Criteria for melanocytic lesions in LC-OCT.

BACKGROUND: Line-field confocal optical coherence tomography (LC-OCT) is an emerging diagnostic tool with imaging depth reaching ~400 µm and a novel three-dimensional (3D) cube providing cellular resolution. As far as we are aware, there are only a limited number of papers that have reported diagnostic criteria for melanocytic lesions using this technique, and none of them have been multicentric. OBJECTIVES: Our aim was to establish the diagnostic criteria for melanocytic lesions using LC-OCT and identify the most significant architectural and cytologic features associated with malignancy. METHODS: A retrospective evaluation of 80 consecutive melanocytic lesions from a prospective multicentric data set spanning three European centres was conducted. We excluded facial, acral and mucosal lesions from the study. Dermoscopic and LC-OCT images were evaluated by a consensus of four observers. Multivariate logistic regression with backward elimination was employed. RESULTS: The main melanoma diagnostic criteria include detecting >10 pagetoid cells in 3D acquisition, irregular 3D epidermal architecture, disrupted dermoepidermal junction (DEJ) and clefting. Significant risk factors were irregular 3D epidermal architecture, >10 pagetoid cells, dendritic cells at DEJ without underlying inflammation. Novel malignancy criteria in vertical view were DEJ disruption and clefting around atypical melanocyte nests. Exclusive melanoma features were epidermal nests, epidermal consumption, dense dermal nests with atypia. Protective features in the absence of any malignancy indicators were DEJ ring pattern, cobblestone, elongated rete ridges (vertical), well-defined DEJ and wave pattern (vertical). CONCLUSIONS: A series of diagnostic criteria for the identification of melanocytic lesions with LC-OCT have been established. Validation of these criteria in clinical practice through future studies is essential to further establish their utility.

Understanding the anatomy of dermoscopy of melanocytic skin tumours: Correlation in vivo with line-field optical coherence tomography.

BACKGROUND: Early melanoma detection is the main factor affecting prognosis and survival. For that reason, non-invasive technologies have been developed to provide a more accurate diagnosis. Recently, line-field confocal optical coherence tomography (LC-OCT) was developed to provide an in vivo, imaging device, with deep penetration and cellular resolution in three dimensions. Combining the advantages of conventional OCT and reflectance confocal microscopy, this tool seems to be particularly suitable for melanocytic lesions. OBJECTIVES: The objective of this study was to identify and describe the correlation between specific dermoscopic criteria and LC-OCT features in three dimensions associated with melanocytic lesions. METHODS: Dermoscopic and LC-OCT images of 126 melanocytic lesions were acquired in three different centres. The following dermoscopic criteria have been considered: reticular pattern, dots and globules, structureless areas, blue-whitish veil, regression structures, negative network, homogeneous pattern, streaks and blotches. RESULTS: 69 (55%) benign and 57 (45%) malignant lesions were analysed. A regular reticular pattern was found associated in the 75% of the cases with the presence of elongated rete ridges with pigmented cells along the basal layer, while atypical reticular pattern showed an irregular organization of rete ridges with melanocytic hyperplasia, broadened and fused ridges and elongated nests. Both typical and atypical dots and globules were found associated with melanocytic nests in the dermis or at the dermoepidermal junction (DEJ), as well as with keratin cysts/pseudocysts. Grey globules corresponded to the presence of melanin-containing dermal inflammatory cells (melanophages) within the papillae. Structureless brown/black areas correlated with alterations of the DEJ. We observed the same DEJ alterations, but with the presence of dermal melanophages, in 36% of the cases of blue/white/grey structureless areas. A description of each LC-OCT/dermoscopy correlation was made. CONCLUSIONS: LC-OCT permitted for the first time to perform an in vivo, 3D correlation between dermoscopic criteria and pathological-like features of melanocytic lesions.

Line-field confocal optical coherence tomography of basosquamous carcinoma: a case series with histopathological correlation.

