HPV16 oncogene expression levels during early cervical carcinogenesis are determined by the balance of epigenetic chromatin modifications at the integrated virus genome.

In cervical squamous cell carcinomas, high-risk human papillomavirus (HRHPV) DNA is usually integrated into host chromosomes. Multiple integration events are thought to be present within the cells of a polyclonal premalignant lesion and the features that underpin clonal selection of one particular integrant remain poorly understood. We previously used the W12 model system to generate a panel of cervical keratinocyte clones, derived from cells of a low-grade premalignant lesion naturally infected with the major HRHPV type, HPV16. The cells were isolated regardless of their selective advantage and differed only by the site of HPV16 integration into the host genome. We used this resource to test the hypothesis that levels of HPV16 E6/E7 oncogene expression in premalignant cells are regulated epigenetically. We performed a comprehensive analysis of the epigenetic landscape of the integrated HPV16 DNA in selected clones, in which levels of virus oncogene expression per DNA template varied ~6.6-fold. Across the cells examined, higher levels of virus expression per template were associated with more open chromatin at the HPV16 long control region, together with greater loading of chromatin remodelling enzymes and lower nucleosome occupancy. There were higher levels of histone post-translational modification hallmarks of transcriptionally active chromatin and lower levels of repressive hallmarks. There was greater abundance of the active/elongating form of the RNA polymerase-II enzyme (RNAPII-Ser2P), together with CDK9, the component of positive transcription elongation factor b complex responsible for Ser2 phosphorylation. The changes observed were functionally significant, as cells with higher HPV16 expression per template showed greater sensitivity to depletion and/or inhibition of histone acetyltransferases and CDK9 and less sensitivity to histone deacetylase inhibition. We conclude that virus gene expression per template following HPV16 integration is determined through multiple layers of epigenetic regulation, which are likely to contribute to selection of individual cells during cervical carcinogenesis.

Lack of correlation between predicted and actual off-target effects of short-interfering RNAs targeting the human papillomavirus type 16 E7 oncogene.

BACKGROUND: When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3' UTRs. It is assumed that the higher the number of predicted transcriptional OTEs, the greater the size of the actual OTE signature and the more detrimental the phenotypic consequences in target-negative cells. METHODS: We tested this general assumption by investigating the OTEs of potential therapeutic siRNAs targeting the human papillomavirus (HPV) type-16 E7 oncogene. We studied HPV-negative squamous epithelial cells, from normal cervix (NCx) and skin (HaCaT), which would be vulnerable to 'bystander' OTEs following transfection in vivo. RESULTS: We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted). The unpredicted OTEs included stimulation of innate immune pathways, as well as indirect (downstream) effects of other OTEs, which affected important cancer-associated pathways. Although most significant OTEs observed were seen in both NCx and HaCaT cells, only 0-5.9% of differentially expressed genes overlapped between the two cell types. CONCLUSION: These data do not support the assumption that actual OTEs correlate well with predicted OTEs.

Diagnosis of bladder cancer by immunocytochemical detection of minichromosome maintenance protein-2 in cells retrieved from urine.

BACKGROUND: We tested the accuracy of immunocytochemistry (ICC) for minichromosome maintenance protein-2 (MCM-2) in diagnosing bladder cancer, using cells retrieved from urine. METHODS: Adequate samples were obtained from 497 patients, the majority presenting with gross haematuria (GH) or undergoing cystoscopic surveillance (CS) following previous bladder cancer. We performed an initial study of 313 patients, followed by a validation study of 184 patients. In all cases, presence/absence of bladder cancer was established by cystoscopy/biopsy. RESULTS: In the initial study, receiver operator characteristic analysis showed an area under the curve of 0.820 (P<0.0005) for the GH group and 0.821 (P<0.01) for the CS group. Optimal sensitivity/specificity were provided by threshold values of 50+ MCM-2-positive cells in GH samples and 200+ cells in CS samples, based on a minimum total cell number of 5000. Applying these thresholds to the validation data set gave 81.3% sensitivity, 76.0% specificity and 92.7% negative predictive value (NPV) in GH and 63.2% sensitivity, 89.9% specificity and 89.9% NPV in CS. Minichromosome maintenance protein-2 ICC provided clinically relevant improvements over urine cytology, with greater sensitivity in GH and greater specificity in CS (P=0.05). CONCLUSIONS: Minichromosome maintenance protein-2 ICC is a reproducible and accurate test that is suitable for both GH and CS patient groups.

