Aim: To examine the potential of p16INK4A as a biomarker for dysplastic squamous and glandular cells of the cervix in tissue sections and ThinPrep™ smears. Methods: Immunocytochemical analysis of p16INK4A expression was performed on 22 normal cervical tissue samples, five cervical glandular intraepithelial neoplasia (cGIN), 38 cervical intraepithelial neoplasia 1 (CIN1), 33 CIN2, 46 CIN3, and 10 invasive cancer cases (eight squamous and two adenocarcinomas). All samples were formalin fixed and paraffin wax embedded, and immunohistochemical analysis was carried out using a mouse monoclonal anti-p16INK4A antibody after antigen unmasking. The staining intensity was assessed using a 0 to 3 scoring system. In addition, the expression status of p16INK4A was examined in 12 normal ThinPrep smears, one smear exhibiting cGIN, and a total of 20 smears exhibiting mild, moderate, and severe dyskaryosis. Human papillomavirus (HPV) detection was carried out using a modified SYBR green assay system. Fluorogenic polymerase chain reaction (PCR) and solution phase PCR were used for specific HPV typing. Results: p16INK4A immunoreactivity was absent in all normal cervical tissues examined. Dysplastic squamous and glandular cells were positive for p16INK4A expression in all cases included in this study, except for one CIN3 case. p16INK4A expression was mainly nuclear in CIN1 cases, and both nuclear and cytoplasmic in CIN2, CIN3, cGIN, and invasive cases. All cases positive for HPV expressed the p16INK4A protein, although not all cases found positive for p16INK4A were HPV positive. In general, the p16INK4A staining intensity was lower in cases negative for HPV or those containing a low risk HPV type. Conclusion: This pattern of overexpression demonstrates the potential use of p16INK4A as a diagnostic marker for cervical squamous and also glandular neoplastic lesions. In addition, the technique can be used to identify individual dyskaryotic cells in ThinPrep smears. Thus, p16INK4A is a useful marker of cervical dyskaryosis.

Some anticancer drugs, but not all, inhibit replication of human immunodeficiency virus (HIV) and thus, exhibit a therapeutic potential. Such drugs, unlike the traditional HIV enzyme inhibitors, could suppress HIV strains that are resistant to inhibitors of viral enzymes, decrease proviral burden in vivo, or reduce reservoirs of infection via killing infected cells. Thus, they may be an effective adjunct therapy or perhaps result in a cure. The incidence of HIV infection and AIDS mortalities continue to increase worldwide, including the United States and parts of Africa, with a parallel increase in a number of other manifestations, including AIDS defining malignancies. The basis for continual spread of HIV presumably in large part stems from the viral resistance to previously successful drugs and the lack of curative antiretroviral drugs. To reverse these trends, other approaches for AIDS therapy must be developed. One possibility is the development of potent anticancer drugs, that exhibit anti-HIV activities. At least four chemically and pharmacologically distinct classes of anticancer drugs, i.e. certain cyclin-dependent kinase inhibitors (CDKIs), topoisomerase 1 enzyme (top 1) inhibitors, non-nucleoside antimetabolites, and estrogen receptor ligands are promising candidates. These drugs, at high doses are used for cancer therapy; at lower concentrations they exhibit anti-HIV activities in cultured cells. While the antiretroviral and the anticancer activities of the cdk inhibitor flavopiridol appear to be mutually exclusive and unrelated in cells and animal model(s) of HIV disease, the top 1 inhibitor 9-nitrocamptothecin, as well as the cdk-inhibitor roscovitine inhibit replication of HIV via selective sensitization of HIV-infected cells to apoptosis. In contrast, the inhibitory effects of these compounds are different from other cancer therapeutics that, at toxic concentrations, activate HIV either in cultured cells (such as certain ingenol and butyrate derivatives) and/or in patients (such as the widely used cyclophosmamide and cisplatin). This quality may lead to the eradication of proviral reservoirs, which is not accomplished by the currently available antiretroviral drugs. In this review, relevant available clinical and in vitro data that either support or discourage using certain anticancer drugs for treatment of HIV disease, and the rationales for developing novel antiretroviral drugs that may target infected cells rather than viral proteins are discussed.

Gene identification studies, centred on a region of overlapping loss of heterozygosity in breast tumours within band 1p31.1, lead to the characterisation of LPHH1, a novel human 7TM gene. The coding sequence of LPHH1 extends over a 60 kb region and comprises in excess of 28 exons. Alternative splicing occurs minimally at five positions, four of which are within the coding sequence. The fifth region of alternative splicing occurs at the extreme 5′ end of the transcript. A clear tissue specific bias in alternative exon selection is observed to some degree at all five positions, including the extreme 5′ region, which raises the possibility of multiple and perhaps tissue specific promoters. One such putative promoter region, which appears to be utilised predominantly in breast cancer cells, has been identified. LPHH1 is highly evolutionarily conserved, with the simplest (19 exon) gene product being 95% identical between human and rat. Comparison of the alternatively spliced exons between three species, where data are available, has so far revealed 100% identity in the encoded peptide sequences, suggesting conservation of a functional aspect of this splicing. Gene expression has been observed in all tissues and cell lines tested, with the exception of lymphoblastoid and multiple myeloma lines, where there appears to be only a very low level of transcription. LPHH1 also appears to be downregulated in human bone marrow. These data are consistent with a role for the gene products in adhesion-mediated signalling. Analysis of a panel of breast tumour cell lines revealed that a number apparently overexpressed the gene whilst others showed very low levels of transcription. In one case, the overexpression correlated with a low level increase in gene copy number in the tumour line. In addition to differences in the overall levels of expression, LPHH1 mRNAs were alternatively spliced to varying degrees with shifts in the major gene product to truncated or altered forms in some lines. No somatic LPHH1 mutations were detected through sequence analysis of four primary breast tumours that showed loss of the adjacent 1p31.1 marker D1S207.

