Kaposi's sarcoma-associated herpesvirus infects endothelial and spindle cells.

Kaposi's sarcoma (KS), a vascular tumour that contains characteristic spindle cells forming slit-like spaces, may have an infectious aetiology. Recently, sequences of a new human herpesvirus, KSHV/HHV-8, have been identified in both HIV-associated and classical KS. We sought to identify the target cell of this virus in KS tumour tissue. Using PCR in situ hybridization (PCR-ISH) we show that KSHV/HHV-8 is present in the flat endothelial cells lining vascular spaces of KS lesions as well as in typical KS spindle cells. These findings show that KSHV/HHV-8 is present in the cell types thought to represent neoplastic cells in these lesions.

Shigatoxin encoding Bacteriophage ϕ24B modulates bacterial metabolism to raise antimicrobial tolerance.

How temperate bacteriophages play a role in microbial infection and disease progression is not fully understood. They do this in part by carrying genes that promote positive evolutionary selection for the lysogen. Using Biolog phenotype microarrays and comparative metabolite profiling we demonstrate the impact of the well-characterised Shiga toxin-prophage ϕ24B on its Escherichia coli host MC1061. As a lysogen, the prophage alters the bacterial physiology by increasing the rates of respiration and cell proliferation. This is the first reported study detailing phage-mediated control of the E. coli biotin and fatty acid synthesis that is rate limiting to cell growth. Through ϕ24B conversion the lysogen also gains increased antimicrobial tolerance to chloroxylenol and 8-hydroxyquinoline. Distinct metabolite profiles discriminate between MC1061 and the ϕ24B lysogen in standard culture, and when treated with 2 antimicrobials. This is also the first reported use of metabolite profiling to characterise the physiological impact of lysogeny under antimicrobial pressure. We propose that temperate phages do not need to carry antimicrobial resistance genes to play a significant role in tolerance to antimicrobials.

Non-isotopic detection of in situ nucleic acid in cervix: an updated protocol.

Recently, sensitive non-isotopic in situ hybridisation (NISH) methodology for the detection of human DNA and human papilloma virus (HPV) DNA in archival paraffin blocks of cervix was described. An amended protocol, now used in this laboratory for detection of these genes by NISH is presented. The amendments include the following: protease digestion at 37 degrees C; tissue dehydration in air rather than ethanol; stringency washing in formamide solution; blocking non-specific binding of avidin alkaline phosphatase with a modified buffer; and increasing the concentration of avidin alkaline phosphatase for detecting low abundance DNA. These changes simplify and increase the sensitivity of the protocol such that "Y" chromosome repeats are visualised in almost all female cells.

Simultaneous in situ genotyping and phenotyping of human papillomavirus cervical lesions: comparative sensitivity and specificity.

The sensitivity and specificity of immunocytochemistry were compared with those of non-isotopic in situ hybridisation (NISH) for the direct detection of human papillomaviruses in biopsy specimens. Four monoclonal antibodies raised to the capsid protein of HPV16 were less specific than NISH: all four reacted with lesions containing HPV33, and HPV18. Absolute discrimination of HPV types, therefore, was not possible with the monoclonal antibodies used in this study. The relative sensitivities of these antibodies were also lower than NISH. Sequential immunocytochemistry and NISH on the same section showed that 2.9-13.0 times as many cells were positive by NISH than by immunocytochemistry using the most sensitive monoclonal antibody. These data indicate that NISH has higher diagnostic specificity and sensitivity than immunocytochemistry using monoclonal antibodies to the HPV16 capsid protein.

Interphase cytogenetics using biotin and digoxigenin labelled probes: III. Increased sensitivity and flexibility for detecting HPV in cervical biopsy specimens and cell lines.

A monoclonal antibody to digoxin enabled sandwich techniques to be used for the detection of hybridised digoxigenin labelled probes in cultured cells and paraffin wax sections. This system has greater flexibility than alkaline phosphatase conjugated polyclonal antidigoxigenin antibody and permits the use of alternative detector enzymes, such as horseradish peroxidase and fluorescence labels. The APAAP detection system that does not require the use of biotin can also be used in situations where endogenous biotin is a problem. The low level of background staining combined with precise substrate deposition of the amplified peroxidase system gives higher sensitivity and resolution. This permits localisation of closely adjacent chromosomal loci in interphase nuclei. The most sensitive peroxidase based digoxigenin detection system visualises two and a half to 12 copies of human papillomavirus (HPV) per nucleus. This system is also suitable for the analysis of low copy number HPV infection of cervical tissues.

