RNA In Situ Hybridization Research Articles

MiRNeye: a microRNA expression atlas of the mouse eye

BackgroundMicroRNAs (miRNAs) are key regulators of biological processes. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution.ResultsIn this report, we present the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridization (ISH) procedures. We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina.ConclusionsThis analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. The combination of the two approaches offers a valuable resource to decipher the contributions of specific miRNAs and miRNA clusters to the development of distinct ocular structures.

Determination of gene expression patterns using in situ hybridization to Drosophila testes.

We describe a whole-mount RNA in situ hybridization (ISH) method optimized for detection of the cellular and subcellular distributions of specific mRNA within Drosophila testes and male genital tract. Digoxygenin (dig)-labeled antisense RNA probes are in vitro transcribed from a template synthesized by (RT)-PCR; the probe length is reduced by hydrolysis. Testes and male genital tracts are dissected from adult flies, fixed and processed for hybridization. Both probe and fixed testes can be stored before use. Extensive post-hybridization washing reduces the background. Detection is through alkaline phosphatase-conjugated anti-dig antibodies followed by a color reaction. This protocol is suitable for low-medium throughput applications with parallel processing of 2-48 samples, and takes 4-5 d to complete. We have used this protocol, which is similar to other RNA ISH protocols, but optimized for whole-mount Drosophila testes, to document the expression of about 1,000 genes in Drosophila melanogaster male genital tract.

Diverse cell-specific expression of myoglobin isoforms in brain, kidney,gill and liver of the hypoxia-tolerant carp and zebrafish

Myoglobin (Mb) is famous as a muscle-specific protein--yet the common carp expresses the gene (cMb1) encoding this protein in a range of non-muscle tissues and also expresses a novel isoform (cMb2) in the brain. Using a homologous antibody and riboprobes, we have established the relative amounts and cellular sites of non-muscle Mb expression in different tissues. The amounts of carp myoglobin (cMb) in supernatants of different tissues were just 0.4-0.7% relative to that of heart supernatants and were upregulated by two-to-four fold in liver, gill and brain following 5 days of hypoxic treatment. Brain exhibited both cMb proteins in western analysis, whereas all other tissues had only cMb1. We have also identified cells expressing cMb protein and cMb mRNA using immunohistology and RNA in situ hybridisation (RNA-ISH), respectively. Mb was strongly expressed throughout all cardiac myocytes and a subset of skeletal muscle fibres, whereas it was restricted to a small range of specific cell types in each of the non-muscle tissues. These include pillar and epithelial cells in secondary gill lamellae, hepatocytes, some neurones, and tubular epithelial cells in the kidney. Capillaries and small blood vessels in all tissues exhibited Mb expression within vascular endothelial cells. The cMb2 riboprobe located expression to a subset of neurones but not to endothelial cells. In zebrafish, which possesses only one Mb gene, a similar expression pattern of Mb protein and mRNA was observed. This establishes a surprisingly cell-specific distribution of Mb within non-muscle tissues in both carp and zebrafish, where it probably plays an important role in the regulation of microvascular, renal and brain function.

A Core Paired-Type and POU Homeodomain-Containing Transcription Factor Program Drives Retinal Bipolar Cell Gene Expression

The diversity of cell types found within the vertebrate CNS arises in part from action of complex transcriptional programs. In the retina, the programs driving diversification of various cell types have not been completely elucidated. To investigate gene regulatory networks that underlie formation and function of one retinal circuit component, the bipolar cell, transcriptional regulation of three bipolar cell-enriched genes was analyzed. Using in vivo retinal DNA transfection and reporter gene constructs, a 200 bp Grm6 enhancer sequence, a 445 bp Cabp5 promoter sequence, and a 164 bp Chx10 enhancer sequence, were defined, each driving reporter expression specifically in distinct but overlapping bipolar cell subtypes. Bioinformatic analysis of sequences revealed the presence of potential paired-type and POU homeodomain-containing transcription factor binding sites, which were shown to be critical for reporter expression through deletion studies. The paired-type homeodomain transcription factors (TFs) Crx and Otx2 and the POU homeodomain factor Brn2 are expressed in bipolar cells and interacted with the predicted binding sequences as assessed by electrophoretic mobility shift assay. Grm6, Cabp5, and Chx10 reporter activity was reduced in Otx2 loss-of-function retinas. Endogenous gene expression of bipolar cell molecular markers was also dependent on paired-type homeodomain-containing TFs, as assessed by RNA in situ hybridization and reverse transcription-PCR in mutant retinas. Cabp5 and Chx10 reporter expression was reduced in dominant-negative Brn2-transfected retinas. The paired-type and POU homeodomain-containing TFs Otx2 and Brn2 together appear to play a common role in regulating gene expression in retinal bipolar cells.

