Gliptin-associated bullous pemphigoid shows peculiar features of anti-BP180 and -BP230 humoral response: Results of a multicenter study.

BACKGROUND: Recently, several case-control studies demonstrated an association between gliptins and bullous pemphigoid (BP) occurrence. However, data on the clinical and immunologic features of gliptin-associated bullous pemphigoid (GABP) are controversial. OBJECTIVE: This study aimed to clinically and immunologically characterize a large cohort of GABP patients to get an insight into the pathophysiology of this emerging drug-induced variant of BP. METHODS: Seventy-four GABP patients were prospectively enrolled and characterized from 9 different Italian dermatology units between 2013 and 2020. RESULTS: Our findings demonstrated the following in the GABP patients: (1) a noninflammatory phenotype, which is characterized by low amounts of circulating and skin-infiltrating eosinophils, is frequently found; (2) immunoglobulin (Ig)G, IgE, and IgA humoral responses to BP180 and BP230 antigens are reduced in frequency and titers compared with those in patients with idiopathic BP; (3) IgG reactivity targets multiple BP180 epitopes other than noncollagenous region 16A. LIMITATIONS: A limitation of the study is that the control group did not comprise only type 2 diabetes mellitus patients with BP. CONCLUSION: GABP patients show peculiar features of anti-BP180 and -BP230 humoral responses, laying the foundation for diagnostic improvements and getting novel insights into understanding the mechanism of BP onset.

Conserved roles of chromatin remodellers in cohesin loading onto chromatin.

Cohesin is a conserved, ring-shaped protein complex that topologically entraps DNA. This ability makes this member of the structural maintenance of chromosomes (SMC) complex family a central hub of chromosome dynamics regulation. Besides its essential role in sister chromatid cohesion, cohesin shapes the interphase chromatin domain architecture and plays important roles in transcriptional regulation and DNA repair. Cohesin is loaded onto chromosomes at centromeres, at the promoters of highly expressed genes, as well as at DNA replication forks and sites of DNA damage. However, the features that determine these binding sites are still incompletely understood. We recently described a role of the budding yeast RSC chromatin remodeler in cohesin loading onto chromosomes. RSC has a dual function, both as a physical chromatin receptor of the Scc2/Scc4 cohesin loader complex, as well as by providing a nucleosome-free template for cohesin loading. Here, we show that the role of RSC in sister chromatid cohesion is conserved in fission yeast. We discuss what is known about the broader conservation of the contribution of chromatin remodelers to cohesin loading onto chromatin.

Multiple Skin Squamous Cell Carcinomas in Junctional Epidermolysis Bullosa Due to Altered Laminin-332 Function.

Variably reduced expression of the basement membrane component laminin-332 (α3aß3γ2) causes junctional epidermolysis bullosa generalized intermediate (JEB-GI), a skin fragility disorder with an increased susceptibility to squamous cell carcinoma (SCC) development in adulthood. Laminin-332 is highly expressed in several types of epithelial tumors and is central to signaling pathways that promote SCC tumorigenesis. However, laminin-332 mutations and expression in individuals affected by JEB-GI and suffering from recurrent SCCs have been poorly characterized. We studied a JEB-GI patient who developed over a hundred primary cutaneous SCCs. Molecular analysis combined with gene expression studies in patient skin and primary keratinocytes revealed that the patient is a functional hemizygous for the p.Cys1171* mutant allele which is transcribed in a stable mRNA encoding for a ß3 chain shortened of the last two C-terminal amino acids (Cys1171-Lys1172). The lack of the Cys1171 residue involved in the C-terminal disulphide bond to γ2 chain did not prevent assembly, secretion, and proteolytic processing of the heterotrimeric molecule. Immunohistochemistry of SCC specimens revealed accumulation of mutant laminin-332 at the epithelial-stromal interface of invasive front. We conclude that the C-terminal disulphide bond is a structural element crucial for laminin-332 adhesion function in-vivo. By saving laminin-332 amount, processing, and signaling role the p.Cys1171* mutation may allow intrinsic pro-tumorigenic properties of the protein to be conveyed, thus contributing to invasiveness and recurrence of SCCs in this patient.