BACKGROUND: Basosquamous carcinoma (BSC) is a rare and potentially aggressive cutaneous neoplasm combining histopathological features of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Line-field confocal optical coherence tomography (LC-OCT) is a new, non-invasive imaging technique featuring excellent resolution and penetration. To date, studies about the use of LC-OCT in the BCC and SCC fields are available, but similar investigations are lacking in the BSC field. OBJECTIVE: The goal of the present study was to identify/describe LC-OCT criteria of BSC. METHODS: Consecutively enrolled BSCs were imaged with dermoscopy and LC-OCT prior to surgical excision. Dermoscopic and LC-OCT images were evaluated, and histopathological slides were reviewed. RESULTS: Six BSCs from six patients [four (66.7%) males and two (33.3%) females; mean age 76.5 (62-96) years] were included. Identified LC-OCT criteria for BSC included BCC-associated (dermal lobules with millefeuille pattern, dilated vessels, bright cells within the epidermis, bright cells within lobules, stromal stretching, stromal brightness) and SCC-associated features (acanthosis, hyperkeratosis, disarranged epidermal architecture, broad strands, elastosis and glomerular vessels). Interruption of the dermal-epidermal junction and ulceration represented overlapping criteria. CONCLUSION: Line-field confocal-OCT is a new promising technique that may support the non-invasive recognition of BSC through the simultaneous detection of BCC-associated and SCC-associated features. We hypothesize that the use of LC-OCT might be helpful not only in the diagnostic setting but also in the follow-up surveillance for an early identification of recurrences. Further larger studies are needed to prove this hypothesis.

Line-field confocal optical coherence tomography for actinic keratosis and squamous cell carcinoma: a descriptive study.

BACKGROUND: Early and accurate diagnosis of cutaneous squamous cell carcinomas (SCCs) and actinic keratoses (AK) is fundamental to reduce their associated morbidity and to select the correct treatment. Line-field confocal optical coherence tomography (LC-OCT) is a new imaging device that can characterize healthy skin and basal cell carcinoma, but no large studies on keratinocyte cell tumours have yet been published. AIM: To identify and describe LC-OCT criteria associated with SCC and AK, and to compare LC-OCT findings in these tumours. METHODS: A retrospective observational multicentre study was conducted. Lesions were imaged with the LC-OCT device before surgery and examined histologically. LC-OCT criteria for AK/SCC were identified and their presence was evaluated in all study lesions. Univariate and multivariate analyses were performed to compare AK and SCCs, and to investigate differences between in situ and invasive tumours. RESULTS: In total, 158 patients with 50 AK and 108 SCCs (62 in situ and 46 invasive) were included. Cytological and architectural alterations were found in most lesions, and differences were found between AK and SCCs. Although the visualization of the dermoepidermal junction (DEJ) was often hampered by hyperkeratosis and acanthosis, an outlined DEJ without broad strands was observed in almost all AK and almost all in situ SCCs, but in only three invasive SCCs (P < 0.001) when the DEJ was detectable. CONCLUSION: Our results suggest that LC-OCT can help clinicians in the identification of AK and SCC and their differentiation, providing a real-time and noninvasive examination. Further studies are needed to confirm our data.

Illness and drug modifiable factors associated with violent behavior in homeless people with severe mental illness: results from the French Housing First (FHF) program.

OBJECTIVE: The aim of this study was to investigate the factors associated with violent behavior in a large multicenter sample of Homeless Schizophrenia (SZ) and Bipolar Disorder (BD) (HSB) subjects. METHODS: This multicenter study was conducted in 4 French cities: Lille, Marseille, Paris and Toulouse. Violent behavior was defined by at least one episode of verbal or physical violence in the last 6 months. RESULTS: Overall, 675 HSB patients, mean aged 38 years and 82.5% men were included, 458 SZ (68.4%) and 212 BD (31.6%). During the 6 months before evaluation, 213 (34.3%) committed at least one physical or verbal violence. In multivariate analysis, violence has been associated with younger age (aOR = 0.96[0.94-0.99], p = .001), number of nights in the street (aOR = 1.01[1.01-1.01]), BD diagnosis (aOR = 1.63[1.01-2.65], p = .04), higher current illness severity (CGI score) (aOR = 1.32[1.07-1.64], p = .01), higher rates of current manic episode (aOR = 2.24[1.32-3.81], p = .002), current alcohol use disorder (aOR = 2.05 [1.33-3.15], p = .001), antisocial personality disorder (aOR = 2.51[1.55-4.07], p < .001) and with antidepressant consumption (aOR = 2.01[1.01-4.04], p = .04). No specific antipsychotic or mood stabilizer has been associated with decreased rates of violent behavior, however clozapine, lithium and carbamazepine remained poorly prescribed. CONCLUSION: In case of violent behavior in HSB subjects, clinicians should focus in priority on the treatment of mania, antidepressant iatrogenic effect and alcohol use disorder by pharmacological and non-pharmacological treatments. Clozapine, lithium and carbamazepine should be chosen as the treatments of reference in this population but may be hard to manage in some cases. The current clinical trial number is NCT01570712.