The pattern of viral persistence in monkeys intra-tracheally infected with Simian varicella virus

In situ PCR (ISPCR) and in situ hybridisation (ISH) was performed on 32 tissues from 10 monkeys, intra-tracheally (IT) infected with simian varicella virus (SVV) and 5 tissues from 3 uninfected control animals. The results showed persistence of SVV DNA up to 2 years post-infection (pi) and the localisation of SVV to be confined to neurons except at time points 9 and 10 months pi where SVV positive satellite cells were also detected. There was no evidence for transcription of SVV ORFs 63 and 21 in the ganglia of the one IT infected and 2 naturally infected monkeys investigated using RNA ISH.

Human papillomavirus: cause of epithelial lacrimal sac neoplasia?

Epithelial tumours of the lacrimal sac are rare but important entities that may carry grave prognoses. In this study the prevalence and possible role of human papillomavirus (HPV) infection in epithelial tumours of the lacrimal sac were evaluated. Five papillomas and six carcinomas of the lacrimal sac were investigated for the presence of HPV using the polymerase chain reaction (PCR) technique. Fifteen specimens of dacryocystitis were included in the PCR reactions as controls. Furthermore, DNA in situ hybridization (ISH) and RNA ISH were performed. Low-risk HPV types 6 or 11 were identified in all four lacrimal sac papillomas suitable for PCR analysis and in situ hybridization. Four of six lacrimal sac carcinomas harboured HPV. One carcinoma was positive for HPV 11 only, two carcinomas had concomitant infection with HPV 6 or 11 and high-risk HPV 16, and the remaining carcinoma was positive for HPV 16. All specimens of dacryocystitis were betaglobin-positive and HPV-negative. Using DNA ISH, two papillomas and a single carcinoma showed evidence for vegetative HPV 11 DNA replication, whereas no HPV 16 DNA replication was found in the five carcinomas tested. HPV 11 RNA was demonstrated in two papillomas. By analysing 11 epithelial lacrimal sac papillomas and carcinomas using PCR, DNA ISH and RNA ISH, we found HPV DNA in all investigated transitional epithelium tumours of the lacrimal sac. HPV RNA was present in two of eight epithelial lacrimal sac tumours positive for HPV DNA. As RNA degrades fast in paraffin-embedded tissue, only a small fraction of DNA-positive tumours can be expected to be RNA-positive. We therefore suggest that HPV infection is associated with the development of lacrimal sac papillomas and carcinomas.

Semiautomated Multiplexed Quantum Dot-Based in Situ Hybridization and Spectral Deconvolution

Gene expression profiling has identified several potentially useful gene signatures for predicting outcome or for selecting targeted therapy. However, these signatures have been developed in fresh or frozen tissue, and there is a need to apply them to routinely processed samples. Here, we demonstrate the feasibility of a potentially high-throughput methodology combining automated in situ hybridization with quantum dot-labeled oligonucleotide probes followed by spectral imaging for the detection and subsequent deconvolution of multiple signals. This method is semiautomated and quantitative and can be applied to formalin-fixed, paraffin-embedded tissues. We have combined dual in situ hybridization with immunohistochemistry, enabling simultaneous measurement of gene expression and cell lineage determination. The technique achieves levels of sensitivity and specificity sufficient for the potential application of known expression signatures to biopsy specimens in a semiquantitative way, and the semiautomated nature of the method enables application to high-throughput studies.

