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Biomedical subjects

V Uhlmann

Publications and source records attributed to V Uhlmann.

12 recordsLinked to original sources
PubMed2002

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 300,00 copies/ng total RNA. CONCLUSIONS: The data confirm an association between the presence of measles virus and gut pathology in children with developmental disorder.

Adolescent↗
PubMed2001

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, kappa light chain, lambda 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/2,000 cell genome equivalents (sensitivity of 1 viral genome in 10(6) 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.

Aged↗
PubMed2000

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, Kaposi's sarcoma (KS), malignant lymphoma (Hodgkin's and non-Hodgkin's), 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 Kaposi's 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.

AIDS-Related Complex↗
PubMed2000

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.

Humans↗
PubMed2000

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 Castleman's 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 Castleman's disease and examines the correlation between HHV-8 and Castleman's disease lymph node angiogenesis. METHODS: Sixteen formalin fixed, paraffin wax embedded samples from patients with Castleman's 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 Castleman's disease and two solitary Castleman's 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 Kaposi's sarcoma initiates) and endothelial cells in Castleman's disease might finally explain the link between intranodal Kaposi's sarcoma and Castleman's disease.

Adult↗
PubMed1999

Cyclin D1 expression and HHV8 in Kaposi sarcoma.

BACKGROUND: Human herpesvirus 8 (HHV8) appears to be the agent responsible for Kaposi sarcoma. The mechanism remains undetermined but may involve cell cycle regulating genes including D type cyclins which are pivotal in cell cycle progression. Recent HHV8 genetic analysis has revealed the presence of a v-cyclin which is homologous to D type cyclins. AIMS: First, to assess whether there is an independent relation between endogenous cyclin D1 expression in Kaposi sarcoma and HHV8 status; second to determine whether v-cyclin mRNA expression varies with Kaposi sarcoma stage. METHODS: Cyclin D1 immunohistochemistry was performed on 17 paraffin embedded Kaposi sarcoma samples from 16 patients. HHV8 status was assessed in 15 of these using nested polymerase chain reaction (PCR) to ORF 26 and the newly described technique of TaqMan PCR. An additional 10 fresh Kaposi sarcoma samples (early and nodular) were examined for HHV8 v-cyclin RNA. RESULTS: One case, which did not contain amplifiable HHV8, showed strong cyclin D1 staining. The remaining cases were negative or weakly staining; v-cyclin transcript load was higher in early Kaposi sarcoma. CONCLUSIONS: While endogenous cyclin D1 expression is independent of HHV8 status, v-cyclin transcription is higher in early lesions, supporting the "viral hit" hypothesis.

Cyclin D1↗
PubMed1998

Molecular analysis of ras oncogenes in CIN III and in stage I and II invasive squamous cell carcinoma of the uterine cervix.

AIM: To examine the prevalence of genital type human papilloma virus (HPV) and mutations at codons 12, 13, and 61 in H, Ki, and N-ras in CIN III and early invasive squamous cell carcinomas of the cervix. METHODS: Prevalence of HPV was examined in 20 CIN III and 20 stage I and II cervical carcinomas, using non-isotopic in situ hybridisation (NISH) and solution phase polymerase chain reaction (PCR). In addition, mutations at codons 12, 13, and 61 were examined in H, Ki, and N-ras in these CIN III and early invasive squamous cell carcinomas, to assess the prevalence of ras gene point mutations and to define where in the pathobiology of squamous cell carcinoma such events occur. A non-isotopic PCR/RFLP assay was used to define these mutations. RESULTS: Of the 20 CIN IIIs examined, 19 contained HPV 16 DNA sequences by PCR and NISH. Dual infection was not uncovered. The 20 early (stage I and II) invasive squamous cell carcinomas showed predominant HPV 16 positivity (17/20), with one case HPV 18 positive, confirmed on PCR and NISH. Activating mutations were not identified in any of the CIN III cases. Only one stage I, HPV 16 positive carcinoma showed an activating mutation in H-ras codon 12, which was not present in adjacent normal ectocervical mucosa from the same patient. CONCLUSIONS: ras Activation does not appear to occur in conjunction with HPV infection, particularly of HPV 16 infected high grade cervical intraepithelial neoplasia, or to occur commonly in early cervical squamous cell carcinoma. The postulated model of HPV linked carcinogenesis suggests malfunctional control of viral transcription as a necessary component of neoplastic progression. It is also clear that host gene alterations are equally necessary for HPV linked carcinogenesis to occur.

Carcinoma, Squamous Cell↗
PubMed1998

A novel, rapid in cell RNA amplification technique for the detection of low copy mRNA transcripts.

Growing interest now focuses on improvements of in situ polymerase chain reaction (PCR) technology for the detection of DNA and RNA cellular sequences. In this study, reverse transcription PCR in situ hybridisation (RT PCR-ISH) was developed and used to determine gene expression of pyruvate dehydrogenase in a cell model system, using human peripheral blood lymphocytes (PBLs). The success of in cell RNA amplification depends on the type of cell/tissue fixation, cell permeabilisation, and the efficiency of reverse transcription and cDNA amplification. This paper presents new approaches to overcome the critical aspects of fixation, permeabilisation, and reverse transcription when performing in cell RNA amplification. A novel fixative, "Permeafix", possessing fixative and permeabilisation properties, was used for cell fixation procedures. "Permeafix" obviated the need for pre-amplification proteolysis, facilitating entry of PCR reagents to target sequences within the cell. In addition, a simple on step RNA in cell amplification protocol using recombinant Thermus thermophilus (rTth) DNA polymerase, which reverse transcribes mRNA efficiently to cDNA and then catalyses cDNA amplification, was used. The value of a semi-junctional primer system for in cell gene expression studies, without the need to perform DNase digestion, is demonstrated.

DNA Primers↗