Cost effectiveness of two therapeutic regimens of infliximab in ankylosing spondylitis: economic evaluation within a randomised controlled trial.

OBJECTIVE: To determine the incremental cost-effectiveness ratios (ICERs) of two therapeutic regimens of infliximab for ankylosing spondylitis (AS). METHODS: 230 patients with active AS who were participating in a randomised controlled trial comparing two infliximab infusion modalities-every 6 weeks (Q6) and on demand (DEM)-were included in an economic evaluation within the trial. Data were collected by phone every 3 months for 1 year. Direct and indirect costs were calculated from a payer perspective. Health-related quality of life was assessed with a general health rating scale. ICERs were calculated for one 20% improvement (ASAS20), for one partial remission and for one quality-adjusted life year (QALY) gained. RESULTS: The Q6 regimen was significantly more efficacious than the DEM regimen but also more costly (euro22 388 vs euro17 596; p<0.001), because it required significantly more infliximab infusions per patient (8.4 vs 6.2). The ICERs of the Q6 to DEM regimen were euro15 841 for one ASAS20 response, euro23 296 for one partial remission and euro50 760 for one QALY gained. CONCLUSION: The administration of infliximab every 6 weeks is cost effective as compared with a DEM regimen; however, the ICER is close to the acceptability threshold of euro50 000 for one QALY gained. TRIAL REGISTRATION NUMBER: NCT 00439283.

Affinity chromatography for purification of the modular protein growth factor receptor-bound protein 2 and development of a screening test for growth factor receptor-bound protein 2 Src homology 3 domain inhibitor using peroxidase-linked ligand.

Growth factor receptor-bound protein 2 (Grb2) is an adapter protein involved in the Ras-dependent signaling pathway that plays an important role in human cancers initiated by oncogenic receptors. Grb2 is constituted by one Src homology 2 domain surrounded by two SH3 domains, and the inhibition of the interactions produced by these domains could provide an antitumor approach. In evaluating chemical libraries, to search for potential Grb2 inhibitors, it was necessary to elaborate a rapid test for their screening. We have developed, first, a batch method based on the use of an affinity column bearing a Grb2-SH3 peptide ligand to isolate highly purified Grb2. We subsequently describe a very rapid 96-well screening of inhibitors based on a simple competition between purified Grb2 and a peroxidase-coupled proline-rich peptide.

Design of peptoid analogue dimers and measure of their affinity for Grb2 SH3 domains.

This paper describes the design of the highest affinity ligands for Grb2 SH3 domains reported so far. These compounds were designed by combining N-alkyl amino acid incorporation in a proline-rich sequence with subsequent dimerization of the peptoid sequence based on structural data and molecular modeling. Optimization of the linker size is discussed, and the N-alkyl amino acid incorporation into both monomeric halves is reported. Because the affinity for Grb2 of the optimized compounds was too high to be measured using the fluorescent modifications that they induce on the Grb2 emission spectrum, a competition assay was developed. In this test, Grb2 is pulled down from a cellular extract by the initial VPPPVPPRRR peptide bound to Sepharose beads. In the presence of competitors, the test quantifies the amount of Grb2 displaced from the beads. It has enabled us to determine a K(i) value in the 10(-10) M range for the highest affinity Grb2 peptoid analogue dimer.

The cytoplasmic/transmembrane domain of dipeptidyl peptidase IV, a type II glycoprotein, contains an apical targeting signal that does not specifically interact with lipid rafts.

We investigated the signals involved in the apical targeting of dipeptidyl peptidase IV (DPP IV/CD26), an archetypal type II transmembrane glycoprotein. A secretory construct, corresponding to the DPP IV ectodomain, was first stably expressed in both the enterocytic-like cell line Caco-2 and the epithelial kidney MDCK cells. Most of the secretory form of the protein was delivered apically in MDCK cells, whereas secretion was 60% basolateral in Caco-2 cells, indicating that DPP IV ectodomain targeting is cell-type-dependent. A chimera (CTM-GFP) containing only the cytoplasmic and transmembrane domains of mouse DPP IV plus the green fluorescent protein was then studied. In both cell lines, this chimera was preferentially expressed at the apical membrane. By contrast, a secretory form of GFP was randomly secreted, indicating that GFP by itself does not contain cryptic targeting information. Comparison of the sequence of the transmembrane domain of DPP IV and several other apically targeted proteins does not show any consensus, suggesting that the apical targeting signal may be conformational. Neither the DPP IV nor the CTM-GFP chimera was enriched in lipid rafts. Together these results indicate that, besides the well-known raft-dependent apical targeting pathway, the fate of the CTM domain of DPP IV may reveal a new raft-independent apical pathway.

