Telomerase activity and telomerase subunits gene expression patterns in neuroblastoma: a molecular and immunohistochemical study establishing prognostic tools for fresh-frozen and paraffin-embedded tissues.
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PURPOSE: We have recently demonstrated that telomerase activity (TA) is an independent prognostic factor in neuroblastomas. In the present study, the prognostic impact of TA and gene expression of the three major telomerase subunits is evaluated by molecular and immunohistochemical techniques in fresh-frozen and paraffin-embedded tissues. PATIENTS AND METHODS: One hundred thirty-three neuroblastomas of all stages were analyzed for TA. The TA levels of 75 neuroblastoma cases were correlated with gene expression of telomerase subunits hTRT, human telomerase RNA (hTR), and telomerase protein 1 (TP1) by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), using an innovative approach on the LightCycler instrument (Roche Diagnostics, Mannheim, Germany). For selected cases, the applicability of RT-PCR and immunohistochemistry for hTRT expression analysis was investigated in paraffin-embedded tissues. TA and subunit expression patterns were correlated with traditional prognostic indicators and disease outcome. RESULTS: TA was present in a total of 39 (29.3%) of 133 neuroblastomas and in 31 (29.8%) of 104 initial neuroblastomas without cytotoxic pretreatment. TA was significantly correlated with both event-free and overall survival (P <.0001). Furthermore, we found a significant correlation between expression levels of TA and hTRT (P <.0001) as well as hTR (P <.001). Multivariate analysis revealed only TA and tumor stage but not serum lactate dehydrogenase, MYCN amplification, or age at diagnosis as independent prognostic factors. CONCLUSION: The significant correlation with clinical outcome strongly recommends that analysis of TA be incorporated into the clinical investigation of each individual neuroblastoma at the time of diagnosis. Because the mere presence or absence of TA without further quantification is sufficient basis for predicting disease outcome, the telomeric repeat amplification protocol assay could be complemented with but not replaced by analysis of hTRT or hTR expression. (+info)
Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed paraffin-embedded tissues: experience with 63 markers.
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Formalin fixation produces cross-links between the proteins and the fixative that alter the ability of some antibodies to recognize antigens. We used formalin-fixed, paraffin-embedded tissues to compare two different antigen retrieval methods for 63 antibodies used in the diagnosis of infectious and neoplastic diseases of animal species. Eighty-four percent of the antibodies needed some type of antigen retrieval for optimal results. Of those antibodies, 67.7% were monoclonal and 32.3% were polyclonal. Steam heat was the method of choice for 31 antibodies. Ten antibodies reacted only with steam heat, but 9 antibodies did not react when steam heat was used. Optimal results were obtained with enzyme digestion for 22 antibodies. Only 10 antibodies yielded optimal results without antigen retrieval; 64% of these antibodies were polyclonal. All antibodies against cytokeratins were optimally retrieved with proteinese K. Antigen retrieval appears to be necessary for the majority of antibodies when used with formalin-fixed, paraffin-embedded tissues. (+info)
Duplex PCR for differential identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin- fixed paraffin-embedded tissues from cattle.
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We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex. (+info)
CyA-mediated renal interstitial and vascular lesions in the rat under low-sodium diet.
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Nephrotoxicity of CyA was analyzed histologically in rats fed a low-sodium diet. CyA was subcutaneously administered daily at a dose of 15 mg/kg for 10 or 35 days with or without prior uninephrectomy (UNT) in male Sprague-Dawley rats receiving a low-sodium diet (0.03% sodium). CyA-administered rats showed impaired renal function as well as tubulo-interstitial lesions, such as edema, tubular basement membrane changes, and tubular atrophy, in the cortex, especially in the subcapsular portion, within 10 days. On day 35, the tubulo-interstitial lesions were advanced with mild focal interstitial fibrosis. These lesions were mild in the UNT group compared to the non-UNT group. Immunohistochemically, CyA treatment caused an increase in number of renin-positive cells in the afferent arteriolar wall at juxtaglomerular area. These cells lost the expression of calponin, which is a marker of mature smooth muscle cells. In addition, in afferent arterioles and interlobular arteries, electron-dense fibrous bodies were found in the smooth muscle cells on days 10 and 35. Immunoelectron microscopically, these bodies showed scattered positive staining for calponin and alpha-actinin, were negative or only peripherally positive for alpha-SMA and vimentin, and were completely negative for desmin. This study revealed that CyA could cause interstitial lesions starting in the subcapsular portion of the renal cortex and vascular lesions of the preglomerular artery. Increases in number of renin granules and formation of cytoplasmic fibrous bodies in smooth muscle cells could be the forerunner of severe arteriolar wall damage. (+info)
The expression and distribution of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages.
