Operational classification of cutaneous squamous cell carcinomas based on unsupervised clustering of real cases by experts.

BACKGROUND: There is currently no staging system for cutaneous squamous cell carcinoma (cSCC) that is adapted to decision-making and universally used. Experts have unconscious ability to simplify the heterogeneity of clinical situations into a few relevant groups to drive their therapeutic decisions. Therefore, we have used unsupervised clustering of real cases by experts to generate an operational classification of cSCCs, an approach that was successful for basal cell carcinomas. OBJECTIVE: To generate a consensual and operational classification of cSCCs. METHOD: Unsupervised independent clustering of 248 cases of cSCCs considered difficult-to-treat. Eighteen international experts from different specialties classified these cases into what they considered homogeneous clusters useful for management, each with freedom regarding clustering criteria. Convergences and divergences between clustering were analysed using a similarity matrix, the K-mean approach and the average silhouette method. Mathematical modelling was used to look for the best consensual clustering. The operability of the derived classification was validated on 23 new practitioners. RESULTS: Despite the high heterogeneity of the clinical cases, a mathematical consensus was observed. It was best represented by a partition into five clusters, which appeared a posteriori to describe different clinical scenarios. Applicability of this classification was shown by a good concordance (94%) in the allocation of cases between the new practitioners and the 18 experts. An additional group of easy-to-treat cSCC was included, resulting in a six-group final classification: easy-to-treat/complex to treat due to tumour and/or patient characteristics/multiple/locally advanced/regional disease/visceral metastases. CONCLUSION: Given the methodology based on the convergence of unguided intuitive clustering of cases by experts, this new classification is relevant for clinical practice. It does not compete with staging systems, but they may complement each other, whether the objective is to select the best therapeutic approach in tumour boards or to design homogeneous groups for trials.

Sclerodermiform basal cell carcinomas vs. other histotypes: analysis of specific demographic, clinical and dermatoscopic features.

BACKGROUND: Among the various types of basal cell carcinoma, the sclerodermiform variant has a high risk of recurrence and local invasiveness. A systematic description of the dermatoscopic features associated with specific body localization is lacking. OBJECTIVES: To describe the clinical and dermoscopic features of sclerodermiform basal cell carcinoma (BCC) according to localization in the body confronting with superficial and nodular types. METHODS: Clinical and dermoscopic images of sclerodermiform, nodular and superficial BCCs were retrospectively evaluated to study the location in the various body districts, maximum diameter, clinical appearance of the lesion, features of edges and presence or absence of specific dermatoscopic criteria of BCCs. RESULTS: We examined 291 histopathologically proven BCCs showing that in nodular BCCs, classical arborizing vessels were more frequently found in the body macro-area (trunk and limbs; n = 46, 97.9%) than in the head/neck area (n = 43, 82.7%); within sclerodermiform BCCs, short arborizing vessels were found more frequently in the head/neck district (n = 35, 49.3%) than in the body (n = 6, 23.1%; P-value 0.02); within nodular BCCs, multiple blue-grey dots and globules were more frequently found on the trunk (n = 23, 48.9%) than in the head/neck district (n = 12, 23.1%; P-value 0.01). In sclerodermiform BCCs, ulceration was found more frequently in the head/neck district (n = 38, 53.5%) than in the body (n = 4, 15.4%; P-value > 0.01), and in superficial BCCs, ulceration was found more frequently in the head/neck district (n = 5, 38.5%) than in the body (n = 8, 9.8%; P-value 0.02). CONCLUSION: Our study shows that superficial BCC are found frequently in the head/neck district dermoscopically characterized by ulceration and arborizing vessels; nodular BCCs are more frequently found in the body than in the head/neck district, and the dermoscopic pattern is characterized by the combination of three features: (i) classical arborizing vessels, (ii) multiple blue-grey dots and (iii) globules. Instead, sclerodermiform BCC is preferentially located in areas at high-moderate risk of recurrence; if pink-white areas and/or fine arborizing vessels are seen, clinicians should consider this diagnosis. Furthermore, location-specific dermatoscopic criteria have been described.

Clinical and dermoscopic features of genital pigmented Bowen disease.