Epstein-Barr Virus (EBV) detection and typing by PCR: a contribution to diagnostic screening of EBV-positive Burkitt's lymphoma

BackgroundEpstein-Barr virus (EBV) is associated to the etio-pathogenesis of an increasing number of tumors. Detection of EBV in pathology samples is relevant since its high prevalence in some cancers makes the virus a promising target of specific therapies. RNA in situ hybridization (RISH) is the standard diagnostic procedure, while polymerase chain reaction (PCR)-based methods are used for strain (EBV type-1 or 2) distinction. We performed a systematic comparison between RISH and PCR for EBV detection, in a group of childhood B-cell Non-Hodgkin lymphomas (NHL), aiming to validate PCR as a first, rapid method for the diagnosis of EBV-associated B-cell NHL.MethodsEBV infection was investigated in formalin fixed paraffin-embedded tumor samples of 41 children with B-cell NHL, including 35 Burkitt's lymphoma (BL), from Rio de Janeiro, Brazil, by in situ hybridization of EBV-encoded small RNA (EBER-RISH) and PCR assays based on EBNA2 amplification.ResultsEBV genomes were detected in 68% of all NHL. Type 1 and 2 accounted for 80% and 20% of EBV infection, respectively. PCR and RISH were highly concordant (95%), as well as single- and nested-PCR results, allowing the use of a single PCR round for diagnostic purposes. PCR assays showed a sensitivity and specificity of 96% and 100%, respectively, with a detection level of 1 EBV genome in 5,000–10,000 EBV-negative cells, excluding the possibility of detecting low-number EBV-bearing memory cells.ConclusionWe describe adequate PCR conditions with similar sensitivity and reliability to RISH, to be used for EBV diagnostic screening in high grade B-NHL, in "at risk" geographic regions.

Lack of BIC and microRNA miR‐155 expression in primary cases of Burkitt lymphoma

We previously demonstrated high expression of primary-microRNA BIC (pri-miR-155) in Hodgkin lymphoma (HL) and lack of expression in most non-Hodgkin lymphoma subtypes including some Burkitt lymphoma (BL) cases. Recently, high expression of BIC was reported in BL in comparison to pediatric leukemia and normal peripheral-blood samples. In this study, we extended our series of BL cases and cell lines to examine expression of BIC using RNA in situ hybridization (ISH) and quantitative RT-PCR (qRT-PCR) and of miR-155 using Northern blotting. Both BIC RNA ISH and qRT-PCR revealed no or low levels of BIC in 25 BL tissue samples [including 7 Epstein-Barr virus (EBV)-positive cases] compared to HL and normal controls. In agreement with these findings, no miR-155 was observed in BL tissues. EBV-negative and EBV latency type I BL cell lines also showed very low BIC and miR-155 expression levels as compared to HL cell lines. Higher levels of BIC and miR-155 were detected in in vitro transformed lymphoblastoid EBV latency type III BL cell lines. An association of latency type III infection and induction of BIC was supported by consistent expression of BIC in 11 and miR-155 in 2 posttransplantation lymphoproliferative disorder (PTLD) cases. In summary, we demonstrated that expression of BIC and miR-155 is not a common finding in BL. Expression of BIC and miR-155 in 3 latency type III EBV-positive BL cell lines and in all primary PTLD cases suggests a possible role for EBV latency type III specific proteins in the induction of BIC expression.

Human Papillomaviruses Associated with Epidermodysplasia Verruciformis in Non-Melanoma Skin Cancers: Guilty or Innocent?

