Polyphenol-Rich Blackcurrant Juice Prevents Endothelial Dysfunction in the Mesenteric Artery of Cirrhotic Rats with Portal Hypertension: Role of Oxidative Stress and the Angiotensin System.

Chronic liver diseases with portal hypertension are characterized by a progressive vasodilatation, endothelial dysfunction, and NADPH oxidase-derived vascular oxidative stress, which have been suggested to involve the angiotensin system. This study evaluated the possibility that oral intake of polyphenol-rich blackcurrant juice (PRBJ), a rich natural source of antioxidants, prevents endothelial dysfunction in a rat model of cirrhosis induced by chronic bile duct ligation (CBDL), and, if so, determined the underlying mechanism. Male Wistar rats received either control drinking water or water containing 60 mg/kg gallic acid equivalents of PRBJ for 3 weeks before undergoing surgery with CBDL or sham surgery. After 4 weeks, vascular reactivity was assessed in mesenteric artery rings using organ chambers. Both the acetylcholine-induced nitric oxide (NO)- and endothelium-dependent hyperpolarization (EDH)-mediated relaxations in mesenteric artery rings were significantly reduced in CBDL rats compared to sham rats. An increased level of oxidative stress and expression of NADPH oxidase subunits, COX-2, NOS, and of the vascular angiotensin system are observed in arterial sections in the CBDL group. Chronic intake of PRBJ prevented the CBDL-induced impaired EDH-mediated relaxation, oxidative stress, and expression of the different target proteins in the arterial wall. In addition, PRBJ prevented the CBDL-induced increase in the plasma level of proinflammatory cytokines (interleukin [IL]-1α, monocyte chemotactic protein 1, and tumor necrosis factor α) and the decrease of the anti-inflammatory cytokine, IL-4. Altogether, these observations indicate that regular ingestion of PRBJ prevents the CBDL-induced endothelial dysfunction in the mesenteric artery most likely by normalizing the level of vascular oxidative stress and the angiotensin system.

Quantitative and qualitative normative dataset for intraepidermal nerve fibers using skin biopsy.

BACKGROUND: Skin biopsy is the most relevant tool to diagnose small-fiber neuropathy. A well-documented normal dataset for intraepidermal nerve fiber in the distal leg is required to improve its diagnostic value. METHODS: Three hundred healthy subjects were enrolled in the study, after clinical and biological screening to exclude neurological and systemic pathologies. A distal leg biopsy was taken and intraepidermal nerve fiber density after protein gene product-9.5 immunocytochemistry with brightfield microscopy was determined. Morphological variations of intraepidermal nerve fibers, previously described in small-fiber neuropathies, were analyzed. One hundred biopsies were also analyzed at the ultrastructural level. FINDINGS: The median number of fibers was lower in men compared to women and decreased with age. Using statistical modeling taking into account age and gender, we calculated the 5th percentile of intraepidermal nerve fiber density as follows: 7.6156-0.0769 x age (years) + 1.5506 x gender (woman = 1; man = 0). We observed a low frequency of large swellings or horizontal branchings but an increasing frequency of small swellings of intraepidermal nerve fibers and irregular distribution along the dermal-epidermal junction with age. Axonal diameter of unmyelinated fibers of the papillary dermis did not vary with age or gender. Ultrastructural analysis also showed that fiber endings in close apposition to Merkel cells should not be mistaken for small-fiber swellings. CONCLUSIONS: Our dataset allows accurate calculation of the normal density of intraepidermal nerve fibers for each year of age and provides original morphological observations that improve the diagnostic value of skin biopsy in the distal leg for small-fiber neuropathy.

Adenylate Cyclase Type III Is Not a Ubiquitous Marker for All Primary Cilia during Development.