Pigmented Bowen disease (pBD) is an uncommon variant of squamous cell carcinoma in situ. Sometimes it can show clinical and dermoscopic features that are seen in other pigmented lesions of the skin and mucosa, making the diagnosis difficult. We report six cases of pBD occurring on the anogenital area, and discuss the importance of dermoscopy for improving the diagnostic accuracy in pBD.

Nanofat 2.0: experimental evidence for a fat grafting rich in mesenchymal stem cells.

Different strategies have been developed in the last decade to obtain fat grafts as rich as possible of mesenchymal stem cells, so exploiting their regenerative potential. Recently, a new kind of fat grafting, called "nanofat", has been obtained after several steps of fat emulsification and filtration. The final liquid suspension, virtually devoid of mature adipocytes, would improve tissue repair because of the presence of adipose mesenchymal stem cells (ASCs). However, since it is probable that many ASCs may be lost in the numerous phases of this procedure, we describe here a novel version of fat grafting, which we call "nanofat 2.0", likely richer in ASCs, obtained avoiding the final phases of the nanofat protocol. The viability, the density and proliferation rate of ASCs in nanofat 2.0 sample were compared with samples of nanofat and simple lipoaspirate. Although the density of ASCs was initially higher in lipoaspirate sample, the higher proliferation rate of cells in nanofat 2.0 virtually filled the gap within 8 days. By contrast, the density of ASCs in nanofat sample was the poorest at any time. Results show that nanofat 2.0 emulsion is considerably rich in stem cells, featuring a marked proliferation capability.

Evaluation of cutaneous surface parameters in psoriatic patients.

PURPOSE: The purpose of this study was to compare cutaneous surface parameters in lesional and non-lesional skin of psoriatic patients and in corresponding areas of control subjects. METHODS: Sixty-six psoriatic patients (of any grade of severity, with or without arthritis, without any therapy other than systemic biologic drugs) and 28 healthy controls were enrolled in this observational, case-control study. Exclusion criteria were current or past sebo-psoriasis and seborrheic dermatitis, pustular or erithrodermic psoriasis; treatment with immune-suppressive agents, retinoids, or ultraviolet phototherapy in the last 6 months; topical treatment in the last 2 weeks. Corneometry, sebumetry, and pHmetry were evaluated on non-lesional skin of forehead, cheek, chin and volar region of forearm, and on a psoriatic plaque (on elbow or neighboring areas); in controls, the same areas were considered. RESULTS: Corneometry values were significantly lower in psoriatic plaques vs. elbows of controls. Sebumetry showed significantly higher values in non-lesional forearm skin and plaques of psoriatic patients vs. corresponding areas of controls. pH was significantly lower in all areas in psoriasis. No differences were found between patients treated or not with biologics and with or without arthritis. CONCLUSION: Evaluating surface skin parameters in psoriasis is useful to better understand the etiopathogenic mechanism and could suggest new therapeutic approaches.

Avaliação da técnica de coloração AgNOR em testículos de ovinos / Evaluation of the AgNOR staining method in ovine testicles

A coloração pela prata das regiões organizadoras de nucléolos (NORs) é caracterizada por marcar proteínas ligadas ao ácido ribonucleico ribossômico, avaliando a proliferação em células normais ou neoplásicas. Objetivou-se estudar, em testículos de ovinos obtidos em matadouro, a validade do uso da técnica de coloração pela prata (AgNOR) na identificação das regiões organizadoras de nucléolo (NORs) em células saudáveis da linhagem espermatogênica. Utilizaram-se 43 pares de testículos de ovinos mestiços entre seis e 10 meses de idade. Testes de Wilcoxon e Spearman foram empregados, com nível de 5%. As médias das NORs nas células das gônadas direita e esquerda foram, respectivamente: espermatogônia (8,77±1,14 e 9,04±0,96), espermatócitos (4,99±2,00 e 6,20±2,07; P<0,05), Leydig (8,05±2,82 e 7,89±2,29) e Sertoli (8,07±1,88 e 7,61±2,16; P<0,05). Houve correlação (P<0,05) entre os lados para o número de NORs: espermatócitos x Leydig (0,49); espermatócitos x Sertoli (0,49) e Leydig x Sertoli (0,96). Conclui-se ser válido o emprego da técnica AgNOR para avaliar o potencial proliferativo das células saudáveis em testículos de ovinos com prática execução e baixo custo.(AU)

