Prdm5 suppresses Apc(Min)-driven intestinal adenomas and regulates monoacylglycerol lipase expression.

PRDM proteins are tissue-specific transcription factors often deregulated in diseases, particularly in cancer where different members have been found to act as oncogenes or tumor suppressors. PRDM5 is a poorly characterized member of the PRDM family for which several studies have reported a high frequency of promoter hypermethylation in cancer types of gastrointestinal origin. We report here the characterization of Prdm5 knockout mice in the context of intestinal carcinogenesis. We demonstrate that loss of Prdm5 increases the number of adenomas throughout the murine small intestine on an Apc(Min) background. By using the genome-wide ChIP-seq (chromatin immunoprecipitation (ChIP) followed by DNA sequencing) and transcriptome analyses we identify loci encoding proteins involved in metabolic processes as prominent PRDM5 targets and characterize monoacylglycerol lipase (Mgll) as a direct PRDM5 target in human colon cancer cells and in Prdm5 mutant mouse intestines. Moreover, we report the downregulation of PRDM5 protein expression in human colon neoplastic lesions. In summary, our data provide the first causal link between Prdm5 loss and intestinal carcinogenesis, and uncover an extensive and novel PRDM5 target repertoire likely facilitating the tumor-suppressive functions of PRDM5.

Alpha-actinin interactions with syndecan-4 are integral to fibroblast-matrix adhesion and regulate cytoskeletal architecture.

All cells of the musculoskeletal system possess transmembrane syndecan proteoglycans, notably syndecan-4. In fibroblasts it regulates integrin-mediated adhesion to the extracellular matrix. Syndecan-4 null mice have a complex wound repair phenotype while their fibroblasts have reduced focal adhesions and matrix contraction abilities. Signalling through syndecan-4 core protein to the actin cytoskeleton involves protein kinase Cα and Rho family G proteins but also direct interactions with α-actinin. The contribution of the latter interaction to cell-matrix adhesion is not defined but investigated here since manipulation of Rho GTPase and its downstream targets could not restore a wild type microfilament organisation to syndecan-4 null cells. Microarray and protein analysis revealed no significant alterations in mRNA or protein levels for actin- or α-actinin associated proteins when wild type and syndecan-4 knockout fibroblasts were compared. The binding site for syndecan-4 cytoplasmic domain was identified as spectrin repeat 4 of α-actinin while further experiments confirmed the importance of this interaction in stabilising cell-matrix junctions. However, α-actinin is also present in adherens junctions, these organelles not being disrupted in the absence of syndecan-4. Indeed, co-culture of wild type and knockout cells led to adherens junction-associated stress fibre formation in cells lacking syndecan-4, supporting the hypothesis that the proteoglycan regulates cell-matrix adhesion and its associated microfilament bundles at a post-translational level. These data provide an additional dimension to syndecan function related to tension at the cell-matrix interface, wound healing and potentially fibrosis.

Syndecan signaling: when, where and why?

Syndecans are the only family of transmembrane heparan sulphate proteoglycans. Invertebrates all appear to have one Syndecan core protein, but in mammals there are four. Examination of the core protein sequences shows that the cytoplasmic domains are the most conserved. This suggests that Syndecans make important interactions and/or signalling contributions. It has been established that all syndecans can interact with proteins of the actin-associated cytoskeleton, but details of signalling have been harder to ascertain. However, it appears that Syndecans can signal, primarily to the cytoskeleton, and the details are reviewed here. Only for vertebrate syndecan-4 is there substantial detail, and it remains a challenge to dissect the functions and signalling of other vertebrate and invertebrate syndecans.

Fluoroquinolone's effect on growth of human chondrocytes and chondrosarcomas. In vitro and in vivo correlation.