Adenylate cyclase type III (AC3) is localized in plasma membrane of neuronal primary cilium and can be used as a marker of this cilium. AC3 has also been detected in some other primary cilia such as those of fibroblasts, synoviocytes or astrocytes. Despite the presence of a cilium in almost all cell types, we show that AC3 is not a common marker of all primary cilia of different human and mouse tissues during development. In peripheral organs, AC3 is present mainly in primary cilia in cells of the mesenchymal lineage (fibroblasts, chondroblasts, osteoblasts-osteocytes, odontoblasts, muscle cells and endothelial cells). In epithelia, the apical cilium of renal and pancreatic tubules and of ductal plate in liver is AC3-negative whereas the cilium of basal cells of stratified epithelia is AC3-positive. Using fibroblasts cell culture, we show that AC3 appears at the plasma membrane of the primary cilium as soon as this organelle develops. The functional significance of AC3 localization at the cilium membrane in some cells but not others has to be investigated in relationship with cell physiology and expression at the cilium plasma membrane of specific upstream receptors.

Effect of the oestrogen receptor antagonist fulvestrant on the cirrhotic rat lung.

It has been postulated that cirrhosis-related lung vasodilatation and the subsequent hepatopulmonary syndrome are partly explained by an increased estradiol level through an enhanced endothelial formation of nitric oxide (NO). In this study, we assessed whether the oestrogen receptor antagonist fulvestrant (F) improves cirrhosis-related lung abnormalities. Cirrhosis was induced in rats by chronic bile duct ligation (CBDL). Four groups were studied: CBDL, CBDL+F, sham, and sham+F. Histological, immunohistochemical, and Western blot analyses were performed on lung samples. In the lung, the endothelial NO synthase and the nitrotyrosine protein expressions were increased in CBDL as compared to sham rats. Both parameters were significantly reduced by fulvestrant in the CBDL rats. Surprisingly, the level of pVASP (an indirect marker of NO formation and action) was decreased in CBDL rats, and fulvestrant had no effect on this parameter. The level of the vascular endothelial growth factor, the diameter of small lung vessels, and the number of macrophages were increased in CBDL lungs in comparison with sham lungs, and these parameters were unaffected by fulvestrant treatment. In conclusion, fulvestrant may not be relevant to improve lung abnormalities in cirrhosis because NO may not be biologically active and because key events contributing to the lung abnormalities are not affected by fulvestrant.

Probiotics (VSL#3) prevent endothelial dysfunction in rats with portal hypertension: role of the angiotensin system.

AIMS: Portal hypertension characterized by generalized vasodilatation with endothelial dysfunction affecting nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) has been suggested to involve bacterial translocation and/or the angiotensin system. The possibility that ingestion of probiotics prevents endothelial dysfunction in rats following common bile duct ligation (CBDL) was evaluated. METHODS: Rats received either control drinking water or the probiotic VSL#3 solution (50 billion bacteria.kg body wt⁻¹.day⁻¹) for 7 weeks. After 3 weeks, rats underwent surgery with either resection of the common bile duct or sham surgery. The reactivity of mesenteric artery rings was assessed in organ chambers, expression of proteins by immunofluorescence and Western blot analysis, oxidative stress using dihydroethidium, and plasma pro-inflammatory cytokine levels by flow cytometry. RESULTS: Both NO- and EDH-mediated relaxations to acetylcholine were reduced in the CBDL group compared to the sham group, and associated with a reduced expression of Cx37, Cx40, Cx43, IKCa and SKCa and an increased expression of endothelial NO synthase (eNOS). In aortic sections, increased expression of NADPH oxidase subunits, angiotensin converting enzyme, AT1 receptors and angiotensin II, and formation of ROS and peroxynitrite were observed. VSL#3 prevented the deleterious effect of CBDL on EDH-mediated relaxations, vascular expression of connexins, IKCa, SKCa and eNOS, oxidative stress, and the angiotensin system. VSL#3 prevented the CBDL-induced increased plasma TNF-α, IL-1α and MCP-1 levels. CONCLUSIONS: These findings indicate that VSL#3 ingestion prevents endothelial dysfunction in the mesenteric artery of CBDL rats, and this effect is associated with an improved vascular oxidative stress most likely by reducing bacterial translocation and the local angiotensin system.

Targeted apc;twist double-mutant mice: a new model of spontaneous osteosarcoma that mimics the human disease.