The silver staining technique for AgNOR nucleolar organizer regions (NORs) is characterized by marking proteins linked to the ribosomal ribonucleic acid, evaluating cell proliferation. The aim was to study the validity of the AgNOR staining technique in the testicular cell proliferation in crossbred ovine. Forty-three pairs of ovine testicles between 6 and 10 months old were collected. Wilcoxon and Spearman tests were used with a significance level of 5%. The mean NORs count in cells of the right and left gonads were respectively: spermatogonia (8.77±1.14 and 9.04±0.96), spermatocytes (4.99±2.00 and 6.20±2.07, P<0.05), Leydig (8.05±2.82 and 7.89±2.29) and Sertoli cells (8.07±1.88 and 7.61±2.16; P<0.05). There was a correlation between the mean values for the right and left sides for the number of NORs (P<0.05) between Leydig x spermatocytes (0.49); spermatocytes x Sertoli (0.49) and Sertoli x Leydig (0.96). The study demonstrates that the AgNOR staining technique is indicated to evaluate the cell proliferative potential in ovine testis with practical implementation and low cost.(AU)

Comparação entre dois protocolos de tratamento de ceratoconjuntivite seca experimentalmente induzida em coelhos / Comparison of two treatment protocols of experimentally induced keratoconjunctivitis sicca in rabbits

O objetivo deste estudo foi avaliar e comparar a eficácia de dois protocolos de tratamento de ceratoconjuntivite seca (CCS) experimentalmente induzida em coelhos: uma formulação oftálmica tópica composta por álcool polivinílico 1,4%, adicionado com acetilcisteína 10% e pilocarpina 1% (AAP), e outro protocolo com o uso do óleo de semente de linhaça (OL) tópico em forma de colírio, durante 12 semanas. Foram utilizados 15 coelhos machos, adultos, da raça Nova Zelândia, alocados aleatoriamente em três grupos: grupo C (controle), grupo AAP (formulação oftálmica) e grupo L (OL tópica). Os animais foram avaliados semanalmente pelo teste lacrimal de Schirmer, teste de fluoresceína e teste de Rosa Bengala; uma vez por mês, pelo exame de citologia esfoliativa ocular; ao final do experimento, pela análise histopatológica da córnea e conjuntiva. Os resultados demonstraram que houve um aumento maior na produção lacrimal quando utilizada a formulação oftálmica, e uma resolução mais rápida das úlceras de córnea, bem como diminuição no número de células desvitalizadas quando utilizado o óleo de semente de linhaça, além de aumento no número de células caliciformes em ambos os grupos de tratamento. A associação desses dois protocolos pode ser no futuro uma alternativa no tratamento da CCS.

The objective of this study was to evaluate and compare the effectiveness of two treatment protocol of experimentally induced keratoconjunctivitis sicca (KCS) in rabbits, a topical ophthalmic formulation composed by 1.4% povinilic alcohol added with 10% acetylcysteine and 1% pilocarpine (AAP) and another protocol with the topical use of the linseed seed oil (LO) in eye drop form f or 12 weeks. Fifteen male New Zealand white rabbits were aleatory allocated in 3 groups: Group C (Control), Group AAP (ophthalmic formulation) and Group L (LO topical). The animals were evaluated weekly using the Schirmer's tear test, fluorescein test and Rose Bengal test monthly for ocular cytology, and at the end of the experiment for histopathological analysis of cornea and conjunctive. The results demonstrated that there was a larger increase in the tear production when the ophthalmic formulation was us ed and a faster rapid resolution of corneal ulcers and decrease in the number of devitalized cells when linseed seed oil was used, besides an increase in the number of caliciform cells in both treatment groups. The association of those two protocols can be a future alternative in the treatment of KCS.

Effects of repeated ankle plantar-flexions on H-reflex and body sway during standing.