Lipoprotein lipase is frequently overexpressed or translocated in cervical squamous cell carcinoma and promotes invasiveness through the non-catalytic C terminus.

BACKGROUND: We studied the biological significance of genes involved in a novel t(8;12)(p21.3;p13.31) reciprocal translocation identified in cervical squamous cell carcinoma (SCC) cells. METHODS: The rearranged genes were identified by breakpoint mapping, long-range PCR and sequencing. We investigated gene expression in vivo using reverse-transcription PCR and tissue microarrays, and studied the phenotypic consequences of forced gene overexpression. RESULTS: The rearrangement involved lipoprotein lipase (LPL) and peroxisome biogenesis factor-5 (PEX5). Whereas LPL-PEX5 was expressed at low levels and contained a premature stop codon, PEX5-LPL was highly expressed and encoded a full-length chimeric protein (including the majority of the LPL coding region). Consistent with these findings, PEX5 was constitutively expressed in normal cervical squamous cells, whereas LPL expression was negligible. The LPL gene was rearranged in 1 out of 151 cervical SCCs, whereas wild-type LPL overexpression was common, being detected in 10 out of 28 tissue samples and 4 out of 10 cell lines. Forced overexpression of wild-type LPL and PEX5-LPL fusion transcripts resulted in increased invasiveness in cervical SCC cells, attributable to the C-terminal non-catalytic domain of LPL, which was retained in the fusion transcripts. CONCLUSION: This is the first demonstration of an expressed fusion gene in cervical SCC. Overexpressed wild-type or translocated LPL is a candidate for targeted therapy.

Cerebrotendinous xanthomatosis: 11-year treatment with chenodeoxycholic acid in five patients. An electrophysiological study.

We report the electrophysiological follow-up of five cerebrotendinous xanthomatosis patients treated for 11 years with chenodeoxycholic acid (CDCA). Nerve conduction velocity (NCV) was reduced in three cases. P100 latency of visual evoked potentials was delayed in four cases, interpeaks I-III and I-V of brainstem auditory evoked potentials (BAEPs) was increased in two and interpeak N13-20 of upper limb somatosensory evoked potentials (SEPs) was slowed in one. After 4 months of therapy with CDCA, NCV was normal and did not show any significant change during the 11 years of observation. Central motor conduction time of motor evoked potentials (MEPs) and N24-P40 interpeak latency of lower limb SEPs were increased in five and four cases, respectively, in spite of 2/3-year treatment with CDCA. Improvement of evoked potentials, especially of MEPs and SEPs, was slower and continued over the whole 11-year period. The size of xanthomas slightly decreased in some patients during treatment and the clinical manifestations stabilized, avoiding progressive worsening, but there was no significant improvement in neurological deficit. Two sisters of patients who never took CDCA showed progressive worsening of clinical manifestations, upper limb SEPs and BAEPs.

Latency associated promoter transgene expression in the central nervous system after stereotaxic delivery of replication-defective HSV-1-based vectors.

The herpes simplex virus type 1 (HSV-1) latency associated promoter (LAP) has been shown to sustain long-term reporter gene expression within sensory neurones. Its activity within the CNS is, however, less well understood. In this study we characterise the activity of the LAP after stereotaxic delivery of recombinant HSV-1-based vectors to the brain. Two classes of vectors were utilised in these studies: (1) a replication-defective vector lacking the glycoprotein H and thymidine kinase genes, designated CS1, and (2) a virus mutant severely impaired for immediate-early (IE) gene expression which lacks functional VP16, ICP4 and ICP0 genes, designated in1388. Both vectors contain the LacZ gene under the control of the LAP. Following delivery of either vector to the striatum, beta-gal expression was detected within anatomically related CNS regions distal to the site of injection. At these sites the number of beta-gal-positive cells increased with time and remained stable up to 4 weeks p.i. beta-Gal expression could not be detected at the site of injection after delivery of CS1 but beta-gal expression within neurones located at this site was observed after delivery of in1388, indicating reduced toxicity of this severely disabled virus. Transgene expression decreased dramatically with both vectors at later time-points (>4 weeks after delivery), but PCR analysis demonstrated that viral genomes were stably maintained for up to 180 days following delivery, indicating that the loss of beta-gal-positive neurones was not likely to be due to a loss of vector-transduced cells. Moreover, after delivery of an equivalent virus to the rat striatum in situ hybridisation analysis showed a similar decrease in the number of neurones expressing the endogenous LATs with time. These data indicate that although the HSV-1 LAP can drive the expression of foreign genes in a variety of CNS neurones, in these cells there is a slow down-regulation of the viral promoter which eventually results in the loss of detectable transgene expression.