TWIST and adenomatosis polyposis coli (APC) are critical signaling factors in normal bone development. In previous studies examining a homogeneously treated cohort of pediatric osteosarcoma patients, we reported the frequent and concurrent loss of both TWIST and APC genes. On these bases, we created a related animal model to further explore the oncogenic cooperation between these two genes. We performed intercrosses between twist-null/+ and Apc1638N/+ mice and studied their progeny. The Apc1638N/+;twistnull/+ mice developed bone abnormalities observed by macroscopic skeletal analyses and in vivo imaging. Complementary histologic, cellular, and molecular analyses were used to characterize the identified bone tumors, including cell culture and immunofluorescence of bone differentiation markers. Spontaneous localized malignant bone tumors were frequently identified in Apc1638N/+;twist-null/+ mice by in vivo imaging evaluation and histologic analyses. These tumors possessed several features similar to those observed in human localized osteosarcomas. In particular, the murine tumors presented with fibroblastic, chondroblastic, and osteoblastic osteosarcoma histologies, as well as mixtures of these subtypes. In addition, cellular analyses and bone differentiation markers detected by immunofluorescence on tumor sections reproduced most murine and human osteosarcoma characteristics. For example, the early bone differentiation marker Runx2, interacting physically with hypophosphorylated pRb, was undetectable in these murine osteosarcomas, whereas phosphorylated retinoblastoma was abundant in the osteoblastic and chondroblastic tumor subtypes. These characteristics, similar to those observed in human osteosarcomas, indicated that our animal model may be a powerful tool to further understand the development of localized osteosarcoma.

Loss of STOP protein impairs peripheral olfactory neurogenesis.

BACKGROUND: STOP (Stable Tubulin-Only Polypeptide) null mice show behavioral deficits, impaired synaptic plasticity, decrease in synaptic vesicular pools and disturbances in dopaminergic transmission, and are considered a neurodevelopmental model of schizophrenia. Olfactory neurons highly express STOP protein and are continually generated throughout life. Experimentally-induced loss of olfactory neurons leads to epithelial regeneration within two months, providing a useful model to evaluate the role played by STOP protein in adult olfactory neurogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Immunocytochemistry and electron microscopy were used to study the structure of the glomerulus in the main olfactory bulb and neurogenesis in the neurosensorial epithelia. In STOP null mice, olfactory neurons showed presynaptic swellings with tubulovesicular profiles and autophagic-like structures. In olfactory and vomeronasal epithelia, there was an increase in neurons turnover, as shown by the increase in number of proliferating, apoptotic and immature cells with no changes in the number of mature neurons. Similar alterations in peripheral olfactory neurogenesis have been previously described in schizophrenia patients. In STOP null mice, regeneration of the olfactory epithelium did not modify these anomalies; moreover, regeneration resulted in abnormal organisation of olfactory terminals within the olfactory glomeruli in STOP null mice. CONCLUSIONS/SIGNIFICANCE: In conclusion, STOP protein seems to be involved in the establishment of synapses in the olfactory glomerulus. Our results indicate that the olfactory system of STOP null mice is a well-suited experimental model (1) for the study of the mechanism of action of STOP protein in synaptic function/plasticity and (2) for pathophysiological studies of the mechanisms of altered neuronal connections in schizophrenia.

Losartan prevents portal hypertension-induced, redox-mediated endothelial dysfunction in the mesenteric artery in rats.