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The cellular response to hypoxia includes the hypoxia-inducible factor-1 (HIF-1)-induced transcription of genes involved in diverse processes such as glycolysis and angiogenesis. Induction of the HIF-regulated genes, as a consequence of the microenvironment or genetic changes, is known to have an important role in the growth of experimental tumors. Hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) are known to dimerize with the aryl hydrocarbon receptor nuclear translocator in mediating this response. Because regulation of the alpha chain protein level is a primary determinant of HIF activity, our aim was to investigate the distribution of HIF-1alpha and HIF-2alpha by immunohistochemistry in normal and pathological tissues using monoclonal antibodies (mAb). We raised a new mAb to detect HIF-1alpha, designated 122, and used our previously validated mAb 190b to HIF-2alpha. In the majority of solid tumors examined, including bladder, brain, breast, colon, ovarian, pancreatic, prostate, and renal carcinomas, nuclear expression of HIF-1alpha and -2alpha was observed in varying subsets of the tumor cells. HIF-2alpha was also strongly expressed by subsets of tumor-associated macrophages, sometimes in the absence of any tumor cell expression. Less frequently staining was observed in other stromal cells within the tumors and in normal tissue adjacent to tumor margins. In contrast, in normal tissue neither molecule was detectable except within subsets of bone marrow macrophages, where HIF-2alpha was strongly expressed. (+info)
Immunohistochemical and in situ hybridization studies of influenza A virus infection in human lungs.
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Influenza viruses are responsible for acute febrile respiratory disease. When deaths occur, definitive diagnosis requires viral isolation because no characteristic viral inclusions are seen. We examined the distribution of influenza A virus in tissues from 8 patients with fatal infection using 2 immunohistochemical assays (monoclonal antibodies to nucleoprotein [NP] and hemagglutinin [HA]) and 2 in situ hybridization (ISH) assays (digoxigenin-labeled probes that hybridized to HA and NP genes). Five patients had prominent bronchitis; by immunohistochemical assay, influenza A staining was present focally in the epithelium of larger bronchi (intact and detached necrotic cells) and in rare interstitial cells. The anti-NP antibody stained primarily cell nuclei, and the anti-HA antibody stained mainly the cytoplasm. In 4 of these cases, nucleic acids (ISH) were identified in the same areas. Three patients had lymphohistiocytic alveolitis and showed no immunohistochemical or ISH staining. Both techniques were useful for detection of influenza virus antigens and nucleic acids in formalin-fixed paraffin-embedded tissues and can enable further understanding of fatal influenza A virus infections in humans. (+info)
Monoclonal antibody MA454 reveals a heterogeneous expression pattern of MAGE-1 antigen in formalin-fixed paraffin embedded lung tumours.
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Cancer/testis (CT) antigens such as those encoded by the MAGE-gene family are expressed in a wide variety of malignant neoplasms. In normal tissues, expression is generally restricted to testis. Current knowledge of the expression pattern of CT antigens is mainly based on mRNA analysis. Little is known about actual protein expression. We previously developed MA454, a monoclonal antibody (mAb) to MAGE-1 recombinant protein. By employing antigen retrieval techniques, we show that MA454 is reactive on formalin-fixed paraffin-embedded tissues. Immunohistochemical (IHC) analysis of a normal tissue panel revealed staining solely in germ cells of testes. A series of 59 lung tumours was co-typed for MAGE-1 expression by RT-PCR and by immunohistochemistry with MA454. MA454 was positive in 19/59 cases (32%). MAGE-1 mRNA was found in 17 of the 54 cases (32%) available for RT-PCR. Of the 19 MA454-reactive tumours, 15 showed a highly heterogeneous pattern of expression. The other 4 MA454 positive cases revealed immunoreactivity in >25% of tumour areas. Of the 53 cases typed for both, mRNA and protein expression, 48 co-typed whereas 5 cases were discrepant, a likely consequence of heterogeneous MAGE-1 expression. The predominantly focal expression of MAGE-1 suggests that this antigen might not be sufficient as a sole target for immunotherapeutic approaches. (+info)
Expression of topoisomerase IIIalpha in normal and neoplastic tissues determined by immunohistochemistry using a novel monoclonal antibody.
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Topoisomerases are nuclear enzymes that modulate the topological structure of DNA in order to facilitate cellular events such as replication and transcription. These enzymes are also the cellular targets of certain classes of chemotherapeutic agents termed topoisomerase poisons. A new human topoisomerase isoform, IIIa, was discovered in 1996, which is thought to have roles in genome stability and possibly chromosome separation during mitosis. It is possible that novel or existing anti-topoisomerase agents target topoisomerase IIIa, suggesting that this enzyme may have potential as a prognostic marker and chemotherapeutic target. In order to study expression patterns of topoisomerase IIIa we have produced a novel monoclonal antibody to human topoisomerase IIIa (TOPO3a-1A4), and used it to assess topoisomerase IIIa expression in a wide range of normal and neoplastic tissues. We have found that topoisomerase IIIa is expressed in a wide range of tissue types, with especially high concentrations in endothelial cells and stromal fibroblasts. No general relationship was observed between expression of topoisomerase IIIa and proliferation. Expression in neoplastic tissues often paralleled their normal counterparts, although certain tumours showed either increased (e.g. colonic adenoma) or reduced (e.g. gastric carcinoma, small cell carcinoma of bronchus) expression. If topoisomerase IIIa is found to be a target for chemotherapeutic agents, clinical response in different tumour types may be related to topoisomerase IIIa expression, which may be assessed using TOPO3a-1A4. (+info)