I Silva

Potential viral pathogenic mechanism for new variant inflammatory bowel disease

Aims: A new form of inflammatory bowel disease (ileocolonic lymphonodular hyperplasia) has been described in a cohort of children with developmental disorder. This study investigates the presence of persistent measles virus in the intestinal tissue of these patients (new variant inflammatory bowel disease) and a series of controls by molecular analysis. Methods: Formalin fixed, paraffin wax embedded and fresh frozen biopsies from the terminal ileum were examined from affected children and histological normal controls. The measles virus Fusion (F) and Haemagglutinin (H) genes were detected by TaqMan reverse transcription polymerase chain reaction (RT-PCR) and the Nucleocapsid (N) gene by RT in situ PCR. Localisation of the mRNA signal was performed using a specific follicular dendritic cell antibody. Results: Seventy five of 91 patients with a histologically confirmed diagnosis of ileal lymphonodular hyperplasia and enterocolitis were positive for measles virus in their intestinal tissue compared with five of 70 control patients. Measles virus was identified within the follicular dendritic cells and some lymphocytes in foci of reactive follicular hyperplasia. The copy number of measles virus ranged from one to 300 000 copies/ng total RNA. Conclusions: The data confirm an association between the presence of measles virus and gut pathology in children with developmental disorder.

Comparison of HPV detection technologies: Hybrid capture 2, PreTect HPV-Proofer and analysis of HPV DNA viral load in HPV16, HPV18 and HPV33 E6/E7 mRNA positive specimens.

Human papillomavirus (HPV) testing using molecular methods in liquid based cytology (LBC) specimens may be useful as an adjunct to cervical screening by cytology. We compared the positivity rate of the commercially available HPV DNA method hybrid capture 2 (hc2) and the commercially available E6/E7 mRNA method PreTect HPV-Proofer in cytological specimens (n=299). LBC specimens collected (n=299) represented the following cervical cytological disease categories: Normal (n=60), borderline nuclear abnormalities (BNA) (n=34), CIN1 (n=121), CIN2 (n=60), CIN3 (n=24). Overall, 69% (205/299) of the cases were positive by hc2 and 38% (112/299) of the cases were positive by PreTect HPV-Proofer. Concordance rates between the two tests were highest in the high-grade cytology cases (CIN2: 67% and CIN3: 83%) and the normal cytology cases (88%) and lowest in the BNA and CIN1 categories (56% and 52%). HPV DNA viral load analyses were carried out on HPV16 (n=55), HPV18 (n=9) and HPV33 (n=13) samples that were positive by PreTect HPV-Proofer. The sensitivity and specificity of PreTect HPV-Proofer and the hc2 DNA test for the detection of high-grade cytology (i.e. CIN2+) were 71.4% and 75.8% vs 100% and 43.7%, respectively. The relatively low detection rate observed by PreTect HPV-Proofer in the whole range of cytological positive cases, combined with a relatively higher specificity and PPV, suggests that PreTect HPV-Proofer may be more useful than hc2 for triage and in predicting high-grade disease.

Cellular localisation of HHV-8 in Castleman's disease: is there a link with lymph node vascularity?

Aims—Human herpesvirus 8 (HHV-8) has been identified in multicentric Castlemans disease and in angioimmunoblastic lymphadenopathies. However, the presence of the virus does not necessarily indicate an aetiological role in these conditions. This study investigates the cell types infected by HHV-8 in Castlemans disease and examines the correlation between HHV-8 and Castlemans disease lymph node angiogenesis. Methods—Sixteen formalin fixed, paraffin wax embedded samples from patients with Castlemans disease (six multicentric, 10 solitary) were examined for the presence of HHV-8 using the polymerase chain reaction (PCR), non-isotopic in situ hybridisation, PCR in situ hybridisation (PCR-ISH), and real time quantitative TaqMan PCR to HHV-8 open reading frame 26 (ORF-26), and viral (v)-cyclin encoding regions. Vascularity was assessed using CD34, CD31, and factor VIII immunocytochemistry, and lymph nodes were scored as “low” or “high”. Results—Five multicentric Castlemans disease and two solitary Castlemans disease biopsies were positive for HHV-8. HHV-8 was identified in approximately 10% of intranodal B lymphocytes, in endothelial cells, and in subcapsular spindle cell proliferations. The copy number of HHV-8 was low at 10–50 copies/1000 cells. The highest copy number was in subcapsular spindle cells. There was no correlation between vascularity score and HHV-8 status. Conclusion—The preferential localisation of HHV-8 in subcapsular spindle cell proliferations (where early intranodal Kaposis sarcoma initiates) and endothelial cells in Castlemans disease might finally explain the link between intranodal Kaposis sarcoma and Castlemans disease.

