Uniform Membranous Staining Research Articles

Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB.

Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non-canonical mechanisms may be employed. The rod-shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function. A recent study reported that the protein encoded by ORF CT009 interacts with MreB despite low sequence similarity to RodZ. The studies herein expand on those observations through protein structure, mutagenesis and cellular localization analyses. Structural analysis indicated that CT009 shares high level of structural similarity to RodZ, revealing the conserved orientation of two residues critical for MreB interaction. Substitutions eliminated MreB protein interaction and partial complementation provided by CT009 in RodZ deficient Escherichia coli. Cellular localization analysis of CT009 showed uniform membrane staining in Chlamydia. This was in contrast to the localization of MreB, which was restricted to predicted septal planes. MreB localization to septal planes provides direct experimental observation for the role of MreB in cell division and supports the hypothesis that it serves as a functional replacement for FtsZ in Chlamydia.

Expression of Folate Receptor-α (FRA) in Gynecologic Malignancies and its Relationship to the Tumor Type

An immunohistochemical evaluation for folate receptor-α (FRA) was undertaken to evaluate expression in gynecologic malignancies involving ovary, endometrium, and the fallopian tube. Commercial tissue microarrays were assessed using an optimized manual immunohistochemical method using MAb 26B3, a newly described monoclonal antibody. A positive result was defined as ≥5% of the sample demonstrating membranous staining. A semiquantitative staining algorithm, defined as the M-score, was used to analyze staining intensity between sample histotypes. MAb 26B3 showed uniform membranous staining and high levels of expression of FRA in ovarian, fallopian tube, and endometrial cancers. All serous ovarian cancers analyzed (70) were positive for FRA expression and no relationship to stage or grade was found. However, a significant difference for FRA expression, between serous and mucinous ovarian carcinomas, was demonstrated (P=0.014). In addition, approximately 90% of all endometrial adenocarcinomas were positive for FRA expression but, unlike ovarian serous carcinomas, a statistically significant relationship to grade was observed (P=0.0029). Although normal ovary is completely devoid of FRA immunoreactivity, normal fallopian tube and cortical serous/tubal inclusion cysts demonstrated uniform and intense FRA staining of columnar epithelium supporting the hypothesis that serous ovarian carcinoma is similar to the tubal epithelium. The data presented further support the hypothesis that FRA expression in gynecologic tumors is due to the cell of origin normally expressing this receptor. This is possibly due to an associated growth advantage, rather than the process of tumorigenesis resulting in aberrant expression of FRA per se.

Inhibitor of differentiation is overexpressed with progression of benign to malignant lesions and related with carcinoembryonic antigen–related cell adhesion molecule 1 distribution in mammary glands

The purpose of the study was to investigate the expression and association of inhibitor of differentiation (Id-1) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in benign, premalignant, and malignant lesions of human mammary glands. The study included 97 cases of benign, premalignant, and malignant lesions of human mammary glands including normal terminal duct lobular units, usual ductal hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma that were surgically excised at the Second Hospital of Shangdong University. Immunohistochemistry was used to determine the expression of Id-1 and CEACAM1. The Id-1 expression was increased with the progression of benign to malignant transformation (P < .05) and positively related with CEACAM1 different expression patterns (r = 0.279, P < .01) in benign, premalignant, and malignant lesions: apical membranous staining in benign, and cytoplasmic and uniform membranous staining in premalignant and malignant lesions. A positive correlation was found between Id-1 expression and morphologic classification of benign to premalignant and malignant lesions (r = 0.641, P < .0001). The CEACAM1 expression patterns showed a significance between benign, premalignant, and malignant lesions (P < .05). The Id-1 expression is increased with the progression of benign to malignant transformation and promotes the CEACAM1 expression; the CEACAM1 expression patterns are changed by movement from apical membrane to bilateral membrane and cytoplasm. That the Id-1 overexpression promotes the transformation of CEACAM1 expression patterns may occur at the early stage in the breast carcinogenesis; and the Id-1 should be regarded as the transforming factor, which may regulate the transformation of CEACAM1 expression patterns.