Clinical and in vitro studies have demonstrated that fluoroquinolones are toxic to chondrocytes; however, the exact mechanism of fluoroquinolone arthropathy is unknown. We investigated the toxicity of ciprofloxacin on normal cartilage and on cartilaginous tumors. Normal human cartilage, enchondroma, and chondrosarcoma explants were cultured either alone or with the addition of ciprofloxacin at 1, 10, or 20 mg/L of medium. Samples were collected up to twenty-one days after treatment and were processed for electron microscopy and conventional light microscopy. The specimens were characterized morphologically with use of conventional light microscopy, electron microscopy, and immunohistochemistry to identify extracellular matrix, cell proliferation, and apoptosis. Cultures of normal chondrocytes expressed type-II collagen. Electron microscopy revealed a large amount of glycogen in the cells; the presence of fat droplets, rough endoplasmic reticulum, and prominent Golgi apparatus; and a proteoglycan layer surrounding the cells. With prolonged ciprofloxacin treatment and with increased doses, there was an increase in dilated rough endoplasmic reticulum, the appearance of phagosomes, and disintegrated bundles of vimentin filaments. The treated chondrocytes showed a decrease in cell proliferation, but there was no induction of apoptosis or effect on the expression of extracellular matrix proteins. Ciprofloxacin-treated chondrosarcoma cultures and tissue samples showed changes in cartilage matrix composition. Ultrastructural analysis demonstrated clumped glycogen, dilation of endoplasmic reticulum, numerous abnormal lysosomes containing degeneration products, and a decreased proteoglycan deposit surrounding the tumor cells. Treated chondrosarcoma cells and tissue specimens did not proliferate, and apoptosis was induced. In contrast, the in vitro growth of other noncartilaginous malignant tumors like osteosarcoma and liposarcoma was unaffected by ciprofloxacin. Our results indicate that ciprofloxacin is toxic to chondrocytes. In vitro and in vivo treated chondrosarcomas are the most affected.

Utility of CK7 and CK20 immunohistochemistry in the detection of synchronous breast and colon carcinoma in a pleural effusion: a case report and supporting survey of archival material.

We present a case of synchronous breast and colon carcinoma in a pleural effusion, to our knowledge the first such reported case in the English-language literature. The patient was a 55-yr-old white female with known metastatic breast and colon carcinoma who developed a malignant pleural effusion which demonstrated two strikingly different populations of malignant cells by immunohistochemical study of cell block material. One cell population demonstrated a cytokeratin (CK)7+/CK20-/ER+ phenotype, while the other demonstrated a CK7-/CK20+/ER- phenotype, consistent with breast and colon origin, respectively. An immunohistochemical survey of archival breast and colon primary and metastatic carcinomas confirmed the established CK7+/CK20- phenotype of breast and CK7-/CK20+ phenotype of colon primary carcinomas, and the maintenance of this phenotype in metastases thereof. A survey of benign and malignant mesothelial lesions confirmed the absence of staining for estrogen receptor, but showed 6/10 cases weakly positive for CK20, which has not been described in other published series. This unusual case graphically illustrates the utility of cytokeratin subset immunohistochemistry in effusion cytology.

Cytokeratin 20 immunoreactivity in renal oncocytomas.

Cytokeratins (CKs) are a group of 20 antigenically distinct intermediate filaments, generally confined to epithelia and their neoplasms. Immunostaining for CKs, in particular coordinate staining for CK7 and CK20, has become a useful tool in diagnostic pathology. Although studies defining CK distribution in neoplasms identify 0--7.7% of renal cell carcinomas (RCCs) positive for CK20, none has described the incidence of CK20 immunopositivity in renal oncocytomas (ROs). Distinction between RCC and RO may be difficult but this distinction is clinically significant, prompting us to establish the incidence of CK20 positivity in RO. We selected fifteen surgical cases of RO from our archives and studied their immunoreactivity for CKs including CK7 and CK20; 12/15 (80%) were positive for CK20, with variation in the number of cells staining. There was also variation in the distribution of CKs within the cells, including diffuse cytoplasmic, perinuclear, and a punctate or dot-like pattern. Such punctate staining corresponds to cytoplasmic balls of intermediate filaments and has been described with CAM 5.2 in RO and CK20 in Merkel cell carcinomas. Our findings suggest that CK20 immunohistochemistry is a useful tool for distinguishing RCCs from ROs. (J Histochem Cytochem 49:919-920, 2001)

Plasmacytoma with aberrant expression of myeloid markers, T-cell markers, and cytokeratin.