H7 influenza strains have pandemic potential. AS03-adjuvanted H7N1 A/mallard/Netherlands/12/2000 split-virion vaccine formulations were evaluated as model H7-subtype vaccine and tested after H7N9 emerged in China, and caused severe human disease with high mortality.In this phase I/II, observer-blind, randomized trial in US and Canada, 420 healthy adults (21–64 years) were randomized to receive 1 of 4 H7N1 vaccine formulations (3.75 or 7.5 μg hemagglutinin adjuvanted with either AS03A or AS03B), 15 μg unadjuvanted H7N1 hemagglutinin, or saline placebo, given as 2-dose series. Immunogenicity was assessed using hemagglutination-inhibition (HI) and microneutralization (MN) assays, at day 42 (21 days post-dose 2), month 6, and month 12 (HI only) for the per-protocol cohorts (398, 379 and 368 participants, respectively). Safety is reported up to month 12.Beneficial AS03 adjuvant effect was demonstrated. Committee for Medical Products for Human Use, and Center for Biologics Evaluation and Research (CBER) criteria were met for all adjuvanted formulations at day 42 (H7N1 HI assay); seroprotection (SPR) and seroconversion rates (SCR) were 88.5–94.8%, mean geometric increase (MGI) 19.2–34.9, and geometric mean titers (GMT) 98.3–180.7. Unadjuvanted H7N1 vaccine did not meet CBER criteria. In adjuvanted groups, antibody titers decreased over time; month 12 SPRs and GMTs were low (2.0–18.8% and 8.1–12.2). MN antibodies showed similar kinetics, with titers persisting at higher range than HI at month 6. All adjuvanted groups showed cross-reactivity against H7N9, with HI responses similar to H7N1.The most frequent solicited symptom in adjuvanted groups was injection site pain (71.2–86.7%); grade 3 solicited symptoms were infrequent. Nine participants reported 17 serious adverse events; none were considered causally related to vaccination.Adjuvanted H7N1 vaccine formulations had an acceptable safety profile and induced an antibody response after 2 doses with cross-reactivity to H7N9.ClinicalTrials.gov: NCT01934127

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. . . . . . . . . .

Detection of measles virus in children with ileo-colonic lymphoid nodular hyperplasia, enterocolitis and developmental disorder

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.

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, GD45RAA, 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 PGR, but was detected by PGR-ISH (sensitivity of I 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 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 PBL 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.

Background & Aims: It is unclear whether fat digestion is required for the induction of gastrointestinal sensations and whether different fats have different effects. We investigated the effect of fat digestion and of medium-chain triglycerides (MCTs; C < 12) and long-chain triglycerides (LCTs; C > 16) on gastrointestinal sensations. Methods: In a double-blind study, 15 healthy subjects were studied on 5 occasions during which LCT or MCT emulsions (2 kcal/min), with or without 120 mg tetrahydrolipstatin (THL, lipase inhibitor), or sucrose polyester (SPE, nondigestible fat) were infused intraduodenally in randomized order. After 30 minutes, the proximal stomach was distended in 1 mm Hg steps/min. Intensity of gastrointestinal sensations (on a 0–10 visual analog scale), plasma cholecystokinin (CCK) levels, and gastric volumes were assessed throughout. Results: LCT and MCT increased gastric volume at baseline pressure compared with SPE, and LCT more than MCT. THL entirely abolished this effect (volumes [mL]: LCT, 213 ± 19; LCT-THL, 39 ± 3; MCT, 155 ± 12; MCT-THL, 43 ± 5; SPE, 44 ± 5). Only LCT increased plasma CCK levels (pmol/L per 30 minutes: LCT, 21 ± 2; LCT-THL, 9 ± 1; MCT, 9 ± 1; MCT-THL, 11 ± 1; SPE, 9 ± 1). During distentions, intragastric volumes were greater during infusion of LCT and MCT than during the respective THL conditions or SPE, but plasma CCK levels did not change. The intensity of sensations increased (hunger decreased) more with LCT than with MCT. During infusion of THL or SPE, the effects were smaller than during LCT or MCT. Conclusions: Fat digestion is required for the modulation of gastrointestinal sensations during gastric distention. The effects of fat depend on the fatty acid chain length and are not entirely explained by release of CCK.GASTROENTEROLOGY 2001;120:1100-1107

Activation of ret/IPTC-1 has been documented in a minority of papillary thyroid carcinomas (PTC). In a recent study, the authors' group detected the presence of ret/IPTC-1 in association with a background of florid lymphocytic thyroiditis (LT) in 58% of cases of PTC studied, which prompted them to examine the incidence of RET/PTC-1 expression in 27 examples of various forms of nonlymphomatous lymphoid infiltration of the thyroid by using TaqMan RT-PCR. Overall, 21 cases (78%) were found to express the chimeric transcript of ret/IPTC-1. Eighteen cases of Hashimoto thyroiditis were positive (95%), and, of these, three had concomitant PTC while the remainder had no histologic evidence of associated malignancy. Three cases of lymphocytic thyroiditis demonstrated activated ret/PTC-1 (43%), two having associated PTC. These data suggest either that ret/PTC-1 is an indicator of follicular thyroid cell activation or that ret/PTC-1 activation is an early event in malignant transformation. If the latter is the case, it may be that, in a defined subset of the cell population, ret/IPTC-1 activation elicits an autoimmune response, which, while possibly curtailing the development of PTC in the majority of cases, results in destruction of the thyroid parenchyma.

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.

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.