Herpes simplex virus type 1 promoter activity during latency establishment, maintenance, and reactivation in primary dorsal root neurons in vitro.

A neonatal rat dorsal root ganglion-derived neuronal culture system has been utilized to study herpes simplex virus (HSV) latency establishment, maintenance, and reactivation. We present our initial characterization of viral gene expression in neurons following infection with replication-defective HSV recombinants carrying beta-galactosidase and/or green fluorescent protein reporter genes under the control of lytic cycle- or latency-associated promoters. In this system lytic virus reporter promoter activity was detected in up to 58% of neurons 24 h after infection. Lytic cycle reporter promoters were shut down over time, and long-term survival of neurons harboring latent virus genomes was demonstrated. Latency-associated promoter-driven reporter gene expression was detected in neurons from early times postinfection and was stably maintained in up to 83% of neurons for at least 3 weeks. In latently infected cultures, silent lytic cycle promoters could be activated in up to 53% of neurons by nerve growth factor withdrawal or through inhibition of histone deacetylases by trichostatin A. We conclude that the use of recombinant viruses containing reporter genes, under the regulation of lytic and latency promoter control in neuronal cultures in which latency can be established and reactivation can be induced, is a potentially powerful system in which to study the molecular events that occur during HSV infection of neurons.

Autosomal recessive paraparesis with amyotrophy of hands and feet and white matter lesions.

We report two siblings with a hitherto undescribed syndrome of autosomal recessive spastic paraparesis accompanied by amyotrophy of hands and feet, and mental deterioration. Laboratory tests showed signs of lower motoneuron involvement in the four limbs, more accentuated in the distal regions. Brain MR showed bilateral symmetrical white matter lesions. We discuss the nosological status of this syndrome in relation to other similar forms of "complicated" spastic paraparesis.

Palatal myoclonus and unusual MRI findings in a patient with membranous lipodystrophy.

We describe an Italian male patient, deceased at 29 years of age, affected with a syndrome characterized by childhood-onset seizures, mental disorders, motor dysfunction and bilateral palatal myoclonus. Skeletal X-ray examination showed diffuse osteopenia of the tubular bones, and cyst-like lesions in the carpal, metacarpal and tarsal bones bilaterally and in the proximal end of the right femur. Skin biopsy showed subcutaneous and adipose tissue containing membranocystic structures. Cerebral MR and CT scans showed fronto-temporal atrophy, altered signal of the white matter and mineralization of the caudate and dentate nuclei. These findings strongly recall polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy, but in the present case, bone alterations were not prominent; moreover, palatal myoclonus has never previously been described in this syndrome.

Ataxia-telangiectasia: somatosensory, brainstem auditory and motor evoked potentials in six patients.

Six patients with ataxia-telangiectasia were given neurophysiological examinations. The patients had progressive sensorimotor axonal neuropathy that had begun at about age eight years. Brainstem auditory evoked potentials (BAEPs) showed central alternations in two of four cases. Somatosensory (BAEPs) and motor evoked potentials (MEPs) were altered in four of five cases. In the advanced stage of the disease the neurophysiological findings, except BAEPs and MEPs, resembled those in Friedreich's ataxia. MEP and BAEP results may therefore be useful for differential diagnosis.

Motor evoked potentials by magnetic stimulation in hereditary and sporadic ataxia.

We report the study of motor evoked potentials by magnetic stimulation in 26 subjects with hereditary or sporadic ataxia. The subjects included 15 cases of late onset cerebellar ataxia (12 classified as olivopontocerebellar atrophy (OPCA), 3 as spinocerebellar atrophy (SCA)) and 11 cases of early onset cerebellar ataxia (4 Friedreich's ataxia (FA) and 7 unclassifiable in Friedreich's ataxia (NFA)). All subjects with FA and SCA had delayed central motor conduction times, more accentuated in corticospinal tracts directed to lumbar motoneurons. A similar but less marked slowing was observed in about half of the subjects with OPCA and NFA. In the last two groups the anomalies are more frequent in hereditary than in sporadic forms.

Early-onset benign limb-girdle myopathy with contractures and facial involvement affecting a father and daughter.

We describe a father and daughter with early-onset benign limb-girdle myopathy and contractures of elbows and hands, resembling Bethlem disease. Muscle biopsy showed a pattern of dystrophy with non specific mitochondrial changes. In both patients there was unusual facial muscle weakness. We discuss the nosologic position of Bethlem myopathy and suggest that facial involvement may be an additional feature of this disease.