Temporal Variation in the Distribution of Hyaluronic Acid, CD44s, and CD44v6 in the Human Endometrium Across the Menstrual Cycle

Tissues undergoing rapid growth and regeneration contain hyaluronic acid (HA) as a prominent component of the extracellular matrix. The physiologic role of HA is partly mediated by its relationship with CD44, its major cell surface receptor. Given the extensive remodeling of the endometrium during the menstrual cycle, the authors sought to determine whether these changes are related to the levels of HA, CD44s, and CD44v6 in the endometrium. Archival paraffin embedded cell blocks from 10 cases of proliferative endometrium and 20 cases of secretory endometrium were retrieved from the surgical pathology files. Specimens from the secretory phase were subdivided into three categories: early secretory (day 15-18), mid-secretory (day 19-23), and late secretory (day 24-28). All cases were stained for hyaluronic acid, CD44s, and CD44v6. Sections from umbilical cord, tonsil, and squamous cell carcinoma served as positive controls for HA, CD44s, and CD44v6, respectively. Positive staining was defined as droplet to diffuse intracytoplasmic or extracellular staining for HA and uniform membranous staining for CD44. During the proliferative phase, the endometrial glands and the stroma were both negative for CD44s and CD44v6 in all cases. In the secretory phase, the endometrial glands were negative for CD44s in all cases, but CD44v6 was expressed in 12 (60%) of cases. In contrast, the stromal cells expressed CD44s in 18 (90%) cases and were negative for CD44v6 in all cases. HA staining was present in the endometrial stroma throughout the menstrual cycle but was most intense (3+) and diffuse during the midsecretory phase. There was perivascular staining for HA throughout the cycle; it was most intense adjacent to the spiral arterioles in the secretory phase. These data indicate temporal and geographic differences in HA and CD44 staining in the endometrium in concert with the menstrual cycle. The timing of peak staining of HA and CD44s in the stroma and the upregulation of CD44v6 in secretory glands are coincident with the period in which the endometrium is most receptive to embryo implantation. Whether these changes are mere hormonal consequences or actually help modulate the cyclical changes in the endometrium warrants further study.

Differentiation of mesothelioma from adenocarcinoma in serous effusions: The role of hyaluronic acid and CD44 localization

Differentiating cells of mesothelial origin from adenocarcinoma (ACA) based on morphology alone can be a diagnostic challenge, especially in cytological specimens. Malignant mesothelioma (MM) is characterized by accumulation of abundant intracellular hyaluronic acid (HA), a feature that is not reported in ACA. The purpose of this study was to evaluate the significance of cellular HA using an HA-specific binding peptide (HABP) and the expression of its principal receptor, the standard CD44 molecule (CD44S). Archival paraffin-embedded cell blocks of serous fluids from 28 cases of reactive mesothelial cells, 14 cases of MM, 20 cases of metastatic ovarian carcinomas, 17 cases of metastatic breast carcinomas, 12 cases of metastatic lung ACA, and 12 cases of metastatic gastrointestinal ACA were stained with HA using a biotinylated HABP and CD44S. Positive staining was defined as droplet to diffuse cytoplasmic staining for HA and uniform membranous staining for CD44S. All MMs and 93% (26/28) of the benign mesothelial cells were positive for intracytoplasmic HA vs. none of ACAs. CD44S was expressed in 100% (28/28) of mesothelial hyperplesia, 86% (12/14) of MMs, 70% (14/20) of ovarian carcinomas, 29% (5/17) of breast carcinomas, 25% (3/12) of gastrointestinal ACAs, and 8% (1/12) of lung ACAs. In MM and reactive mesothelial cells, CD44S stained cell membranes diffusely with highlights on the villous surfaces and in ACA it was focal and confined to cell membranes. Immunostaining with HA is a reliable marker that can distinguish between cells of mesothelial origin (reactive mesothelial cells and MM) and ACA. The CD44S staining pattern of cells of mesothelial origin is of diagnostic significance. CD44 may prove useful in conjunction with other stains in the differential diagnosis of mesothelioma and ADA.

Use of E-cadherin and CD44 aids in the differentiation between reactive mesothelial cells and carcinoma cells in pelvic washings.

The presence of malignant cells in peritoneal washings is an independent prognostic factor in the evaluation of gynecologic malignancies. The differentiation between reactive mesothelial cells and carcinoma cells can be a diagnostic challenge based on morphology alone. The expression of some cell adhesion molecules may be helpful in the differential diagnosis. To evaluate the specificity of 2 transmembrane cell adhesion proteins (E-cadherin and CD44) in the differentiation of mesothelial cells from carcinoma cells in pelvic washings, formalin fixed, paraffin embedded cell blocks of pelvic washings from 19 cases of metastatic ovarian adenocarcinoma and 16 cases of benign peritoneal washings were immunostained with monoclonal antibodies to E-cadherin and CD44. The staining patterns were evaluated blindly by three observers. Positive staining was defined as uniform membranous staining for each marker. Fourteen benign peritoneal washings (87.5%) demonstrated immunoreactivity with anti-CD44. On the contrary, only four adenocarcinomas (21.1%) demonstrated anti-CD44 immunoreactivity. E-cadherin expression was identified in only 2 benign peritoneal washings (12.5%) whereas 16 adenocarcinomas (84.2%) strongly expressed E-cadherin. The differences in immunostaining for both CD44 and E-cadherin between benign and malignant peritoneal washings were statistically significant. The combination of positive staining for E-cadherin and negative staining for CD44 was 100% specific for metastatic adenocarcinoma, whereas a combination of negative staining for E-cadherin and positive staining for CD44 was 100% specific for reactive mesothelial cells. Both E-cadherin and CD44 reliably distinguish reactive mesothelial cells from adenocarcinoma. The combination of E-cadherin/CD44 is highly specific and is a useful diagnostic adjunct with which to distinguish benign reactive mesothelial cells from adenocarcinoma in pelvic washings.

