explored.     

Cytological Changes Associated With Tubo-Endometrioid Metaplasia of the Uterine Cervix

Two women who had undergone previous cervical surgery for the treatment of glandular intraepithelial neoplasia (GIN) and cervical intraepithelial neoplasia (CIN), were found to have severely dyskaryotic cells of glandular and metaplastic type in follow-up cervical smears. A third patient was found to have similar abnormal cells in a routine screening smear. All of the patients were subjected to either hysterectomy or cervical conization and in all cases histological examination showed tubo-endometrioid glands in the endocervix, well away from the uterine isthmus, with no associated endometrial stromal tissue. All of the cervical smears were reviewed and cytological features that facilitate the distinction between tubo-endometrioid metaplasia and squamous or glandular dyskaryosis were identified. These features include the smaller size of affected cells, more marked nuclear hyperchromasia, inconspicuous nucleoli, the formation of glandular structures, the lack of discrete squamous dyskaryosis and the absence of the typical 'feathering' noted with GIN. the possibility of tubo-endometrioid metaplasia should be considered when atypical glandular or metaplastic cells are noted in cervical smears, particularly, but not exclusively, in women who have been treated for CIN or GIN. In the presence of these changes clinicians should rebiopsy the cervix before embarking on further unnecessary surgery which may adversely affect fertility and pregnancy, particularly in younger patients.