Characterization of a di-leucine-based signal in the cytoplasmic tail of the nucleotide-pyrophosphatase NPP1 that mediates basolateral targeting but not endocytosis.

Enzymes of the nucleotide pyrophosphatase/phosphodiesterase (NPPase) family are expressed at opposite surfaces in polarized epithelial cells. We investigated the targeting signal of NPP1, which is exclusively expressed at the basolateral surface. Full-length NPP1 and different constructs and mutants were transfected into the polarized MDCK cell line. Expression of the proteins was analyzed by confocal microscopy and surface biotinylation. The basolateral signal of NPP1 was identified as a di-leucine motif located in the cytoplasmic tail. Mutation of either or both leucines largely redirected NPP1 to the apical surface. Furthermore, addition of the conserved sequence AAASLLAP redirected the apical nucleotide pyrophosphatase/phosphodiesterase NPP3 to the basolateral surface. Full-length NPP1 was not significantly internalized. However, when the cytoplasmic tail was deleted upstream the di-leucine motif or when the six upstream flanking amino acids were deleted, the protein was mainly found intracellularly. Endocytosis experiments indicated that these mutants were endocytosed from the basolateral surface. These results identify the basolateral signal of NPP1 as a short sequence including a di-leucine motif that is dominant over apical determinants and point to the importance of surrounding amino acids in determining whether the signal will function as a basolateral signal only or as an endocytotic signal as well.

High-throughput fluorogenic assay for determination of botulinum type B neurotoxin protease activity.

Botulinum neurotoxins are responsible for botulism, a flaccid muscular paralysis caused by inhibition of acetylcholine release at the neuromuscular junction. This occurs by cleavage of conserved proteins involved in exocytosis such as synaptobrevin by the zinc metallopeptidase activity of the light chain of some botulinum neurotoxins. Botulism, for which there is presently no therapy available, is a relatively widespread disease that may result in death. Consequently, the development of drugs able to inhibit the hydrolytic activity of these neurotoxins is of great interest. Design and screening of such inhibitors could be largely facilitated by using high-throughput assays. With this aim, a novel in vitro test for quantifying the proteolytic activity of botulinum type B neurotoxin was developed. The substrate is the 60--94 fragment of human synaptobrevin-1 which was modified by introduction of the fluorescent amino acid l-pyrenylalanine in position 74 and a p-nitrophenylalanyl residue as quenching group in position 77. The cleavage of Syb 60-94 [Pya(74), Nop(77)] by the toxin active chain occurs selectively between residues 76 and 77 as in the case of the unmodified synaptobrevin and is directly quantified by measuring the strong fluorescence of the formed metabolite Syb 60-76 [Pya(74)]. This is the easiest, quickest, and cheapest assay described to date for measuring the proteolytic activity of botulinum type B neurotoxin. It can be easily automated for high-throughput screening. Moreover, amounts of about 3.5 pg/ml of botulinum type B neurotoxin could be detected by this method.

Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2.

Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein-protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96-induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to König's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT-Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT-Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.

Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation.

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.

Apical secretion and sialylation of soluble dipeptidyl peptidase IV are two related events.

The role of glycans in the apical targeting of proteins in epithelial cells remains a debated question. We have expressed the mouse soluble dipeptidyl peptidase IV (DPP IV ectodomain) in kidney (MDCK) and in intestinal (Caco-2) epithelial cell lines, as a model to study the role of glycosylation in apical targeting. The mouse DPP IV ectodomain was secreted mainly into the apical medium by MDCK cells. Exposure of MDCK cells to GalNac-alpha-O-benzyl, a drug previously described as an inhibitor of mucin O-glycosylation, produced a protein with a lower molecular weight. In addition this treatment resulted in a decreased apical secretion and an increased basolateral secretion of mouse DPP IV ectodomain. When expressed in Caco-2 cells, the mouse DPP IV ectodomain was secreted mainly into the basolateral medium. However, BGN was still able to decrease the amount of apically secreted protein and to increase its basolateral secretion. Neuraminidase digestion showed that the most striking effect of BGN was a blockade of DPP IV sialylation in both MDCK and Caco-2 cells. These results indicate that a specific glycosylation step, namely, sialylation, plays a key role in the control of the apical targeting of a secreted DPP IV both in MDCK and Caco-2 cells.