Humoral and cell-mediated immunity in rabbits immunized with live non-replicating avipox recombinants expressing the HIV-1SF2 env gene.

The canarypox (CP) and fowlpox (FP) viruses, which are unable to replicate productively in non-avian species, have been utilized as live vectors carrying the HIV-1SF2 env gene with the putative immunosuppressive (IS) region complete (CPIS+ and FPIS+) or deleted (CPIS- and FPIS-). To determine if these avipox-env recombinants could be utilized to elicit a specific immune response against HIV-1, six groups of rabbits were immunized with CPIS+, CPIS-, FPIS+, FPIS- constructs or their non-engineered wild-type CPwt or FPwt counterparts. After a primary inoculation and successive boosters, env-specific humoral and cell-mediated immunity were demonstrated by ELISA, immunoblots and lymphoproliferation assays. Antibody titres and neutralization activities were higher in CP- than FP-inoculated rabbits, the CPIS+ always showing a similar immunogenic capacity to CPIS-. Evidence is also presented indicating that rabbit sera possess group-specific antibodies, which were, however, unable to cross-neutralize divergent HIV-1 strains. Although the protective capacity against HIV-1 experimental infection has not yet been determined in these animals, our results suggest that these recombinants might represent promising and safer candidate vaccines against HIV-1.

Dementia, myoclonus, peripheral neuropathy, and lipid-like material in skin biopsy during psychotropic drug treatment.

Chronic treatment of humans with several drugs is associated with lesions resembling lipidosis in different tissues. Recently, a Creutzfeldt-Jacob-like syndrome has been observed during tricyclic antidepressant therapy, but no evidence of interaction of these drugs with lysosomal function has been reported during such treatment. We report a case of dementia, myoclonus, peripheral neuropathy, and lipid storage in the skin due to antidepressant drug therapy, in which the discontinuation of drugs resulted in an improvement of clinical and electrophysiologic signs together with reduction of morphological evidence of lipid lysosomal storage.

Gating of trigemino-facial reflex from low-threshold trigeminal and extratrigeminal cutaneous fibres in humans.

Changes in the size of the test components (R1 and R2) of the trigemino-facial reflex were studied after electrical subliminal conditioning stimulation were applied to the trigeminal, median and sural nerves. After conditioning activation of the trigeminal nerve (below the reflex threshold), the early R1 reflex component showed phasic facilitation, peaking at about 50 ms of interstimulus delay, followed by a long-lasting inhibition recovering at 300-400 ms. The same conditioning stimulation resulted in a monotonic inhibition of the late R2, starting at 15-20 ms, with a maximum at 100-150 ms and lasting 300-400 ms. Intensity threshold for both the R1 and R2 changes ranged from 0.90 to 0.95 times the perception threshold. A similar longlasting inhibition of the R2 reflex response was also seen after conditioning stimulation applied to low-threshold cutaneous afferents of the median and sural nerves. The minimum effective conditioning-test interval was 25-30 ms and 40-45 ms respectively and lasted 600-700 ms. By contrast the early R1 reflex response exhibited a slight long-lasting facilitation with a time course similar to that of the R2 inhibition. The threshold intensity to obtain facilitation of the R1 and inhibition of the R2 test responses after conditioning volley in the median and sural nerves was similar and ranged from 0.9 to 1.2 times the perception threshold. These results demonstrate that low-threshold cutaneous afferents from trigeminal and limb nerves exert powerful control on trigeminal reflex pathways, probably via a common neural substrate. There is evidence that, in addition to any post-synaptic mechanism which might be operating, presynaptic control is a primary factor contributing to these changes.

Evoked potentials in cerebrotendinous xanthomatosis and effect induced by chenodeoxycholic acid.

Evoked potentials are reported in 10 patients with cerebrotendinous xanthomatosis, eight of whom had peripheral neuropathy. Four subjects showed delayed N13 to N20 interpeak latencies for arm somatosensory evoked potentials, and five showed moderately prolonged I to III and I to V interpeak latencies of brain-stem auditory evoked potentials. Six of seven patients showed marked delay and desynchronization of visual evoked potentials. All five patients undergoing transcutaneous magnetic stimulation of the motor cortex presented greatly delayed central motor conduction time, especially of the lower limbs. After treatment with chenodiol (750 mg/d for at least 2 years), there was a significant improvement in nerve conduction velocities, N13 to N20 interpeak latencies, and visual evoked potential latencies. Brain-stem auditory evoked potentials remained unchanged.