Detection of low copy human papilloma virus DNA and mRNA in routine paraffin sections of cervix by non-isotopic in situ hybridisation.

In analysing human papilloma virus (HPV) infection of the cervix in formalin fixed paraffin sections by non-isotopic in situ hybridisation two main problems were found: detachment of sections from the glass during hybridisation and probe detection; inadequate sensitivity and inability to assess sensitivity of the in situ procedure. The first problem was investigated by assessing the efficiency of various tissue adhesives individually and in combination. The second problem was addressed by optimising conditions for DNA unmasking, hybridisation, and biotinylated probe detection. Sensitivity of the final in situ procedure developed was assessed by using the detection of pHY2.1 repeats as a built-in control. Extrapolation of data showed that less than 10 copies of HPV DNA can be visualised by these procedures. HPV nucleic acid, mainly in the form of DNA, was detected not only in koilocytic nuclei but also in suprabasal cells in condylomas and CIN lesions. HPV mRNA was also visualised in the cytoplasm (and probably also nuclei) of the same cell types. These non-isotopic in situ procedures give results comparable to those obtained with radiolabelled probes, but they are less time consuming and provide better morphological resolution.

Interphase cytogenetics using biotin and digoxigenin labelled probes I: relative sensitivity of both reporter molecules for detection of HPV16 in CaSki cells.

This study was undertaken to develop technology for the detection of nucleic acid using two different DNA probe reporter molecules, the ultimate aim being to differentially label two nucleic acids within the same nucleus. Digoxigenin and biotin were used to label DNA probes. The absolute and relative sensitivity of digoxigenin and biotin labelled DNA probes for detecting integrated human papilloma virus 16 (HPV16) was investigated in CaSki cells by non-isotopic in situ hybridisation (NISH). Several methods for the detection of labelled probes were also investigated. The optimal sensitivity of digoxigenin labelled probe was equivalent to that of biotin when alkaline phosphatase was used as the final detector. The median number of discrete viral signals discernible in each cell with the most sensitive detection system was seven to eight with both labelled probes. The average number of HPV16 genomes in each CaSki cell, derived by dot blot hybridisation, was about 270. The calculated absolute sensitivity of NISH for viral detection in this system is complex because of variation of signal size and number. Nevertheless, one signal per nucleus equates to as little as 30 to 40 viral copies, and probably much less. The ability to distinguish up to 15 discrete signals with both digoxigenin and biotin labelled probes in the nuclei of CaSki cells indicates that these methods will be useful in interphase cytogenetics in material routinely fixed in aldehyde.

Interphase cytogenetics using biotin and digoxigenin labelled probes II: Simultaneous differential detection of human and papilloma virus nucleic acids in individual nuclei.

A method was developed for the simultaneous detection of viral and human DNA in contrasting colours in routine formalin fixed, paraffin wax embedded biopsy specimens. This was achieved by non-isotopic in situ hybridisation (NISH) with a biotinylated Y chromosome probe and digoxigenin labelled probe for human papilloma virus type 6 (HPV 6). The tissues studied were peripheral lymphocytes, tonsil, and penile warts. The hybridisation signals produced by biotinylated probes were visualised in red using streptavidin peroxidase and those produced by digoxigenin labelled probes as a blue/black colour using anti-digoxigenin alkaline phosphatase. In lymphocytes and tonsil 95-100% of cells had a detectable Y chromosome; in warts only 60-70% of infected keratinocytes near the skin surface had a demonstrable Y chromosome. This suggests that this chromosome is lost or occluded in cell maturation. In simultaneous double hybridisation with both probes, HPV and Y sequences were demonstrable within the same nucleus in penile warts. This technique permits the simultaneous differential detection of two nuclei acid sequences in interphase nuclei and will have application in analysis of putative dual HPV infections and in determining the intranuclear spatial relations between nucleic acids in interphase nuclei.

In situ human papillomavirus (HPV) genotyping of cervical intraepithelial neoplasia in South African and British patients: evidence for putative HPV integration in vivo.