BACKGROUND & AIMS: Advanced stages of portal hypertension are characterized by generalized vasodilatation and a hyperdynamic syndrome that leads to complications such as hepatopulmonary syndrome. We assessed the endothelial function--particularly the formation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)--in rats following common bile duct ligation (CBDL) to determine the underlying mechanisms of these processes. METHODS: Reactivity of mesenteric artery rings from male Wistar rats was determined in organ chambers. The expression levels of connexins (Cx) (Cx37, Cx40, Cx43), intermediate and small conductance Ca(2+)-activated K(+) channels (IK(Ca), SK(Ca)), endothelial NO synthase (eNOS), NADPH oxidase subunits, and nitrotyrosines were assessed by immunohistochemistry in mesenteric and pulmonary arteries. The vascular formation of reactive oxygen species (ROS) was evaluated using dihydroethidine. Control rats or those that had undergone CBDL were given either the NADPH oxidase inhibitor apocynin or the angiotensin II receptor type 1 antagonist losartan. RESULTS: Decreased EDHF-mediated relaxations to acetylcholine and red wine polyphenols were observed in CBDL rats, compared with controls, whereas the level of NO-mediated relaxation was similar. Impaired EDHF-mediated relaxations were associated with reduced vascular expression of Cx37, Cx40, Cx43, IK(Ca) and SK(Ca); increased expression of eNOS and NADPH oxidase subunits; and increased vascular formation of ROS and peroxynitrites. These effects were prevented by exposure to apocynin or losartan. CONCLUSIONS: CBDL is associated with reduced EDHF-mediated relaxations in the mesenteric artery, whereas NO-mediated relaxations persisted. These findings indicate that impaired EDHF-mediated relaxation involves an excessive vascular oxidative stress, most likely following activation of angiotensin II type 1 receptors.

Macrophage imaging by USPIO-enhanced MR for the differentiation of infectious osteomyelitis and aseptic vertebral inflammation.

The purpose of this study was to prospectively evaluate USPIO-enhanced MR imaging for the differentiation of vertebral infectious osteomyelitis and sterile inflammation. Vertebral osteomyelitis and sterile vertebral inflammation were induced in two groups of six rabbits each. MRI examinations were performed including unenhanced and gadolinium-enhanced fat-saturated SE T1w sequences. Once endplate enhancement was observed on the T1 gadolinium-enhanced MR sequence, a second MRI examination (SE T1w sequence) was performed 24 h after USPIO administration (45 micromol Fe/kg). MR imaging was correlated with histopathological findings (macrophage immunostaining and Perls Prussian blue staining). On gadolinium-enhanced T1 sequences, a significant SNR increase in vertebral endplates was present in both groups without significant difference between the two groups (P = 0.26). On USPIO-enhanced T1 sequences, a significant SNR increase was only observed in the infection group (P = 0.03) with a significant difference in SNR between the infection and the sterile-inflammation groups (P = 0.002). Infected areas presented replacement of bone marrow by an intense macrophage infiltration, some being iron-loaded. Sterile inflammation showed a replacement of bone marrow by inflammatory tissue with only rare macrophages without any Perls blue staining. USPIO-enhanced MR imaging can distinguish infectious osteomyelitis from sterile vertebral inflammation due to different macrophage distributions in the two lesions.

Structural determinants of antimicrobial and antiplasmodial activity and selectivity in histidine-rich amphipathic cationic peptides.

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria, fungi, and parasites; and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using D-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function.

Recovery from chronic demyelination by thyroid hormone therapy: myelinogenesis induction and assessment by diffusion tensor magnetic resonance imaging.

The failure of the remyelination processes in multiple sclerosis contributes to the formation of chronic demyelinated plaques that lead to severe neurological deficits. Long-term cuprizone treatment of C57BL/6 mice resulted in pronounced white matter pathology characterized by oligodendrocyte depletion, irreversible demyelination and persistent functional deficits after cuprizone withdrawal. The use of a combination of in vivo diffusion tensor magnetic resonance imaging (DT-MRI) and histological analyses allowed for an accurate longitudinal assessment of demyelination. Injection of triiodothyronine (T(3)) hormone over a 3 week interval after cuprizone withdrawal progressively restored the normal DT-MRI phenotype accompanied by an improvement of clinical signs and remyelination. The effects of T(3) were not restricted to the later stages of remyelination but increased the expression of sonic hedgehog and the numbers of Olig2(+) and PSA-NCAM(+) precursors and proliferative cells. Our findings establish a role for T(3) as an inducer of oligodendrocyte progenitor cells in adult mouse brain following chronic demyelination.

Macrophage activity in infected areas of an experimental vertebral osteomyelitis model: USPIO-enhanced MR imaging--feasibility study.