Plasmacytomas are localized neoplastic proliferations of monoclonal plasma cells. When multifocal, the process is referred to as multiple myeloma. These lesions exhibit a pattern of antigen expression and cytomorphology that usually leads to a ready diagnosis. However, potentially troublesome variations in immunophenotype occur. We describe a case of a plasmacytoma from a patient who presented with sudden onset of pain and a lytic lesion of the left proximal humerus. Hematoxylin and eosin-stained sections showed a lymphoproliferative lesion composed of large lymphoid cells, some with plasmacytoid and immunoblastic features. The lesion also showed significant mitotic activity. Immunohistochemical staining was positive for CD45 (LCA), CD56 (N-CAM), CD43 (MT1), and cytokeratin CAM5.2. There was also clonal staining for lambda light chains. In addition, flow cytometric analysis showed positivity for myeloid markers such as CD13, CD33, CD38, and CD138. Significant negative markers include CD20 (L26), CD45RO (UCHL-1), and CD79alpha. The unusual phenotypic features of this plasmacytoma illustrate potential diagnostic pitfalls. It is important to fully study such lesions to correctly classify them, because this has significant impact on prognosis and management.

Loss of high-molecular-weight cytokeratin antigenicity in prostate tissue obtained by transurethral resections.

OBJECTIVE: Staining of prostatic basal cells for the expression of high-molecular-weight cytokeratin has been suggested as a way of distinguishing benign from malignant prostate glands. We evaluated the utility of high-molecular-weight cytokeratin in the diagnosis of malignancy in prostate specimens obtained in various ways. DESIGN: Prostate tissues obtained from needle biopsies, transurethral resections, and total prostatectomies were immunostained with monoclonal antibody 34betaE12, an antibody directed against high-molecular-weight cytokeratins. RESULTS: Antiserum to high-molecular-weight cytokeratin only stained the basal cells in normal glands in 3 (12%) of 25 specimens obtained by transurethral resection. Other antigens, such as the alternate 10-nm filament protein vimentin, were unaffected and were detected in 100% of these specimens. However, keratin antigenicity in transurethral biopsies could be restored in these specimens by antigen retrieval in a low pH citrate buffer using a microwave heat technique. Keratin staining in needle biopsies and total prostatectomies was unaffected. CONCLUSION: In summary, our results indicate the technique of transurethral resection results in a specific loss of keratin antigenicity. This limits the utility of anticytokeratin 34betaE12 in interpreting transurethral resections without the application of antigen retrieval.

Comparison of glycoprotein expression between ovarian and colon adenocarcinomas.

OBJECTIVE: Tumor-associated antigens may be expressed as surface glycoproteins. These molecules undergo qualitative and quantitative modifications during cell differentiation and malignant transformation. During malignant transformation, incomplete glycosylation is common, and certain glycosylation pathways are preferred. These antigens might help distinguish between ovarian and colonic adenocarcinomas in the primary and metastatic lesions. Different cytokeratins have been proposed as relatively organ-specific antigens. DESIGN: We used monoclonal antibodies against T1, Tn, sialosyl-Tn, B72.3, CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 to detect tumor-associated glycoproteins and keratin proteins in ovarian and colonic carcinomas. RESULTS: CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 can distinguish between colonic and serous or endometrioid adenocarcinomas of the ovary in both primary and metastatic lesions. Mucinous ovarian adenocarcinomas differed in that they express carcinoembryonic antigen and cytokeratins 7 and 20 and weakly express CA125. The other glycoprotein antigens were equally expressed by ovarian and colonic adenocarcinomas and therefore were of no use in distinguishing between these 2 entities. CONCLUSION: A panel of monoclonal antibodies against cytokeratins 7 and 20 antigens, CA125, and carcinoembryonic antigen is useful in differentiating serous and endometrioid adenocarcinomas of the ovary from colonic adenocarcinomas. Mucinous ovarian adenocarcinomas cannot be distinguished from colonic adenocarcinomas using immunohistochemistry.