Abbreviations: AK, actinic keratoses; EV, epidermodysplasia verruciformis; HPV, human papillomavirus; ISH, in situ hybridization; NMSC, non-melanoma skin cancers; OTR, organ transplant recipients; SCC, squamous cell carcinomas; UVR, ultraviolet radiation

Expression profile and molecular genetic regulation of cyclin D1 expression in epithelioid sarcoma

Epithelioid sarcoma is a distinctive, aggressive soft tissue tumor typically presenting as a subcutaneous or deep dermal mass in the distal extremities of young adults. Molecular genetic data of well-characterized cases of epithelioid sarcoma are sparse. A recent cytogenetic study of epithelioid sarcoma by conventional metaphase comparative genomic hybridization reported recurrent gains at chromosome 11q13, a region containing many genes, including the cyclin D1 gene. Cyclin D1 is a positive cell cycle regulator that is overexpressed in a variety of neoplasms, including mantle cell lymphoma and breast carcinoma. The objective of this study was to examine cyclin D1 expression in epithelioid sarcoma. Of 24 cases evaluated, 23 (96%) displayed cyclin D1 nuclear expression using immunohistochemical evaluation. Eight cases, which expressed cyclin D1 by immunohistochemistry, were evaluated by fluorescence in situ hybridization (FISH) and RNA in situ hybridization (RISH) for amplification of the cyclin D1 gene and messenger RNA (mRNA) expression, respectively. Seven of eight cases showed a typical eusomic state. One case showed pseudoamplification due to aneusomy/polysomy. There was no evidence of cyclin D1 gene amplification or messenger RNA overexpression detected by FISH or RNA in situ hybridization analyses, respectively. Our data clearly demonstrate that cyclin D1 protein is upregulated in epithelioid sarcoma, suggesting a role for this cell cycle regulator in the pathogenesis of epithelioid sarcoma. The high level of cyclin D1 protein expression in epithelioid sarcoma appears to be regulated by translational and/or post-translational mechanisms.

Expression of HERV-K, a Human Endogenous Retrovirus, in Hematopoietic Cells.

Background: In contrast to other human endogenous retrovirus families which are highly defective, HERV-K proviruses possess all viral gene types of exogenous retroviruses. In malignancy (tumor cell lines, breast cancer, melanoma as well as leukemias and lymphomas) but also in many normal tissues, including placenta HERV-K mRNA expression has been detected. In hematopoietic stem cells, an interaction of HERV-K with elements from vectors which are used for gene therapy may lead to unforeseen consequences. The purpose of this study was to localize HERV-K mRNA expression in hematopoietic stem cells of different sources and to evaluate a potential association of HERV-expression with hematologic diseases and to define a possible risk of HERV-expression for transplantation.Methods: Quantitative real time PCR (RTQPCR) was used for detecting HERV-K expression in MNC from blood and/or bone marrow samples of patients with leukemias ( 10 MDS, 16 MPD, 14 AML and 1 CML blast crisis) and MNC from blood (n=9) and lymph nodes (n=3) from lymphoma patients. In addition, 8 blood samples and 2 bone marrow samples from healthy donors as well as 9 cord blood samples and 20 apheresates from lymphoma patients in remission containing PBSC were analyzed. Analysis of the same HERV-K gene (pol) in placenta and maternal blood as well as in genomic DNA from all above mentioned sources served as control. In HERV-K overexpressing samples expression of HERV in specific cells was tested by RNA in situ hybridisation (RISH), and combined staining with a CD34 specific monoclonal antibody.Results: Relative mRNA levels of HERV-K in apheresates as well as in blood and bone marrow samples of patients with MDS, MPD and AML from subtypes with less than 1% CD34 positive cells were in the range of less than 2% in relation to housekeeping genes. However, in bone marrow samples of two AML and a CML blast crisis (all of them with more than 50% CD34+ cells) as well as in CD34+ cells from cord blood (but not from apheresates or normal bone marrow) as well as in three lymph nodes of patients with B-cell lymphomas, an overexpression of HERV-K was detected and confirmed by RISH. Enhanced expression of HERV-K was detected in cord blood derived apoptotic CD34+ cells, whereas the relative amount of HERV in genomic DNA did not change in samples from different sources.Conclusions. Our data indicate that HERV-K may be activated in lymph nodes of lymphomas and in CD34+ cells from bone marrow of leukemia patients and from cord blood but not in stem cell apheresates from peripheral blood. In cord blood and malignancy expression appears to be associated with apoptosis. Similar amounts of HERV-K in different samples of genomic DNA of the same cells and the absence of HERV-K activation in normal bone marrow and PBSC indicate that activation of HERV-K is the result of regulation of gene expression rather than amplification. A possible specific role of HERV in the pathogenesis of hematologic diseases and in hematopoiesis (cord blood) remains to be established. Differences between peripheral blood stem cells and cord blood may support a critical stance regarding the use of cord blood derived stem cells for transplantation and gene therapy.