Expression profile of human herpesvirus 8 (HHV-8) in pyothorax associated lymphoma and in effusion lymphoma

Aims—Pyothorax associated lymphoma (PAL) occurs in a clinical setting of longstanding pyothorax or chronic inflammation of the pleura. Like primary effusion lymphoma, it has an association with Epstein-Barr virus (EBV), and is confined to the pleural cavity, but has differing morphological and phenotypic features. Human herpesvirus 8 (HHV-8) has been consistently reported in primary effusion lymphoma. This study examines the immunophenotype of two European cases of PAL, investigates the presence of HHV-8 and its expression profile, and assesses whether PAL is similar to other effusion lymphomas. Methods—Material was obtained from two European cases of PAL. Immunocytochemical analysis was performed using antibodies against CD45, CD20, CD79a, CD45RAA, CD3, CD43, CD45RO (UCHL1), CD30, BCL-2, CD68, epithelial membrane antigen (EMA), BCL-6, p53, Ki-67, κ light chain, λ light chain, and the EBV antigens latent membrane protein 1 (LMP-1) and EBV encoded nuclear antigen 2 (EBNA-2). The cases were examined for HHV-8 by means of polymerase chain reaction in situ hybridisation (PCR-ISH), solution phase PCR, in situ hybridisation (ISH), and real time quantitative TaqMan PCR to HHV-8 open reading frame 26 (ORF-26) and viral (v) cyclin encoding regions. The expression profile of HHV-8 in PAL and in BC-1 and BC-3 cells was assessed by RNA TaqMan PCR to the HHV-8 genes encoding v-cyclin, v-IL-6, and G protein coupled receptor (GPCR). Results—Both cases expressed CD24, CD20, CD79a, BCL-2, light chain restriction, and high Ki-67 staining. EBV was identified by EBER-ISH in one case. HHV-8 was not identified by solution phase PCR, but was detected by PCR-ISH (sensitivity of 1 viral genome copy/cell) in 35% of the cells and by TaqMan PCR, which showed 50–100 HHV-8 copies/2000 cell genome equivalents (sensitivity of 1 viral genome in 106 contaminating sequences). HHV-8 v-IL-6, v-cyclin, and GPCR encoded transcripts were identified using RNA TaqMan PCR. v-IL-6 was high in PAL and in BC-1 and BC-3 cells. Conclusion—The presence of HHV-8 in one of two patients with PAL raises interesting questions in relation to the pathobiology of the condition. Clearly, the results indicate that HHV-8 is not an obligate pathogen, necessary for the effusion phenotype, but might contribute to it by its secretion of specific cytokines.

Improved in situ detection method for telomeric tandem repeats in metaphase spreads and interphase nuclei.

Peptide nucleic acid technology (PNA) has become an extremely useful tool and promises to impact on molecular biology and diagnostics. These synthetic DNA analogues pair with DNA and RNA molecules according to Watson and Crick base pairing rules. This paper describes a sensitive and quick fluorescent in situ hybridisation (ISH) technique to determine DNA telomere repeat sequences (TTA GGG)n using epifluorescence microscopy. Telomeres are special, repeated structures at the end of each eukaryotic chromosome and serve as protective caps to prevent DNA rearrangements and fusion of chromosomes. A model system has been developed, using stimulated peripheral blood lymphocytes, which facilitates simultaneous detection of telomeres in metaphase as well as in interphase nuclei. A fluorescein isothiocyanate labelled PNA probe (18 mer) directed against complementary telomeric sequences at the end of each chromosome is used. In addition, a simple, easy to perform PNA-ISH protocol is described that overcomes common hybridisation problems encountered using DNA and RNA oligoprobes. Furthermore, the usefulness of a chromogenic immunocytochemical detection system is shown for PNA-ISH.

Localisation of HHV-8 in AIDS related lymphadenopathy.