The study investigated relations between effects of repeated ankle plantar-flexion movements exercise on the soleus Hoffmann (H) reflex and on postural body sway when maintaining upright stance. Ten young volunteers performed five sets of ankle plantar-flexions of both lower limbs. Assessment of the feet centre-of-pressure (COP) displacement and H-reflex tests were carried out in quiet stance before, during and after the exercise. H-max and M-max responses were obtained in 8 subjects and reported as the peak-to-peak amplitudes of the right soleus muscle electromyographic waves. Mean dispersion of COP along the antero-posterior direction increased significantly during the exercise; whilst the overall H-reflex response indicated a reduction without a concomitant modification in the M-max response. H-reflex responses, however, varied between participants during the first sets of exercise, showing two main trends of modulation: either depression or early facilitation followed by reduction of the H-reflex amplitude. The extent of reflex modulation in standing position was correlated to the concentric work performed during the exercise (r=0.85; p<0.01), but not to the antero-posterior COP dispersion. These results suggest that during a repeated ankle plantar-flexions exercise, modulation of the H-reflex measured in upright stance differs across individuals and is not related to changes of postural sway.

[Pain in Parkinson's disease]. / Syndromes douloureux de la maladie de Parkinson.

INTRODUCTION: Pain may be a presenting symptom of Parkinson's disease or may occur during the motor fluctuation stages of the disease. The complexity and pathophysiology of pain in Parkinson's disease still remains poorly understood. OBJECTIVE: To characterize clinically the different painful presentations of Parkinson's disease, their relationship to the stage of the disease and their connections with motor fluctuations and treatment. METHODS: We reviewed painful syndromes in 388 consecutive parkinsonian patients of the Lausanne Movement Disorders Registry, based on an itemized questionnaire used prospectively to characterize the pain by its description, topography, date of appearance and possible relationship with motor fluctuations. Among these patients with clinically-diagnosed dopa-responsive Parkinson's disease, 269, i.e. 67 percent presented sensory or painful syndromes. Among them, 94 percent had muscular pain: stiffness (85 percent), cramps, pseudo-cramps, spasms (3 percent) or various myalgias (7 percent); 51 percent presented osteo-ligamentar "rheumatologic" pain, articular (23 percent), periarticular (3 percent) or spinal (31 percent), but less defined and localized neurogenic painful syndromes were less frequent (8 percent), such as paresthesia (6 percent), dysesthesia (<1 percent), burning sensation (2 percent), itching (<1 percent), ill defined discomfort (6 percent) or a feeling of heaviness (1 percent), with segmental (86 percent), axial (54 percent), radicular or pseudo-radicular (14 percent), acral distal (4 percent) or less frequently anorectal or visceral distribution. Restless legs or akathisia were occasional (10 percent). Headaches and facial pain were less frequent (1 percent), we did not encounter phantom pain. More than one quarter were present at the beginning of the disease, only (3 percent) of them resolved during the development of the disease. About one-third were clearly linked with motor fluctuations, the majority occurring in off phase (34 percent). We did not find any correlation with age, gender, duration or stage of disease, L dopa equivalent dose, depression, insomnia or autonomic dysfunction. CONCLUSION: Painful syndromes are found in two thirds of patients with Parkinson's disease, with mainly pain of muscular origin, followed by osteoarticular and neurogenic painful syndromes, a quarter of the patients experience pain in early phases of the disease and a third in relation with motor fluctuations.

Endogenous activation of group-I metabotropic glutamate receptors is required for differentiation and survival of cerebellar Purkinje cells.