Loss of Runx3 function in leukocytes is associated with spontaneously developed colitis and gastric mucosal hyperplasia

RUNX transcription factors are key regulators of lineage-specific gene expression and might be involved in autoimmune diseases. Runx3 plays a role during the development of sensory neurons and T cells and regulates transforming growth factor beta (TGF-beta) signaling in dendritic cells. Here, we report that at 4 weeks of age, Runx3 knockout (KO) mice spontaneously develop inflammatory bowel disease (IBD) characterized by leukocyte infiltration, mucosal hyperplasia, formation of lymphoid clusters, and increased production of IgA. Additionally, at a considerably older age (8 months), the KO mice also develop progressive hyperplasia of the gastric mucosa associated with disturbed epithelial differentiation and cellular hyaline degeneration. Analysis of cytokines in the colonic mucosa of Runx3 KO mice revealed a mixed T helper 1/T helper 2 response. By using immunohistochemistry and RNA in situ hybridization, Runx3 expression in the gastrointestinal tract is detected in lymphoid and myeloid populations but not in the epithelium. The data indicate that loss of leukocytic cell-autonomous function of Runx3 results in IBD and gastric lesion in the KO mice. IBD in humans is viewed as a complex genetic disorder. Several susceptibility loci were identified on different human chromosomes including the chromosomal region 1p36 where RUNX3 resides. It is thus tempting to speculate that mutations in RUNX3 may constitute an IBD risk factor in humans.

Tissue microarrays for high-throughput molecular pathology.

SummaryModern research technologies, including DNA, protein, and antibody microarrays identify a steadily growing number of clues that are useful in molecular disease classification, drug development, and the prediction of response to treatment. Subsequent validation of the clinical importance of such candidate genes or proteins requires large-scale analysis of human tissues. To date, this analysis constitutes an important bottleneck in the process of discovery because tissue analysis by the conventional slide-by-slide strategy is slow and expensive. To overcome these limitations, tissue microarray (TMA) technology has been developed. TMA allows for the simultaneous analysis of up to 1,000 tissue samples in a single experiment, using all types of in-situ analyses including immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and RNA in situ hybridization (RNA-ISH). TMA technology has the potential to greatly facilitate the translation of basic research into clinical practice. Potential applications include the establishment of associations between molecular changes and clinical endpoints, testing of potential therapeutic targets using tissue samples from specific cancer patients, standardization of molecular detection of targets, and rapid translation of results from cell lines and animal models to human cancer. Because of its beneficial economic aspects and ability to differentiate ethnic differences in tumor biology, TMA applications may become particularly important in developing countries.

Epstein-Barr virus in gastric carcinomas and gastric stump carcinomas: a late event in gastric carcinogenesis

Background: To determine at what stage during gastric carcinogenesis Epstein-Barr virus (EBV) enters the gastric epithelial cells, the presence of EBV was investigated in two pathogenetically related but distinct forms...

In vivo transcription of the Epstein-Barr virus (EBV) BamHI-A region without associated in vivo BARF0 protein expression in multiple EBV-associated disorders.