Topical Plant Polyphenols Prevent Type I Interferon Signaling in the Skin and Suppress Contact Hypersensitivity.

Human keratinocytes were recently shown to respond to anti-EGFR (epidermal growth factor receptor) drugs with activation of an interferon-κ-driven autocrine loop, leading to enhanced expression of innate antiviral effectors and of the pro-inflammatory chemokines CXCL10 (C-X-C motif chemokine 10) and CCL2 (C-C motif ligand 2). Here we showed active type I interferon signaling in the skin lesions of cancer patients undergoing treatment with the anti-EGFR drug cetuximab. Strong nuclear positivity for Interferon Regulatory Factor 1 and phosphorylated Signal Transducer and Activator of Transcription 1, enhanced interferon-κ expression and CXCL10 was associated to the epidermal compartment. Notably, 50 micromolar resveratrol and quercetin fully suppressed the low constitutive levels of type I interferon signaling and prevented its activation by the anti-EGFR cetuximab or gefitinib in cultured keratinocytes. In sensitized mice undergoing DNFB (2,4-dinitro-1-fluorobenzene)-induced contact hypersensitivity, local administration of gefitinib prior to elicitation further amplified hapten-induced type I interferon activation, tissue edema, and infiltration by T cells, whereas resveratrol or quercetin suppressed this inflammatory cascade. Overall, these data suggest that topical application of resveratrol or quercetin could be potentially effective in preventing pathological conditions due to overactivation of type I IFN (interferon)-driven circuits in the skin, including the inflammatory manifestations of anti-EGFR drug-induced skin-targeted toxicity.

Interleukin-17 and interleukin-22 promote tumor progression in human nonmelanoma skin cancer.

Interleukin-17 (IL-17) and IL-22 have been reported to play critical roles in autoimmunity and inflammation but information about their role in cancer is limited. In this study, we investigated the role of IL-17 and IL-22 in the progression of human skin basal-cell carcinoma (BCC) and squamous-cell carcinoma (SCC). We found that both tumor types are infiltrated with an high number of IL-17(+) and IL-22(+) T lymphocytes, as demonstrated by immunohistochemistry and by FACS analysis performed on peritumoral T-cell lines isolated from skin biopsies. In vitro studies demonstrated that proliferation and migration of the BCC- and SCC-cell lines M77015 and CAL27 were increased by IL-17 and IL-22. Moreover, IL-17, alone or in combination with TNF-α, was able to induce the production of two cytokines important for tumor progression, IL-6 and IL-8, in CAL27. We also showed that IL-17 upregulated NF-κB signaling, while IL-22 activated the STAT3 pathway and the antiapoptotic AKT protein in M77015 and CAL27. Finally, in vivo experiments demonstrated that IL-17 and IL-22 enhanced tumor growth in nude mice injected with CAL27. Altogether, our findings indicate that high levels of IL-22 and IL-17 in the BCC and SCC microenvironment promote tumor progression.

Ion channels expression and function are strongly modified in solid tumors and vascular malformations.

BACKGROUND: Several cellular functions relate to ion-channels activity. Physiologically relevant chains of events leading to angiogenesis, cell cycle and different forms of cell death, require transmembrane voltage control. We hypothesized that the unordered angiogenesis occurring in solid cancers and vascular malformations might associate, at least in part, to ion-transport alteration. METHODS: The expression level of several ion-channels was analyzed in human solid tumor biopsies. Expression of 90 genes coding for ion-channels related proteins was investigated within the Oncomine database, in 25 independent patients-datasets referring to five histologically-different solid tumors (namely, bladder cancer, glioblastoma, melanoma, breast invasive-ductal cancer, lung carcinoma), in a total of 3673 patients (674 control-samples and 2999 cancer-samples). Furthermore, the ion-channel activity was directly assessed by measuring in vivo the electrical sympathetic skin responses (SSR) on the skin of 14 patients affected by the flat port-wine stains vascular malformation, i.e., a non-tumor vascular malformation clinical model. RESULTS: Several ion-channels showed significantly increased expression in tumors (p < 0.0005); nine genes (namely, CACNA1D, FXYD3, FXYD5, HTR3A, KCNE3, KCNE4, KCNN4, CLIC1, TRPM3) showed such significant modification in at least half of datasets investigated for each cancer type. Moreover, in vivo analyses in flat port-wine stains patients showed a significantly reduced SSR in the affected skin as compared to the contralateral healthy skin (p < 0.05), in both latency and amplitude measurements. CONCLUSIONS: All together these data identify ion-channel genes showing significantly modified expression in different tumors and cancer-vessels, and indicate a relevant electrophysiological alteration in human vascular malformations. Such data suggest a possible role and a potential diagnostic application of the ion-electron transport in vascular disorders underlying tumor neo-angiogenesis and vascular malformations.