Epstein-Barr virus-associated adult respiratory distress syndrome in a patient with AIDS: a case report and review.

BACKGROUND: Epstein-Barr virus (EBV) infection has been associated with fatal pneumonitis in immunocompetent patients. We present a case of fatal adult respiratory distress syndrome caused by EBV infection in a patient with acquired immunodeficiency syndrome (AIDS), to our knowledge the first such reported case, along with a survey of archival autopsy cases to assess baseline expression of EBV in AIDS patients. DESIGN: The case patient's autopsy material was studied exhaustively for infectious agents by culture, histochemistry, and immunohistochemistry, with negative results. Formalin-fixed paraffin-embedded lung, spleen, lymph node, and liver tissue were further studied by in situ hybridization using a probe for EBV early RNA (EBER, Kreatech). The same method was applied to lymphoid tissues from eight other archival AIDS autopsy cases. Case patient tissues were also examined by electron microscopy. RESULTS: Strikingly numerous lymphocytes were positive for EBV early RNA in the case patient's spleen, lymph nodes, and hepatic portal areas. In addition to positive lymphocytes in the lung, EBV-infected pneumocytes were also present. Electron microscopy also demonstrated viral material in lymphocytes and pneumocytes. Of the archival cases studied, only one spleen was found to have rare positive lymphocytes. CONCLUSION: Primary or reactivation EBV infection may represent a previously underreported cause of morbidity and mortality in AIDS patients. Autopsy tissues from AIDS patients do not routinely show overexpression of EBV early RNA by in situ hybridization, making this technique ideal for assessing the contribution of EBV to terminal events in these patients.

Noninvasive prenatal diagnosis. Use of density gradient centrifugation, magnetically activated cell sorting and in situ hybridization.

OBJECTIVE: To develop a noninvasive method suitable for clinical prenatal diagnosis. STUDY DESIGN: Fetal nucleated erythrocytes were separated from peripheral blood of 17 healthy pregnant women using small magnetically activated cell sorting columns (MiniMACS) following density gradient centrifugation and dual antibody labeling methods. The protocol was designed to compare the efficacy of antitransferrin receptor (CD71)/antiglycophorin A (GPA) antibodies with antithrom-bospondin receptor (CD36)/anti-GPA antibodies in identifying nucleated erythrocytes in maternal blood. Cytospin preparations of the isolated cells were subjected to in situ hybridization with specific DNA probes for the Y chromosome and chromosome 21 to confirm the fetal origin. RESULTS: After MiniMACS the enrichment factors for the CD71/GPA- and CD36/GPA-positive cells from maternal blood were similar, and the percentages of fetal cells recovered did not differ. Seven of seven male pregnancies were correctly identified. One case of trisomy 21 was detected. CONCLUSION: The in situ hybridization analysis of fetal nucleated erythrocytes isolated from maternal blood using single density gradient centrifugation, anti-CD71/anti-GPA immunostaining and MiniMACS could be an accurate, sensitive and noninvasive method for prenatal diagnosis.

Expression of the RET/PTC fusion gene as a marker for papillary carcinoma in Hashimoto's thyroiditis.

Hashimoto's thyroiditis is an inflammatory disease of the thyroid gland with autoimmune etiology. Patients afflicted with Hashimoto's have a higher risk of thyroid malignancies such as papillary thyroid carcinoma. In the present study, we investigated the frequency of papillary thyroid carcinoma specific genes in patients diagnosed with Hashimoto's disease. The newly identified oncogenes RET/PTC1 and RET/PTC3 provide useful and specific markers of the early stages of papillary carcinoma as they are highly specific for malignant cells. Using a sensitive and specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we found messenger RNA (mRNA) expression for the RET/PTC1 and RET/PTC3 oncogenes in 95% of the Hashimoto's patients studied. All Hashimoto's patients presenting without histopathologic evidence of papillary thyroid cancer showed molecular genetic evidence of cancer. These data suggest that multiple, independent occult tumors exist in these patients at high frequency.

Expression of very low density lipoprotein receptor in the vascular wall. Analysis of human tissues by in situ hybridization and immunohistochemistry.