We have applied subtype-selective antagonists of metabotropic glutamate (mGlu) receptors mGlu1 or mGlu5 [7-(hydroxy-imino) cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) or 2-methyl-6-(phenylethynyl)pyridine (MPEP)] to mixed rat cerebellar cultures containing both Purkinje and granule cells. The action of these two drugs on neuronal survival was cell specific. Although CPCCOEt (1, 10, 30 microm) reduced the survival of Purkinje cells, MPEP (3 or 30 microm) selectively reduced the survival of granule cells. Both effects required an early exposure of cultures to antagonists [from 3 to 6 d in vitro (DIV) for CPCCOEt, and from 3 to 6 or 6 to 9 DIV for MPEP]. Addition of MPEP from 6 to 9, 9 to 13, or 13 to 17 DIV also induced profound morphological changes in the dendritic tree and dendritic spines of Purkinje cells, suggesting that endogenous activation of mGlu5 receptors is required for the age-dependent refinement of Purkinje cell phenotype. In in vivo studies, an early blockade of mGlu1 receptors induced in rats by local injections of LY367385 (20 nmol/2 microl), local injections of mGlu1 antisense oligonucleotides (12 nmol/2 microl), or systemic administration of CPCCOEt (5 mg/kg, s.c.) from postnatal day (P) 3 to P9 reduced the number and dramatically altered the morphology of cerebellar Purkinje cells. In contrast, mGlu5 receptor blockade induced by local injections of antisense oligonucleotides reduced the number of granule cells but also produced substantial morphological changes in the dendritic tree of Purkinje cells. These results provide the first evidence that the development of cerebellar neurons is under the control of mGlu1 and mGlu5 receptors, i.e., the two mGlu receptor subtypes coupled to polyphosphoinositide hydrolysis.

Increased ACTH and cortisol secretion after interleukin-alpha injection in the common marmoset (Callithrix jacchus jacchus).

We have studied the effect of intravenous injection of interleukin-1 (dose range: from 0.25 to 4.5 microg/kg of body weight) on plasma ACTH and cortisol levels in the marmoset, a primate paradygm of peripheral glucocorticoid resistance. Blood sampling were collected and body temperature recorded 0, 15, 30, 60, 120, 180, 240 and 300 min after injection. Interleukin-1 stimulated secretion of ACTH in a dose-dependent fashion. Maximal secretion occurred 120 min after injection, and lasted up to 240 min. Plasma ACTH levels returned to baseline 300 min after interleukin-1 injection. Plasma cortisol levels were related to ACTH levels. Body temperature elevation, which occurred 10-15 min after injection was dose-dependent, and lasted 3 h. Results suggest that the pyrogenic effect of interleukin is associated, in the marmoset, with integrated activation of the hypothalamic-pituitary-adrenal axis. In light of the proneness of marmosets to hyperimmune disorders, our data are consistent with the hypothesized central biological role of IL-1, as well as the pathophysiological relevance of the neuro-endocrine-immune cross-talk during the acute phase response.

Selective activation of mGlu4 metabotropic glutamate receptors is protective against excitotoxic neuronal death.

Activation of group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, and mGluR8) has been established to be neuroprotective in vitro and in vivo. To disclose the identity of the receptor subtype(s) that exert(s) the protective effect, we have used group III agonists in combination with mGluR4 subtype-deficient mice (-/-). In cortical cultures prepared from wild-type (+/+) mice and exposed to a toxic pulse of NMDA, the selective group III agonist (+)-4-phosphonophenylglycine [(+)-PPG] reversed excitotoxicity with an EC(50) value of 4.9 microm, whereas its enantiomer (-)-PPG was inactive. This correlated closely with the potency of (+)-PPG in activating recombinant mGluR4a. In cortical neurons from -/- mice, (+)-PPG showed no protection against the NMDA insult up to 300 microm, whereas group I/II mGluR ligands still retained their protective activity. Classical group III agonists (l-2-amino-4-phosphonobutyrate and l-serine-O-phosphate) were also substantially neuroprotective against NMDA toxicity in +/+ and heterozygous (+/-) cultures but were inactive in -/- cultures. Interestingly, -/- cultures were more vulnerable to low concentrations of NMDA and showed higher extracellular glutamate levels compared with +/+ cultures. We have also examined neurodegeneration induced by intrastriatal infusion of NMDA in wild-type or mGluR4-deficient mice. Low doses of (R,S)-PPG (10 nmol/0.5 microl) substantially reduced NMDA toxicity in +/+ mice but were ineffective in -/- mice. Higher doses of (R,S)-PPG were neuroprotective in both strains of animals. Finally, microdialysis studies showed that intrastriatal infusion of NMDA increased extracellular glutamate levels to a greater extent in -/- than in +/+ mice, supporting the hypothesis that the mGluR4 subtype is necessary for the maintenance of the homeostasis of extracellular glutamate levels.