Comprehensive analysis of PTEN status in Sezary syndrome.

Sézary syndrome (SS) is an incurable leukemic variant of cutaneous T-cell lymphoma characterized by recurrent chromosomal alterations, among which, chromosome 10q deletion is very frequent. In this study, we investigated the PTEN status, on locus 10q23, in 44 SS patients; our findings show that PTEN is deleted in 36% of SS cases, whereas PTEN downregulation is observed in almost all of the samples evaluated by quantitative reverse-transcriptase polymerase chain reaction and Western blotting analysis. Neither DNA sequence mutation nor promoter hypermethylation were found at the PTEN locus, but we demonstrate that PTEN level can be also reduced by a group of miRs previously found upregulated and of prognostic relevance in SS; particularly, miR-21, miR-106b, and miR-486 were able to control PTEN abundance either in vitro or in vivo. Finally, because reduced PTEN activates the PI3/AKT-mediated pathway of cell growth and survival, we demonstrate that PTEN deficiency is associated with activated AKT in skin resident but not circulating SS cells, suggesting that the cutaneous milieu may strongly contribute to the SS cell growth. To our knowledge, this is the first study fully exploring the PTEN status in a large cohort of SS patients, unveiling potential elements of clinical utility in this malignancy.

Stimulation of 5-HT7 receptor during adolescence determines its persistent upregulation in adult rat forebrain areas.

Brain serotonin 7 (5-HT7) receptors play an important functional role in learning and memory, in regulation of mood and motivation, and for circadian rhythms. Recently, we have studied the modulatory effects of a developmental exposure (under subchronic regimen) in rats with LP-211, a brain-penetrant and selective 5-HT7 receptor agonist. We aimed at further deciphering long-term sequelae into adulthood. LP-211 (0.250 mg/kg i.p., once/day) was administered for 5 days during the adolescent phase (postnatal days 43-45 to 47-49). When adult (postnatal days >70), forebrain areas were obtained for ex vivo immunohistochemistry, whose results prompted us to reconsider the brain connectivity maps presented in our previous study (Canese et al., Psycho-Pharmacol 2015;232:75-89.) Significant elevation in levels of 5-HT7 receptors were evidenced due to adolescent LP-211 exposure, in dorsal striatum (which also shows an increase of dopaminergic D2 auto-receptors) and-unexpectedly-in piriform cortex, with no changes in ventral striatum. We observed that functional connectivity from a seed on the right hippocampus was more extended than reported, also including the piriform cortex. As a whole, the cortical loop rearranged by adolescent LP-211 exposure consisted in a hippocampus receiving connections from piriform cortex and dorsal striatum, the latter both directly and through functional control over the 'extended amygdala'. Such results represent a starting point to explore neurophysiology of 5-HT7 receptors. Further investigation is warranted to develop therapies for sleep disorders, for impaired emotional and motivational regulation, for attentive and executive deficit. The 5-HT7 agonist LP-211 (0.250 mg/kg i.p., once/day) was administered for 5 days during adolescence (postnatal days 43-45 to 47-49) in rats. When adult (postnatal days >70), a significant elevation in levels of 5-HT7 receptors were evidenced in dorsal striatum and-unexpectedly-in piriform cortex.

Alcohol dependence and serotonin transporter functional polymorphisms 5-HTTLPR and rs25531 in an Italian population.