In South Africa asymptomatic wart virus infection diagnosed by morphological criteria occurs in 16-20% of all ethnic groups; the incidence in black women is 66%. To identify human papillomavirus (HPV) types the prevalence of HPV in cervical intraepithelial neoplasia (CIN) in South African women (n = 72) with age matched British women (n = 73) was compared by non-isotopic in situ hybridisation (NISH) using digoxigenin labelled probes for HPV 6, 11, 16, 18, 31, 33 and 35 on archival biopsy specimens. A higher proportion of British biopsy specimens (68%) contained HPV than those from South Africa (50%) in CIN 2 and 3; this difference was due to HPV 16. Thirty six per cent of the positive biopsy specimens from South African women also contained HPV 33/35 compared with 16% in the United Kingdom. There was no difference in HPV detection with age in either group. These data indicate that HPV types vary geographically, with "minor" HPV types being more common in South Africa. Three qualitatively distinct NISH signals were observed; a diffuse (type 1) signal in superficial cells, mainly koilocytes; a punctate signal (type 2) in basal/"undifferentiated" cells in CIN 3; and combined type 1 and 2 signals in CIN with wart virus infection (type 3). The punctate signal may represent HPV integration.

In situ evidence for HPV 16, 18, 33 integration in cervical squamous cell cancer in Britain and South Africa.

In a previous study three types of HPV signal were described in CIN. It was suggested that a type 1 signal represented episomal HPV while a type 2 signal represented integrated HPV; and a type 3 signal was indicative of both episomal and integrated HPV. To test this hypothesis 91 squamous cell cancers (SCC) of the cervix from Britain and South Africa were examined for HPV 6, 11, 16, 18, 31, 33, 35. Of the South African group (n = 69) 64% contained HPV types 16 (n = 29) and 18 (n = 15). The SCC in the British group (n = 22) contained HPV 16 and HPV 33 in 12 and three cases, respectively. Of the HPV positive biopsy specimens, 86% showed a type 2 signal in keratinising and non-keratinising tumours and the remainder a type 3 signal. Type 3 signal was present only in keratinising tumours. The presence of punctate signal in 100% of HPV containing SCC, together with localisation of HPV signal to sister chromatids in tumour cell mitotic figures in vivo, provides further evidence for type 2, and the punctate component of type 3 signal representing viral integration.

Screw loosening in an in vitro mid fibular osteotomy model under dynamic loading conditions.

The purpose of this study was to evaluate the effect of cyclic loading on screw fixation in an experimental bone plating model. The test specimens consist of plated porcine fibulae subjected to cyclic compressive, bending and torsional loading. Breakaway torque measurements of orthopedic screws are found to be significantly less than the screws tightening torque. The breakaway torque for a given screw and tapped bone hole is found to be consistent after repeated tightening, and is proposed as a viable approach to quantify bone screw loosening. After cyclic loading at moderate levels no screw loosening was identified, but instead an apparent paradoxical tightening, as observed in breakaway torque measurements of the screws. Cyclic loading of a plated fibular fracture was not found to cause screw loosening unless accompanied by gross failure as found under excessive load levels.

Clinical trial of telepathology as an alternative modality in breast histopathology quality assurance.

Telepathology is a potential alternative to conventional histopathology. A clinical trial using a robotic telepathology system was conducted to assess the clinical and technical utility and effectiveness of telepathology in the U.K. breast screening pathology quality assurance program. Eighty-seven cases of breast disease were chosen at random from a series of 192 cases from the U.K. Breast Screening Pathology National Quality Assurance Scheme (NEQAS) collection. There were 20 benign, 23 carcinoma in situ (CIS), and 44 invasive malignant cases. The diagnostic accuracy of telepathology (TP) compared with conventional light microscopic (LM) diagnosis was 98.8%; this included a single case deferred for LM examination. The figure was similar when compared with expert consensus diagnosis (CD). In invasive tumor typing, TP accuracy was 95.4% (42/44 cases), the difference being attributable to slide color fading and would have had no impact on patient management. The accuracy of TP versus LM and expert consensus in tumor grading was 91.3% for carcinoma in situ (21/23 cases), a discordance with no relevance to patient management. TP grading of invasive tumor compared with LM diagnosis, had an accuracy of 86.4% (38/44) with a clinically significant accuracy of 97.7% (43/44). The time taken for TP diagnosis averaged 3.9 minutes per case by the end of the study. This data demonstrates that telepathology diagnostic accuracy is comparable to conventional microscopy and may therefore be envisaged as an alternative to conventional light microscopy for more rapid proficiency testing in breast screening (and perhaps other) quality assurance schemes.

