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.

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.

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.

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.

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.