The recently cloned very low density lipoprotein (VLDL) receptor binds triglyceride-rich, apolipoprotein-E-containing lipoproteins with high affinity. The observation that VLDL receptor mRNA is abundantly expressed in extracts of tissues such as skeletal muscle and heart, but not liver, has led to the hypothesis that this receptor may facilitate the peripheral uptake of triglyceride-rich lipoproteins. However, little information is available concerning the types of cells that express this receptor in vivo. As expression of the VLDL receptor in the vascular wall might have important implications for the uptake and transport of triglyceride-rich lipoproteins, and perhaps facilitate the development of atherosclerosis in hypertriglyceridemic individuals, we used in situ hybridization and immunohistochemistry to determine whether VLDL receptor mRNA and protein was expressed in human vascular tissue. We observed expression of the receptor by both endothelial and smooth muscle cells within normal arteries and veins, as well as within atherosclerotic plaques. In the latter, the VLDL receptor was also expressed by macrophage-derived foam cells. The widespread distribution of the VLDL receptor in vascular tissue suggests a potentially important role for this receptor in normal and pathophysiological vascular processes.

Osteoclastic giant cell tumor of the pancreas: an immunohistochemical study.

A case of an osteoclastic giant cell tumor of the pancreas is presented. Immunohistochemical studies were performed, which showed keratin (CAM, AE1) and epithelial membrane antigen positivity in the tumor cells. The findings support an epithelial origin for this tumor.

Proliferative activity in metastatic malignant melanoma: comparison of pre-treatment and post-treatment metastases.

In this study, the proliferative activity of malignant melanoma metastases was assessed before and after isolated limb perfusion chemotherapy by quantitating AgNORs, mitoses and PCNA activity. No significant difference in either AgNOR count, mitotic activity or PCNA index was observed. We conclude that AgNOR count, mitotic activity and PCNA index were not significantly effected by isolated limb perfusion chemotherapy.

Quantitation in immunohistochemistry. A research method or a diagnostic tool in surgical pathology?

Quantification of immunohistochemical methods is feasible by two ways, namely microscope photometry and TV densitometry. The rapid progress of computer technology argues largely in favour of the second system. From the immunohistochemical methods either ABC, the PAP or the APAAP method should be chosen. Diaminobenzidine is the H2-donor of choice for the peroxidase mediated systems, whereas Fast Red TR or Neufuchsin can be used with the APAAP system. The test efficiency, and the precision of measurements should be investigated in each laboratory doing quantitative immunohistochemistry. Low levels of coefficients of variation (CV) should be reached for one field of interest. Examples of introducing the method of quantitative immunohistochemistry in a routine laboratory are discussed. The demonstration of either a steroid receptor or a proliferation marker, such as PCNA or Ki67, are seemingly of the greatest clinical interest.

Argyrophilic nucleolar organizer regions and proliferating cell nuclear antigen/cyclin in Kaposi's sarcoma. Comparison with morphology and with immunoperoxidase staining for factor VIII-related antigen.

The proposal that Kaposi's sarcoma undergoes cellular transformation from early to late stages was studied with argyrophilic nucleolar organizer regions, proliferating cell nuclear antigen/cyclin, and immunoperoxidase staining for factor VIII-related antigen. Staining of argyrophilic nucleolar organizer regions was significantly increased in the plaque/nodular stage compared to the patch stage. The endothelial-selective marker factor VIII-related antigen stained more intensely in patch stage lesions. This was inversely correlated with staining of argyrophilic nucleolar organizer regions. Proliferating cell nuclear antigen/cyclin staining did not correlate with tumor stage or with factor VIII-related antigen. The changes in argyrophilic nucleolar organizer regions and factor VIII-related antigen staining are evidence for cellular transformation in Kaposi's sarcoma.

Detection of human papillomavirus in laryngeal lesions by in situ hybridization.