The in vivo expression of the Epstein-Barr virus (EBV) BamHI-A rightward transcripts (BARTs) as well as the putative BART-encoded BARF0 and RK-BARF0 proteins in various EBV-associated malignancies was investigated. RT-PCRs specific for the different splice variants of the BARTs and both a nucleic acid sequence-based amplification assay and an RT-PCR specific for the BARF0 ORF were used. Abundant transcription of BARTs was found in EBV-associated Hodgkin's lymphomas, Burkitt's lymphomas (BL), T-cell non-Hodgkin's lymphomas, post-transplant lymphoproliferative disorders, AIDS-related lymphomas and gastric carcinomas. Using RNA in situ hybridization (RISH), BARTs were detected within the neoplastic cells of these malignancies. BARTs encoding RK-BARF0 were not detected. The BARTs detected were shown possibly to encode the RPMS1 and BARF0 proteins, based on their splicing. However, BARTs actually harbouring the BARF0 ORF were detected only in specimens containing a relatively large number of EBV-positive cells. New monoclonal antibodies against the BARF0 protein were generated that efficiently recognized prokaryotic and eukaryotic recombinant BARF0. However, the BARF0 protein was not detected in clinical samples, nor in EBV-positive cell lines, even though these were positive for BARTs by RISH and/or BARF0 RNA in vitro analysis. Using immunoblot analysis, no antibodies against baculovirus-expressed BARF0 protein were detected in the sera of nasopharyngeal carcinoma patients, BL patients and Hodgkin's disease patients, patients with chronic EBV infection, infectious mononucleosis patients or EBV-positive healthy donors. Thus, BARTs containing the BARF0 ORF are expressed in vivo but the BARF0 protein cannot be detected and may be expressed only marginally. It is concluded that the BARF0 protein is unlikely to play a role in vivo in EBV-positive malignancies.

No role for Epstein-Barr virus in Dutch hepatocellular carcinoma: a study at the DNA, RNA and protein levels.

Epstein-Barr virus (EBV) has been suggested to play a role in hepatocellular carcinoma (HCC). However, reports on detailed EBV transcript analyses in HCCs are limited. It was shown recently that expression of the transforming BARF1 (BamHI A rightward open reading frame 1) gene of EBV is restricted to latently EBV-infected epithelial malignancies, i.e. nasopharyngeal carcinoma and gastric carcinoma. The aim of this study was to test the presence of EBV in Dutch HCCs. A semiquantitative DNA PCR-enzyme immunoassay (PCR-EIA) for the BamHI W fragment of EBV was used to assess the presence of EBV in frozen and paraffin-embedded tissues of 16 HCCs. In addition, several RNA detection techniques, i.e. nucleic acid sequence-based amplification (NASBA), RT-PCR, RNA in situ hybridization (RISH) and immunohistochemistry (IHC), were applied. Five of 16 HCCs and two of four hepatitis C virus hepatitis samples were weakly positive for EBV DNA by PCR-EIA. Using sensitive RNA transcription techniques, no transcripts were found for BARF1, EBNA-1 and BARTs (BamHI A rightward transcripts) in any of the liver tissues tested. In addition, RISH for EBER1/2 and BARTs and IHC for EBNA-1, LMP-1 and ZEBRA, performed on the paraffin-embedded tissue of the PCR-EIA-positive cases and on adjacent non-neoplastic liver tissues, were negative. The absence of epithelial-specific BARF1 transcripts and other EBV transcripts and proteins in the EBV DNA PCR-positive cases argues strongly against a role for EBV in HCC.

Detecting the expression of human endogenous retrovirus E envelope transcripts in human prostate adenocarcinoma.