Characterization and distribution of an oncofetal antigen (M2A antigen) expressed on testicular germ cell tumours.

M2A antigen is an oncofetal antigen associated with germ cell neoplasia, present in testis on fetal gonocytes and re-expressed on carcinoma in situ (CIS) and germ cell tumours. We developed a panel of monoclonal antibodies (mAb), M2A (IgG2a), D1-26 (IgG2b) and D2-40 (IgG1), to this antigen in order to characterize its structure and study its distribution among germ cell tumours. M2A antigen was purified by sequential lectin and antibody affinity chromatography and characterized as a monomeric Mr 40 000 surface sialoglycoprotein, extensively glycosylated with O-linked carbohydrate structures, but devoid of N-linked sugars. Terminal sialic acid residues were required for reactivity with mAb M2A and D1-26, but not D2-40. Sections of 69 testicular germ cell tumours, fixed in formalin and embedded in paraffin, were stained with mAb D2-40 to examine the distribution of M2A antigen. Uniform membrane staining was observed in seminomas, and focal staining in 69% of embryonal carcinomas, 29% of teratomas and 25% of yolk sac tumours. CIS in the vicinity of all germ cell tumours also displayed uniform membrane staining. The characterization of M2A antigen, and the development of mAb which react with it in conventionally preserved archival specimens, provide important initiatives to study the origin and progression of germ cell neoplasia. © 1999 Cancer Research Campaign

Expression and function of the complement membrane attack complex inhibitor protectin (CD59) in human prostate cancer.

Protectin (CD59) inhibits homologous complement-mediated cytolysis by preventing formation of the membrane attack complex at the point of insertion and polymerization of C9 into cell membranes. The present study investigated the expression and function of CD59 on human prostatic tumor cells in situ and on 5 human prostate cell lines in vitro originating from either metastatic tumors or benign prostate hypertrophy epithelial cells. Immunohistochemical staining of prostate carcinoma tissue with monoclonal antibody (MAb) MEM43 revealed weak to moderately strong expression of CD59 by prostate glandular epithelial cells. Flow cytometry with MEM43 demonstrated that the 5 prostate cell lines expressed different relative quantities of CD59. Indirect immunofluorescence analysis revealed uniform membrane staining of DU145 and PC3 cell lines with no membranous granularity in the staining pattern. Western immunoblots with MAb BRIC 229 showed that PC3 and DU145 cells express CD59 with a m.w. of 18-25 kDa. Treatment of DU 145 and PC3 cells with phosphatidylinositol-specific phospholipase C caused a significant decrease of CD59 expression indicating that the CD59 expressed by prostate cancer cells is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage. PC3 and DU145 cells were completely resistant to human complement-mediated cytolysis but became sensitive to killing in the presence of the CD59-neutralizing MAb YTH53.1. We conclude that malignant and benign human prostate cells express CD59 that is GPI-linked to the cell surface and that CD59 may regulate the immunological response to cancerous prostate cells by protecting the cells from the cytolytic activity of complement.