AIMS: The role of the serotonin transporter gene (SLC6A4) in alcohol dependence (AD) is still unclear. In this paper, we have evaluated the association of the SLC6A4 gene polymorphisms 5-HTTLPR and rs25531 in AD and assessed the polymorphic patterns both in alcoholics and in healthy people of an Italian population. METHODS: Genotyping of the 5-HTTLPR (L/S) and rs25531 (A/G) polymorphisms of the SLC6A4 gene was performed on 403 alcoholics outpatients and 427 blood donors. RESULTS: Comparing AD and control populations and taking into account statistical correction for multiple testing, we found no statistically significant differences for 5-HTTLPR (L/S) and rs25531 polymorphisms in terms of either genotypes or alleles frequencies. By univariate ANOVA, a statistically significant difference was found in the onset of AD: the mean age of onset resulted to be of 25.4 years in males in respect to 28.1 in females. In particular in males, the early AD onset was different, in a statistically significant manner, depending on the presence of at least one S or Lg allele (24.6 years) in respect to La homozygotes (27.5 years) (P = 0.03). CONCLUSIONS: These findings suggest that genetic factors contribute, together with gender and age, to the onset differences in alcohol-dependent phenotypes.

Modulatory effects following subchronic stimulation of brain 5-HT7-R system in mice and rats.

The serotonin receptor 7 (5-HT7-R) plays important functional roles in learning and memory, in regulation of mood and circadian rhythmicity. LP-211 is a new selective agonist, belonging to 1-arylpiperazine category. We report studies aimed to evaluate the modulatory effect of a subchronic regimen on behavioral/molecular parameters. At low dose [0.25 mg/kg intraperitoneally (i.p.)], LP-211 induced a 6-h anticipated wake up in adult mice (with no temporal landmark by constant light), acting as nonphotic stimulus for 'internal clock' resetting. In standard 12:12-h light/dark cycle, a subchronic effect (5-6 days at 0.25 mg/kg, once per day) was observed: delayed wake up, reduced peak of locomotor activity and no evidence for brain cellular proliferation after ex vivo analysis. Other studies in rats were aimed to investigate long-term effects of developmental LP-211 administration into adulthood. Subchronic LP-211 (0.125 mg/kg i.p. once per day during the prepuberal phase) reduced l-glutamate, N-methyl-d-aspartate receptor 1 and dopamine transporter within the ventral striatum. With LP-211 (0.25 mg/kg i.p. once per day during the postpuberal phase), clear reductions were observed in the immunoreactivity of serotonin transporter and dopaminergic D2 receptors in the ventral and dorsal striatum, respectively. Subchronic LP-211 in rats and mice appears to be a suitable tool for studying the role of 5-HT7-R in sleep disorders, emotional/motivational regulations, attentive processes and executive functions.

Clinical subtypes in Parkinson's disease: the impact of MAPT haplotypes.

The H1 haplotype of the MAPT gene influences the risk of PD and has been related to the development of PDD. We evaluated the influence of MAPT haplotypes on the expression of motor features in PD patients. We genotyped, for the MAPT haplotypes H1 and H2, a sample of 181 PD patients with distinct clinical subtypes: tremor dominant and non-tremor dominant (NTD). Our results indicate that the MAPT haplotypes contribute to the expression of motor features of PD. H1 homozygous PD patients are significantly more likely to present a NTD phenotype, a clinical subtype characterized by widespread pathological degeneration, than H2 carriers.

Early inflammatory biomarkers and melanoma survival.

BACKGROUND: Few studies have investigated the role of inflammatory markers in predicting cutaneous melanoma survival. The aim of the study was to identify, if any, early inflammatory markers in the prognosis of all stages of primary cutaneous melanoma. METHODS: We conducted a 10-year cohort study among 2,141 melanoma patients from the same geographic area (Lazio) with primary cutaneous melanoma diagnosed between January 2005 and December 2013. In situ cutaneous melanoma was excluded from the analysis (N = 288), leaving 1,853 cases of invasive cutaneous melanoma. The following hematological markers were obtained from clinical records: white blood cells count (WBC), count and percentages of neutrophils, basophils, monocytes, lymphocytes, and large unstained cells (LUC). Survival probability was estimated by Kaplan-Meier methods, and prognostic factors were evaluated by multivariate analysis (Cox proportional hazards model). RESULTS: In the multivariate analysis, high levels of NLR (>2.1 vs. ≤2.1, HR: 1.61; 95% CI: 1.14-2.29, P = 0.007) and high levels of d-NLR (>1.5 vs. ≤1.5, HR: 1.65; 95% CI: 1.16-2.35, P = 0.005) were independently associated with an increased risk of 10-year melanoma mortality. However, when we stratified by Breslow thickness and clinical stage, we observed that NLR and d-NLR were good markers of prognosis only for patients with Breslow thickness of 2.0 mm and more (NLR, HR: 1.62; 95% CI: 1.04-2.50; d-NLR, HR: 1.69; 95% CI: 1.09-2.62) or clinical stage II-IV (NLR, HR: 1.55; 95% CI: 1.01-2.37; d-NLR, HR: 1.72; 95% CI: 1.11-2.66), independent of other prognostic factors. CONCLUSION: We suggest that a combination of NLR and Breslow thickness may be a useful, cheap, and readily available prognostic marker for cutaneous melanoma survival.