Differential expression of group I metabotropic glutamate receptors in functionally distinct hippocampal interneurons.

Metabotropic glutamate receptors (mGluRs) have been proposed to be involved in oscillatory rhythmic activity in the hippocampus. However, the subtypes of mGluRs involved and their precise distribution in different populations of interneurons is unclear. In this study, we combined functional analysis of mGluR-mediated inward currents in CA1 oriens-alveus interneurons with anatomical and immunocytochemical identification of these interneurons and expression analysis of group I mGluR using single-cell reverse transcription-PCR (RT-PCR). Four major interneuron subtypes could be distinguished based on the mGluR-mediated inward current induced by the application of 100 microm trans-(1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (ACPD) under voltage-clamp conditions and the action potential firing pattern under current-clamp conditions. Type I interneurons responded with a large inward current of approximately 224 pA, were positive for somatostatin, and the majority expressed both mGluR1 and mGluR5. Type II interneurons responded with an inward current of approximately 80 pA, contained calbindin, and expressed mainly mGluR1. Type III interneurons responded with an inward current of approximately 60 pA. These interneurons were fast-spiking, contained parvalbumin, and expressed mainly mGluR5. Type IV interneurons did not respond with an inward current upon application of ACPD, yet they expressed group I mGluRs. Activation of group I mGluRs under current-clamp conditions increased spike frequency and resulted in rhythmic firing activity in type I and II, but not in type III and IV, interneurons. RT-PCR results suggest that activation of mGluR1 in the subsets of GABAergic interneurons, classified here as type I and II, may play an important role in mediating synchronous activity.

The electrogenic sodium bicarbonate cotransporter: developmental expression in rat brain and possible role in acid vulnerability.

The electrogenic sodium bicarbonate cotransporter (NBC) is expressed in glial cells in the brain and plays an important role in the regulation of both intracellular and extracellular pH. Differential vulnerability to acidosis between neurons and glia has been noted and may contribute to infarction after cerebral ischemia. Ionic substitution studies and inhibition of injury by 4, 4'-di-isothiocyanostilbene-2,2'-disulfonic acid suggest that NBC is involved in astrocyte vulnerability to acidic injury. Recently two NBC cDNAs differing in 5'-untranslated and N-terminal coding sequence have been cloned from kidney and pancreas. We cloned one of these cDNAs from rat brain and demonstrate here that the clone is functional by expression in Xenopus oocytes. We determined the developmental and regional expression of NBC in the brain by in situ hybridization. Expression was observed in the spinal cord at embryonic day 17, whereas expression in brain was first seen at approximately postnatal day 0 (P0), increased at P15, and persisted in the adult brain. Expression was widespread throughout the cerebellum, cortex, olfactory bulb, and subcortical structures. Cellular resolution of the in situ hybridization signal and double labeling for glial fibrillary acidic protein were consistent with a glial localization for NBC. Expression of NBC in 3T3 cells that do not normally express this transporter rendered them vulnerable to acid injury. The expression profile suggests that this transporter is critical during the later stages of brain development and could be one of the factors contributing to the different patterns of injury seen in perinatal versus adult cerebral ischemia.

Modulatory action of noradrenergic system on spinal motoneurons in humans.

Previous findings in animals demonstrated that the noradrenergic coeruleospinal system exerts a tonic facilitation on spinal reflexes and that activation of alpha2-autoinhibitory receptors can be responsible for a disfacilitation of the spinal activity. To investigate this issue further, we examined whether this system is also involved in descending facilitatory control of spinal motoneurons in healthy humans. The H-reflex technique was utilized to assay the motoneuronal excitability. The ratio between the maximal reflex response (H) and maximal direct response (M) was determined in each subject and was calculated at 10 min intervals before and after i.v. administration of the alpha2-agonist clonidine (0.5 microg/kg). In all subjects a marked decrease of the H/M ratio, due to depression of the H response, occurred 10 min following the clonidine injection and reached its maximum within 30 min. No significant changes of blood pressure values were provoked by drug injections. These results suggest that an autoinhibitory action may be induced by alpha2-receptor activation of locus coeruleus neurons in humans, and that this device may serve as a mechanism for a myotonolytic action on spinal motoneurons.