PURPOSE: To prospectively evaluate ultrasmall superparamagnetic iron oxide (USPIO) magnetic resonance (MR) imaging for the depiction of macrophages in infected areas of an experimental rabbit vertebral osteomyelitis model. MATERIALS AND METHODS: Lumbar vertebral osteomyelitis was induced in 10 rabbits with intradiscal injection of bacteria in a vertebral disk (test level) versus saline injection in another disk (control level). After a mean interval of 12 days, rabbits were imaged prior to and 24 hours after administration of USPIO. The MR imaging protocol included T1-weighted spin-echo, T2-weighted fast spin-echo, and T2*-weighted gradient-echo sequences. MR findings were compared with histologic findings (macrophage immunostaining and Perls Prussian blue staining). A Wilcoxon signed rank test was used to compare signal-to-noise ratio (SNR) results before and after USPIO administration. RESULTS: T1-weighted MR images of infected vertebral test levels obtained 24 hours after USPIO administration showed a significant increase in SNR (P = .005), whereas T2- and T2*-weighted images showed no significant changes in SNR (P = .14 and P = .87, respectively). Histologic examination results of infected areas demonstrated complete replacement of hematopoietic bone marrow by macrophage infiltration. Perls Prussian blue staining showed that some macrophages were iron loaded. T1- (P = .02), T2- (P = .04), and T2*-weighted (P = .04) images of control vertebrae showed a significant decrease in SNR. Histologic examination results confirmed the persistence of normal hematopoietic bone marrow without macrophage infiltration, which was reflected by more intensive Perls Prussian blue staining compared with that in infected areas. CONCLUSION: MR imaging can depict USPIO-loaded macrophage infiltration present in infected areas in an experimental rabbit model of vertebral osteomyelitis.

P16 expression in relation to human papillomavirus in liquid-based cervical smears.

OBJECTIVE: At present, a simple and reliable cervical cancer screening test remains to be perfected. As overexpression of the protein p16 is correlated with the presence of high-risk HPV in malignant cervical lesions, this protein has been proposed as a surrogate marker of high-risk HPV infection in cervical cancer screening. Since high-risk viral DNA integration is necessary for neoplastic progression, we aimed to examine the expression of p16 in relation to the physical status of HPV (integrated or episomal) on liquid-based cervical smears. METHODS: For each of the 241 liquid-based cervical smear included in our study, we realized a Pap test. Residual cells were processed for in situ hybridization with mucosal HPV DNA probes and for immunocytochemistry with an anti-p16 antibody. Integrated or episomal copies of HPV DNA were detected as dotted or diffuse signals, respectively. RESULTS: In high-grade intraepithelial lesions, both the integrated form of high-risk HPV and overexpression of p16 were detected. However, we observed the presence of some p16-positive/HPV-negative normal and ASCUS smears. Moreover, some p16-negative ASCUS smears and low-grade intraepithelial lesions harbored episomal high-risk HPV. CONCLUSION: If p16 was used as a surrogate marker of high-risk HPV infection, some women would be scored negative in spite of the presence of high-risk HPV. These women are more likely to undergo cancer progression, but no follow-up would be proposed to them in that screening pathway. A possible compromise for the triage of abnormal cervical smears should be a combination of both HPV and p16 testing.

Expression pattern of STOP lacZ reporter gene in adult and developing mouse brain.

Stable tubulin-only polypeptide (STOP) proteins are microtubule-associated proteins responsible for microtubule stabilization in neurons. STOP null mice show apparently normal cerebral anatomy but display synaptic defects associated with neuroleptic-sensitive behavioral disorders. STOP null mice have therefore been proposed as an animal model for the study of schizophrenia. In the present study, the expression pattern of STOP gene in developing and adult brain has been examined by using lacZ gene inserted in the STOP locus, as a reporter gene. beta-Galactosidase (beta-gal) immunostaining was confined to neuronal cells and projections. Strong labeling was observed in the whole olfactory system, cortical layer VII, hippocampus, hypothalamus, cerebellum, habenula, fasciculus retroflexus, and interpeduncular nucleus in adults. Additionally, ventral thalamic nucleus, clusters of positive cells in striatum, and Cajal-Retzius cells of cortical layer I were labeled in young mice. The strong expression of STOP lacZ reporter gene observed in brain is confined to areas that may be involved in the schizophrenia-related symptoms observed in STOP-deficient mice.

p16 expression in relation to human papillomavirus in anogenital lesions.