Effect of stress on hippocampal nociceptin expression in the rat.

Nociceptin/orphanin FQ (N/OFQ) peptide and its receptor are not only ubiquitously expressed in mammalian brain and spinal cord but are also abundant in limbic structures, particularly in the hippocampus. The widespread distribution of N/OFQ reflects the broad spectrum of its biological actions such as nociception, food intake, spontaneous locomotor activity, and learning and memory processes. Since the hippocampus is involved in the control of adrenocortical activity, its role in stress-related phenomena is well characterized. In male Wistar rats, we first examined the effects of acute restraint stress (120 min) on the brain immunohistochemical localization of N/OFQ. The analysis carried out on sections obtained at the onset of stress revealed enhanced expression of N/OFQ in CA1, CA3, and the dentate gyrus as well as increased plasma corticosterone concentrations. Next, we examined whether endogenous glucocorticoid hormone plays a role in the modulation of hippocampal N/OFQ expression in response to stress. To this end, rats were injected with corticosterone (1 mg/kg) or subjected to restraint stress 1 week after adrenalectomy. Two hours after corticosterone administration, plasma glucocorticoid concentrations were comparable to those observed after restraint stress, while N/OFQ expression had significantly increased in all the hippocampal subfields examined. By contrast, in adrenalectomized rats, stress did not modify protein expression. These results confirm that stress can affect N/OFQ expression and that glucocorticoids may constitute hormonal mediators of this complex interplay.

Expression of vascular endothelial growth factor-C in primary cutaneous melanoma predicts sentinel lymph node positivity.

BACKGROUND: Vascular endothelial growth factor-C (VEGF-C), a lymphatic vessel growth factor, has been involved in the formation of lymph nodal metastases in different tumor types. Early evidences indicate that VEGF-C expression in human primary melanoma could be predictive of lymph nodal metastases, whereas the role of lymphangiogenesis is still controversial. METHODS: By immunohistochemical analysis, we investigated VEGF-C or CC chemokine receptor 7 expression, together with the lymphatic and blood vessel network, in 36 patients with primary skin melanomas and metastases at the sentinel lymph node biopsy (SLN-positive), and 26 melanoma patients with negative SLN biopsy (SLN-negative). RESULTS: We found that VEGF-C expression in primary melanoma specimens was significantly associated with SLN-positive (p < 0.001), particularly in thin melanomas. An association between augmented peritumoral lymphatic vessel area and SLN-positive (p < 0.02) was also seen. Conversely, no association between either expression of the CC chemokine receptor 7 in the primary tumor, or intratumoral lymphatic vessel or peritumoral and intratumoral blood vessel area, and SLN-positive was found. CONCLUSIONS: Our results, taking into account the expression of either VEGF-C or related histopathological markers, indicated the possibility to use VEGF-C immunohistochemistry as a marker of metastatic progression, especially in thin cutaneous melanomas.

Monomorphic region of the serotonin transporter promoter gene in New World monkeys.