Background—Many lymph node abnormalities have been described in AIDS. These include opportunistic infections that sometimes result in spindle cell pseudotumours, Kaposis sarcoma (KS), malignant lymphoma (Hodgkins and nonHodgkins), and florid reactive hyperplasia. Among these, reactive hyperplasia is the most common manifestation of AIDS related lymphadenopathy. Aim—To examine whether human herpesvirus 8 (HHV-8), the aetiological agent of KS, can be localised in AIDS related lymphadenopathy and whether its appearance in such nodes is predictive of Kaposis sarcoma development. Methods—A series of human immunodeficiency virus (HIV) positive men (n = 21) with AIDS related lymphadenopathy who at the time of presentation had KS or subsequently developed KS (n = 5) were examined. The prevalence of HHV-8 was assessed in these patients using solution phase polymerase chain reaction (PCR), real time TaqMan quantitative PCR, and in cell amplification techniques (PCR in situ hybridisation (PCR-ISH) and labelled primer driven in cell amplification). Results—Using standard solution phase PCR in a nested format, only two of the 21 patients with AIDS related lymphadenopathy were positive for HHV-8. The lymph node of one of these patients contained KS lesions. Three HHV-8 positive patients were identified using TaqMan PCR (the original two positive patients and one additional patient). All of the positive patients either subsequently developed KS (n = 2) or had KS at the time of diagnosis (n = 1). Two additional patients subsequently developed KS, but were negative for HHV-8 by solution phase PCR and TaqMan PCR. Using PCR-ISH, HHV-8 amplicons were identified in some lymphoid cells (in one patient) and in spindle cells of the KS lesion in another. The positive lymphoid cells were predominantly concentrated in B cell areas of the affected lymph nodes, confirming the B cell tropism exhibited by HHV-8. Conclusions—The presence of HHV-8 in AIDS related lymphadenopathy is predictive of KS development and probably represents seeding of HHV-8 infected B cells from the peripheral blood. These findings support a role for HHV-8 in the pathobiology of KS.

In cell amplification.

The localisation of cellular DNA and RNA sequences in human tissues has greatly facilitated our understanding of disease processes. More than any other technique, in situ hybridisation (ISH) allowed this to be accomplished. However, ISH does not stand alone and must be taken in the context of other developments in cellular and molecular biology that preceded and post-dated its introduction. Today, the repertoire of in situ hybridisation has been extended to include: DNA in situ hybridisation with double stranded DNA probes, oligo DNA probes and peptide nucleic acid probes (PNAs); RNA in situ hybridisation with riboprobes, oligoprobes and cDNA probes; and the newer techniques of in cell amplification, including DNA polymerase chain reaction ISH (DNA PCR ISH), labelled primer driven DNA in situ amplification (LPD ISA), DNA/PNA PCR ISH, RNA reverse transcription PCR ISH (RT PCR ISH), labelled primer driven in situ RNA amplification (LPD ISRA), and isothermal in situ amplification techniques including in situ isothermal replication (IS 3SR). The marriage of PCR technology and in situ hybridisation now allows single copy mammalian gene detection and extremely sensitive mRNA detection. In this review, we analyse the historical background to in cell amplification, the discovery of non-isotopic ISH (NISH), PCR, and early techniques that were used for DNA and RNA amplification. We will then outline detection sensitivity issues and review the major in cell amplification techniques. Like most new molecular biological systems, there has be an explosion of newly described modifications, many of which we will review. Finally, we will present realistic alternative techniques that give equally sensitive results with in cell PCR amplification and discuss the advantages and disadvantages of each system.

Hypothesis. HHV-8 in multiple myeloma: is this the first paracrine model of human tumorigenesis and do Koch's postulates apply?

Human herpesvirus 8 (HHV-8) is now clearly implicated in the pathogenesis of Kaposi’s sarcoma. HHV-8 is also implicated in the pathobiology of eVusion lymphoma and, recently, we and others have demonstrated this virus in eVusion and non-eVusion multiple myeloma, in pyothorax associated lymphoma, and in Castleman’s disease. HHV-8, a a-II herpes virus, is unique in that it carries 16 diVerent genes important in inflammation, the cell cycle, and apoptosis, including orf 16 (a bcl-2 homologue), viral interleukin 6 (v-IL-6), v-cyclin, vMIP I, and vMIP II. vMIP I and vMIP II are inflammatory macrophage proteins, with sequence similarity to the human CC chemokines.