Recent studies have revealed that cervical cancers associated with high-risk human papillomavirus (HPV) showed overexpression of the p16 protein, a cyclin-dependent kinase inhibitor. The expression of this cell cycle regulator in lesions of the anogenital region in association with HPV physical status (episomal or integrated) has not been studied at the present time. In this report, immunohistochemical analysis of p16 and HPV detection by in situ hybridization were performed on 110 formalin-fixed and paraffin-embedded samples of anogenital lesions. The results showed strong diffuse p16 staining in all integrated high-risk HPV-positive lesions, whereas most episomal HPV-positive lesions or HPV-negative lesions showed no p16 immunostaining. However, there were a few HPV-negative lesions or lesions with episomal HPV harboring p16 overexpression. On the other hand, some lesions were p16 negative while showing the presence of high-risk HPV in its episomal form. In conclusion, screening for p16 overexpression in cutaneomucous lesions of the anogenital region allowed good discrimination between HPV-integrated lesions and lesions harboring episomal HPV or no HPV. But p16 overexpression was not always predictive of the presence of high-risk HPV; moreover, absence of p16 immunostaining observed in some high-risk HPV lesions suggested that limiting the screening to p16 would exclude some patients harboring high-risk HPV from any follow-up.

Reelin immunoreactivity in lymphatics and liver during development and adult life.

Reelin, a glycoprotein secreted by Cajal-Retzius cells, is crucial for cortex lamination and cell positioning. Some peripheral reelin expression has also been reported. Here we describe in developing rat and human and in adult rat very definite reelin immunolocalization in two cell types. Reelin was present in endothelial cells of lymphatic vessels, but not of blood vessels. Reelin was also detected in stellate (Ito) cells of the liver as shown by immunoelectron microscopic examination. The immunlocalization observed in adult tissues indicates a broader role of reelin beyond its previously demonstrated role in neuronal development.

Further delineation of the odonto-onycho-dermal dysplasia syndrome.

We report on three boys, two brothers and their maternal cousin, presenting with dry hair, pilar keratosis, severe hypodontia, smooth tongue, onychodysplasia, and keratoderma and hyperhidrosis of palms and soles. Histology of the skin showed orthokeratotic, hyperkeratosis, hypergranulosis, and mild acanthosis in the epidermis. Scanning electron microscopic examination of the hair showed longitudinal depressions in some hair. These features are close to a rare entity: the odonto-onycho-dermal dysplasia but with some differing features.

Retinoic-acid receptor beta expression in melanocytes.

Retinoic acid receptors (RAR) and retinoic X receptors (RXR) are critical for skin homeostasis. In epidermis, RXRalpha and RARgamma isoforms are highly expressed but only weak or no expression of RARbeta has been reported. Here, we re-examined RARbeta in situ expression in comparison with that of the RXRalpha, in both normal skin and melanocytic tumours. In normal skin, RXRalpha was localized in epidermis, sebaceous glands and hair follicles, while RARbeta was detectable only in melanocytes and in stratum granulosum. RXRalpha was never detected in melanocytic tumours, neither in nevi, nor in melanomas. RARbeta was also absent from melanoma cells but was present in nevus cells. These results indicate that melanoma are characterised by simultaneous decrease of RARbeta and absence of RXRalpha that may be responsible for the RA-resistance of most melanoma cell lines.

HPV DNA detection by in situ hybridization with catalyzed signal amplification on thin-layer cervical smears.

Thin layer-based technology in cervical cancer screening now allows both Papanicolaou staining and HPV testing on the same sample. Here, we show that in situ hybridization with catalyzed reporter deposition is a powerful HPV detection method when applied on thin-layer cervical smears, allowing distinction between two staining patterns suggestive of two different physical states of HPV DNA, where diffuse signals are suggestive of episomes and punctate signals are suggestive of viral DNA integration.