Elevation of gamma delta T lymphocytes in peripheral blood and livers of patients with primary sclerosing cholangitis and other autoimmune liver diseases.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease that is possibly an autoimmune disease. Although gamma delta T cells represent a small proportion of the total T-cell population in healthy individuals, there is evidence to suggest a role for these cells in autoimmunity. Accordingly, the aim of this study was to investigate the population of gamma delta T cells in patients with PSC, compared with other chronic liver diseases. An elevation in the percentage and absolute numbers of gamma delta T cells was found in the peripheral blood of patients with PSC (8.66% and 0.13 x 10(-6)/L [P < .01 and < .05, respectively]) and autoimmune hepatitis (AIH) (8.03% and 0.13 x 10(-6)/L [both P < 0.001]) compared with controls (4.10% and 0.06 x 10(-6)/L). We also found an elevation in the percentage and absolute numbers of gamma delta T cells in the portal areas of patients with PSC (10.55% and 4.33 [P < .001 and < .001, respectively]), AIH (7.16% and 4.55 [P = .001 and < .001, respectively]), and primary biliary cirrhosis (PBC) (5.57% and 3.49 [P = .008 and < .001, respectively]) when compared with controls (2.23% and 0.81). These findings suggest a role for gamma delta T cells in the mechanism of immune damage in autoimmune liver diseases.

The discrimination of high-risk HPV types by in situ hybridization and the polymerase chain reaction.

The parameter Tmt has been defined by non-isotopic in situ hybridization and describes the tissue melting temperature (Tmt) of human papillomavirus (HPV) DNA sequences. In this study, multiple in situ hybridization signals for HPV types 16, 31 and 33 in individual archival biopsies hybridized with genomic probes are shown by polymerase chain reactions to be due to cross-hybridization of probe sequences to a single tissue target. Tmt is independent of viral type but depends on the homology between probe and target when using nick-translated whole genomic probes. The difference between Tm and Tmt is not due to the presence of viral capsid protein. Multiple HPV signals in archival material should not therefore be interpreted as indicative of multiple HPV infection unless adequate stringency conditions have been employed or they are present in morphologically distinct areas of the biopsy. Furthermore, extrapolation of calculated DNA homologies to non-isotopic in situ hybridization analysis may not be appropriate. A hybridization signal does not imply probe and target identity: this has implications for HPV typing in clinical material.

Discrimination of closely homologous HPV types by nonisotopic in situ hybridization: definition and derivation of tissue melting temperatures.

It is generally assumed that nucleic acid association during in situ hybridization reactions is similar to that of nucleic acid association in solution. This assumption has been investigated by detecting closely homologous human papillomavirus types 6 and 11 by in situ hybridization as a model for the evaluation of stringency conditions in clinical biopsies. By examining matched and mismatched, labelled and target sequences under various stringency conditions, empirical DNA-DNA stability curves and their derivative equations for tissue melting temperatures (Tmt) were derived. The corresponding values for Tmt are 10-20 degrees C higher than their solution equivalents. These data, supported by polymerase chain reaction experiments, demonstrate that closely homologous viral DNAs cross linked in tissue by formaldehyde fixation do not interact with the corresponding labelled probes as predicted from solution kinetic equations. This not only has theoretical implications but is also relevant to the accuracy of clinical diagnostic testing.

Induction of myocardial heat shock protein 70 during cardiac surgery.

Animal experiments have shown that members of the heat shock protein (HSP) family have cytoprotective properties against ischaemia. In experimentally induced cardiac ischaemia, the induction of HSP70s correlates with reduced infarct size and enhanced myocardial function and endothelial recovery. Direct evidence that increased myocardial HSP70 expression result in cytoprotection during ischaemia has also been obtained using transgenic mice overexpressing either rat or human HSP72. This study examined the induction and expression of myocardial HSP70s after an obligatory period of ischaemia in patients during cardiac surgery. The level of HSP72/HSC73 protein in Tru-cut biopsies of the myocardium, taken before and after an acute ischaemic insult, was examined using a polyclonal antibody. The amount of HSP72 mRNA in the biopsies was also determined by reverse transcriptase polymerase chain reaction (RT-PCR) and correlated HSP72/HSC73 protein expression. In four patients subjected to brief alternating periods of normothermic ischaemia and reperfusion, the amount of myocardial HSP72/HSC73 protein was increased several fold after ischaemic insult. This was accompanied by increased expression of HSP72 mRNA. In contrast, the amounts of myocardial HSP72/HSC73 protein and HSP72 mRNA were unchanged in a patient subjected to a single prolonged period of hypothermic ischaemia. Given the proven myocardial protective properties of HSP72 in experimental models, it is postulated that the observed induction of HSP72 may have a similar function in man.