Genetic variation in the human serotonin system has long been studied because of its functional consequences and links to various neuropsychiatric and behavior-related disorders. Among non-human primates, the common marmosets (Callithrix jacchus) and tufted capuchins monkeys (Cebus apella) are becoming increasingly used as models to study the effects of genes, environments, and their interaction on physiology and complex behavior. In order to investigate the independent functions of and potential interactions between serotonin-related genes, anxiety and neuropsychiatric disorders, we analyzed the presence and variability of the serotonin transporter gene-linked polymorphic region (5-HTTLPR) in marmoset and capuchin monkeys. By PCR and using heterologous primers from the human sequence, we amplified and then sequenced the corresponding 5-HTT region in marmosets and capuchins. The resulting data revealed the presence of a tandem repeat sequence similar to that described in humans, but unlike humans and other Old World primates, no variable length alleles were detected in these New World monkeys, suggesting that if serotonin transporter is involved in modulating behavior in these animals it does so through different molecular mechanisms.

Cyclin B3 implements timely vertebrate oocyte arrest for fertilization.

To ensure successful offspring ploidy, vertebrate oocytes must halt the cell cycle in meiosis II until sperm entry. Emi2 is essential to keep oocytes arrested until fertilization. However, how this arrest is implemented exclusively in meiosis II and not prematurely in meiosis I has until now remained enigmatic. Using mouse and frog oocytes, we show here that cyclin B3, an understudied B-type cyclin, is essential to keep Emi2 levels low in meiosis I. Direct phosphorylation of Emi2 at an evolutionarily highly conserved site by Cdk1/cyclin B3 targets Emi2 for degradation. In contrast, Cdk1/cyclin B1 is inefficient in Emi2 phosphorylation, and this provides a molecular explanation for the requirement of different B-type cyclins for oocyte maturation. Cyclin B3 degradation at exit from meiosis I enables Emi2 accumulation and thus timely arrest in meiosis II. Our findings illuminate the evolutionarily conserved mechanisms that control oocyte arrest for fertilization at the correct cell-cycle stage, which is essential for embryo viability.

Timely diagnosis of cutaneous squamous cell carcinoma: the GP's role.

BACKGROUND: Cutaneous squamous cell carcinoma (SCC) is the second most frequent skin cancer among Caucasians. Advanced cases determine significant tissue destruction and they can metastasize. OBJECTIVES: To evaluate the role of GPs in timely diagnosis of SCC, examining the probability of delay before the diagnosis and treatment of SCC among patients having first seen a GP compared to patients having accessed directly a dermatologist. METHODS: We included a stratified sample of 308 SCC patients treated at a referral center in Italy. Medical records were reviewed and combined with patient interviews. RESULTS: Multivariable analysis has shown that patients who have first seen a GP have a significantly lower likelihood of long patient delay [odds ratio (OR) = 0.45; 95% confidence interval (95% CI) 0.21-0.94; P = 0.04) compared to patients having accessed directly a dermatologist. Treatment delay was not associated with the specialization of the first doctor seen for the lesion (OR = 0.52; 95% CI 0.15-1.84; P = 0.31). CONCLUSION: Our findings highlight the potential role of the GP in facilitating rapid access to appropriate health care.

Reduced Interleukin-17-Expressing Cells in Cutaneous Melanoma.

Characterization of tumor associated lymphocytes (TILs) in tumor lesions is important to obtain a clear definition of their prognostic value and address novel therapeutic opportunities. In this work, we examined the presence of T helper (Th)17 lymphocytes in cutaneous melanoma. We performed an immunohistochemical analysis of a small cohort of primary melanomas, retrospectively selected. Thereafter, we isolated TILs from seven freshly surgically removed melanomas and from three basal cell carcinomas (BCC), as a comparison with a non-melanoma skin cancer known to retain a high amount of Th17 cells. In both studies, we found that, differently from BCC, melanoma samples showed a lower percentage of Th17 lymphocytes. Additionally, TIL clones could not be induced to differentiate towards the Th17 phenotype in vitro. The presence or absence of Th17 cells did not correlate with any patient characteristics. We only observed a lower amount of Th17 cells in samples from woman donors. We found a tendency towards an association between expression by melanoma cells of placenta growth factor, angiogenic factors able to induce Th17 differentiation, and presence of Th17 lymphocytes. Taken together, our data indicate the necessity of a deeper analysis of Th17 lymphocytes in cutaneous melanoma before correlating them with prognosis or proposing Th17-cell based therapeutic approaches.