Human papillomavirus (HPV) is associated with human neoplasms of squamous epithelium. Squamous papillomas and verrucous carcinomas are two types of squamous neoplasms of the larynx that present difficult problems in differential diagnosis. Using in situ hybridization with biotinylated DNA probes, we examined benign squamous papillomas and verrucous squamous carcinomas of the larynx for the presence of HPV. Forty-two biopsy specimens from 18 patients with laryngeal papillomas and 11 biopsy specimens from seven patients with verrucous carcinomas were obtained from the files of Pennsylvania Hospital, Philadelphia, PA. Tissue sections were hybridized with an HPV DNA cocktail. The HPV-positive cases then were subtyped further with DNA probes specific for HPV subtypes 6/11, 16/18, and 31/33/35. All benign squamous papillomas (42 of 42) were positive for HPV subtype 6/11. None of the verrucous carcinomas contained demonstrable HPV (none of 11). Some of the squamous papillomas were recurrences, which shows the persistence of the virus. These results indicate that laryngeal papillomas may be related to HPV, but verrucous carcinomas are not.

Expression of urokinase receptors by human trophoblast. A histochemical and ultrastructural analysis.

BACKGROUND: Through their ability to invade endometrium, remodel the uterine spiral arteries, and sustain placental blood fluidity, trophoblast cells play a central role in establishing and maintaining the integrity of the uteroplacental vasculature. The expression of urokinase receptors by trophoblast may facilitate these processes by focusing plasminogen activator activity to discrete sites on the cell surface and promoting the activation of cell-bound plasminogen. However, although urokinase receptors are expressed by cultured trophoblast, the expression of these receptors by trophoblast in vivo has not been examined. EXPERIMENTAL DESIGN: Immunohistochemistry and immunoelectron microscopy were used to characterize the expression of urokinase receptors by villous and extravillous trophoblast at several points in gestation. RESULTS: Urokinase receptors were expressed in a polarized fashion at the leading edge of migrating extravillous trophoblast cells. Receptors were also abundantly expressed during the first and second trimesters of gestation by villous trophoblast, where they were located on apical villous projections and within intracellular vacuoles, a subset of which were lysosomes. CONCLUSIONS: The polarized expression of urokinase receptors by invasive extravillous trophoblast cells is consistent with a role for these receptors in mediating the extent and directionality of trophoblast migration. In contrast, the expression of urokinase receptors by villous trophoblast, which are not actively invasive in vivo, may serve to facilitate the generation of plasmin at the interface of these cells with maternal plasma, thereby limiting the deposition of fibrin within the placental intervillous spaces. Diminished urokinase receptor expression by villous trophoblast at term may represent a physiologic adaptation to diminish local fibrinolysis and limit hemorrhage at parturition.

The correlation between HIV seropositivity, cervical dysplasia, and HPV subtypes 6/11, 16/18, 31/33/35.

Twenty-one human immunodeficiency virus (HIV) seropositive patients were studied to determine the prevalence of cervical dysplasia, the distribution of human papilloma virus (HPV) subtypes, and the utility of cytologic diagnosis in this population. The patients ranged in age from 18 to 41 years. HIV infection was diagnosed in all patients by ELISA method and confirmed by Western blot. Patients were interviewed to document risk factors for HIV infection and cervical dysplasia. All were examined colposcopically and identifiable lesions were biopsied. In patients with normal colposcopic findings four quadrant biopsies were performed. Cervical and endocervical smears were obtained on all patients at the time of colposcopy and blood was collected for T-cell studies. Eighteen patients (86%) had abnormal cytologic findings. The Pap smear was able to identify 10 of 13 biopsy-proven epithelial abnormalities. In 14 patients there was sufficient tissue for HPV typing by in situ hybridization; the HPV subtypes identified were 6/11 (10 patients), 16/18 (9 patients), and 31/33/35 (9 patients). Dysplasia was present in 11/14 (79%) of the specimens submitted for subtyping. The absolute CD4 cell counts were 342 per mm3 for the dysplasia group and 281 per mm3 for the patients without dysplasia. Patients with dysplasia did not differ significantly from patients without dysplasia in regard to risk factors for cervical dysplasia, including history of STD, tobacco use, multiple sexual partners, age at first coitus, and parity. In addition, history of medication use and risk factors associated with HIV infection were similar in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)