The expression of human endogenous retrovirus (HERV) mRNA and proteins was associated recently with diseases that include human malignancies. The authors report that, in the current study, transcripts encoding the envelope region of an HERV family, HERV-E, were expressed in human prostate carcinoma. RNA was isolated from various prostate tissues and was tested for the expression of various HERV envelope (env) genes by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, RNA in situ hybridization (ISH), and Northern blot analysis. Variants of HERV that appeared in prostate carcinoma tissues were sequenced, and HERV-E was expressed in prokaryotic and eukaryotic systems. In the current study, the authors found that the mRNA of the env gene of one particular family of HERVs, HERV-E, was expressed in some prostate carcinoma tissues (38.8% positive; n = 49 specimens) but not in normal prostate tissues using RT-PCR, RNA ISH, and Northern blot assays. The expression of HERV-E transcripts in prostate tumor epithelial cells was confirmed further by ISH using an HERV-E specific antisense probe. Approximately 50% of the cDNA of HERV-E obtained from prostate carcinoma specimens contained no stop codon and expressed proteins in prokaryotic or eukaryotic expression systems. Furthermore, the expression of both HERV-E and ERV3 (another class of HERV) was detected in the same prostate carcinoma tissues. The expression and distribution of multiple HERV-E endogenous retroviral elements in prostate carcinoma, but not in normal control specimens, suggests that they may serve as novel tumor markers for the early diagnosis and immunotherapy of patients with prostate carcinoma.

Detection of Epstein-Barr virus genome within thymic epithelial tumours in Taiwanese patients by nested PCR, PCR in situ hybridization, and RNA in situ hybridization.

Epstein-Barr virus (EBV) is known to be associated with a variety of tumours, including Burkitt's lymphoma, nasopharyngeal carcinoma, and some carcinomas of other organs with similar lymphoepithelioma-like features. The association between EBV and thymic epithelial tumours is inconclusive, as reports in this regard are not entirely consistent and the methods employed are of different sensitivity and specificity. This study examined 78 thymomas and 21 thymic carcinomas in Taiwanese patients, to detect the viral genome at both DNA and RNA levels. The tissue blocks were first screened by nested polymerase chain reaction (PCR) targeting on the first tandem internal repeats. The positive cases were further submitted for viral localization by in situ PCR insitu hybridization (ISH) and Epstein-Barr-encoded RNA-1 (EBER-1) ISH. None of the thymomas showed a detectable EBV genome. Eight thymic carcinomas were positive for EBV by nested PCR, of which six displayed nuclear signals within the tumour cells by in situ PCR ISH and/or RNA ISH, one displayed signals within the lymphocytes, and one showed no discernible in situ signals. Most of them exhibited a lymphoepithelioma-like morphology. These results show that nested PCR is a sensitive method for screening the EBV genome in thymic epithelial tumours. In situ PCR ISH is reliable for localization of the virus, in addition to EBER-1 RNA ISH. Thymomas are not related to EBV, even in this endemic area. Thymic carcinomas, especially the lymphoepithelioma-like thymic carcinomas, are more often associated with the virus.

Localization of avian leukosis virus subgroup J in naturally infected chickens by RNA in situ hybridization.

The novel subgroup J of avian leukosis virus (ALV-J) has emerged as a significant cause of myeloid neoplasia and weight suppression in broiler chickens. We investigated viral tropism using RNA in situ hybridization (ISH) in naturally infected chickens. Formalin-fixed tissues were collected from 12-day-old embryos (seven infected, two control) and from 0-week-old (four infected, one control), 3-week-old (five infected, one control), 6-week-old (five infected, one control), and 9-week-old (10 infected, two control) chickens naturally infected with ALV-J in ovo. A 636-base antisense riboprobe complementary to the 3' and 5' ends of the pol and env viral genes, respectively, was constructed. Strong positive staining was present in cardiac myocytes, Purkinje fibers, vascular and pulmonary smooth muscle, renal glomeruli, distal tubules, and pituitary glands. Light staining was present in gastrointestinal smooth muscle, thyroid and adrenal glands, and follicular medullae in the cloacal bursa. Staining was not present in any hematopoietic precursors. Tissues from newly hatched chicks exhibited the strongest and most consistent staining, whereas staining in embryos was minimal. RNA ISH confirmed the presence of ALV-J-specific nucleic acid within cytoplasmic inclusions in cardiac myocytes, Purkinje fibers, pituitary glands, and renal glomeruli. Viral tropism for cardiac myocytes and Purkinje fibers may relate pathogenetically to the cardiomyopathy and congestive heart failure described in index chicken flocks infected with ALV-J. Viral tropism for endocrine organs may relate pathogenetically to the weight suppression associated with infection.