CD44 expression in benign and malignant nevomelanocytic lesions

CD44 is an integral membrane glycoprotein that is a principal receptor for hyaluronan and plays a role in cell-extracellular matrix interactions. Recent studies of melanomas in mouse models have suggested that increased CD44 expression by these tumors may relate to metastatic potential. Immunohistochemical expression of CD44 (standard [s] and variant [v6]) in benign and malignant nevomelanocytic lesions was assessed in formalin-fixed, paraffin-embedded tissue and was correlated with histological parameters and prognostic factors. Cases included benign nevi (three junctional, four compound, five intradermal, five blue, six Spitz, one deep penetrating), architecturally disordered (dysplastic) nevi (three, and primary (22) and metastatic melanomas (eight). All of the benign lesions showed diffuse and essentially uniform membrane staining of CD44s in nevomelanocytic cells, regardless of lesion size, depth, or extent of dermal involvement. In contrast, semiquantitative analysis (0 to 3+) of the primary melanomas showed heterogeneous and decreased staining of CD44s, which inversely correlated with lesion size (−0.569) and depth of invasion (−0.622 and −0.617 for Breslow's depth and Clark's level, respectively). These results were significant at P < .05. CD44s expression in metastases paralleled that of their respective primaries. None of the benign nevomelanocytic lesions showed CD44v6 staining. In contrast, all of the malignant nevomelanocytic lesions showed cytoplasmic staining of the tumor cells. Pretreatment with chondroitinase did not alter CD44s staining. CD44s expression by immunohistochemical determination is uniform in benign nevomelanocytic lesions. Malignant melanomas show decreased, heterogeneous staining that inversely correlates with increasing size, depth, and level of invasion. CD44 expression may be a prognostic indicator in malignant melanomas. Tumor staining with anti-chondroitin sulfate monoclonal antibodies suggests that CD44s may be expressed as a chondroitin sulfate proteoglycan in primary melanomas.

Decreased levels of CD44 protein and mRNA in prostate carcinoma. Correlation with tumor grade and ploidy.

CD44, a transmembrane protein, is associated with cell-cell and cell-matrix interaction and with tumor growth and metastasis. Expression of both standard form and variant isoforms of CD44 protein has been associated with aggressive behavior and metastasis in various tumors, but has not been characterized in prostate adenocarcinoma (PAC). The expression of CD44 standard (CD44s) and splice variant v3, v4/5, v6, v7/8, and v10 proteins were studied in 109 PACs and correlated with tumor grade, DNA ploidy, and mRNA levels. Monoclonal antibodies against the various CD44 proteins were applied to microwave irradiated, formalin fixed, paraffin embedded sections. The DNA content of the tumors was evaluated by the Feulgen method with the CAS200 Image Analyzer. Total RNA exhibiting 18s and 28s bands was derived from two benign prostatic tissues and 5 PACs exhibiting decreased levels of CD44 protein by immunohistochemistry. The RNA was analyzed with reverse transcriptase polymerase chain reaction using CD44 specific primers. The basal cells of the benign prostatic acini revealed uniform membranous staining for CD44s, v3, and v6 in 95-97% of cases. Similar staining was observed for v4/5, v7/8, and v10 in 40%, 30%, and 2% of cases, respectively. Secretory epithelial cells of the benign prostatic acini showed predominant expression of CD44s (97% of cases). Staining for CD44 variant proteins (v3, v4/5, v6, v7/8, and v10) in this location ranged from 9-22% of cases. Approximately 70% of the PACs showed significant loss of CD44s expression, which correlated with high tumor grade (Gleason > or = 7) (P = 0.01) and aneuploid status (P = 0.002). In 93-98% of the PACS, there was a complete lack of membranous expression for all CD44 variant isoforms. The metastatic PACS did not show preferential expression of either the standard form or any variant isoform. The cDNA from the normal prostates yielded a prominent CD44 standard form polymerase chain reaction product at 482 base pair (bp) and variant isoforms at approximately 650 and 850 bp. No CD44 products could be amplified from the subset of five PAC cDNAs, even when present at four-fold excess. PACS exhibit down-regulation of CD44 protein expression, which correlates with high tumor grade and aneuploidy. v6 and v3 isoforms were preferentially expressed in the basal cells of benign prostatic acini. Based on a subset of cases, loss of CD44 protein expression is associated with decreased abundance of CD44 mRNA.

Studies on the cytotoxic immune reaction. III. Localization of metabolically dependent cell surface antigens.

Fluorescein-tagged duck anti-rabbit cornea serum was used to demonstrate changes in the localization of metabolically dependent antigens (MDA) on surfaces of resting versus activated corneal endothelial cells. Actively growing cells in culture exhibited uniform membrane staining which was most pronounced at the periphery of the tissue cultures. In the metabolically less-active center, spotty rather than continuous fluorescence was seen. Cells with a very low metabolic activity, e.g., those kept at 4 °C, did not stain at all. This inhibition was reversible, as the cells transferred back to 37 °C were able to bind the antibodies. Cultured cells dispersed with pronase or trypsin reacted with the tagged antiserum and demonstrated membrane fluorescence. However, dispersed cells from excised normal cornea did not stain. The MDA, although not detectable on normal-cell surfaces, were present in these cells, for the isolated cell membrane fraction was capable of absorbing out antibodies to the MDA. The antigenic determinants involved in the reaction with fluorescein-tagged anti-tissue antibodies are thus present but masked in cell membranes of normal tissues &lt;i&gt;in vivo&lt;/i&gt; and can be unmasked by an increase in cellular metabolism and/or cell division but not by treatment with proteolytic enzymes.