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Psoriasis cellular hallmarks, such as the imbalance between Th1/Th17 and Th2 cytokines and the dysregulated expression of vascular endothelial growth factor (VEGF), inducible nitric oxide synthase, (iNOS) and indoleamine 2,3-dioxygenase (IDO), are all detectable in mesenchymal stem cells (MSCs) suggesting that psoriasis originates at mesenchymal level. Aim of the study: In this scenario, MSCs may become the new therapeutic target and interest in the effects of traditionally used drugs, such as Apremilast, on MSCs has greatly increased. MSCs from control subjects (C-MSCs) and from psoriatic patients before (PsO MSCs T0) and after in vivo treatment with Apremilast (PsO-MSCs T12) were isolated and characterized; subsequently, the effects of Apremilast on VEGF, iNOS and IDO expression were evaluated by immunocytochemistry (ICC). The expression of VEGF, iNOS and IDO was also detected in skin sections by immunohistochemistry (IHC). The results indicate that in vivo administration of Apremilast is able to drive the altered profile of PsO-MSCs towards a more physiological pattern. In skin sections, the role of Apremilast is evident in reducing VEGF, iNOS and IDO expression. Apremilast treatment influences the expression of VEGF, iNOS and IDO not only by keratinocytes but also by MSCs, restoring their intrinsic profile and their natural anti-inflammatory action, and decreasing the auto-inflammatory process that underpins the development of psoriasis.

The cancer stem cell theory states that a subset of tumor cells, termed cancer stem cells (CSCs), has the ability to self-renew and differentiate within the tumors. According to this theory, CSCs would be mainly responsible for tumor initiation, progression, resistance to therapy, recurrence, and metastasis. In this study, a culture system was setup to enrich CSCs from bladder cancer (T24), lung cancer (A549), colorectal cancer (CaCo-2), and osteosarcoma (MG63) cell lines, through sphere formation. Magnetic-activated cell sorting was also used to further increase CSC enrichment. Subsequently, molecular characterization of CSC-enriched cell populations and parental cells was carried out, by exploring the expression levels of stem markers and the enzyme nicotinamide N-methyltransferase (NNMT). Results obtained showed a significant upregulation of stem cell markers in CSC-enriched populations, obtained upon sphere formation, compared with parental counterparts. Moreover, NNMT expression levels were markedly increased in samples enriched with CSCs with respect to control cells. Considering the fundamental role played by CSCs in carcinogenesis, reported data strengthen the hypothesis that sustains a pivotal role of NNMT in cancer growth and metastasis. In addition, these findings could represent an important achievement for the development of new and effective anticancer therapies, based on CSC-associated targets.

Background: LL-37 is the only human antimicrobial peptide that belongs to the cathelicidins. The aim of the study was to evaluate the efficacy of LL-37 in the management of MRSA-infected surgical wounds in mice. Methods: A wound on the back of adult male BALB/c mice was made and inoculated with Staphylococcus aureus. Two control groups were formed (uninfected and not treated, C0, infected and not treated, C1) and six contaminated groups were treated, respectively, with: teicoplanin, LL-37, given topically and /or systemically. Histological examination of VEGF expression and micro-vessel density, and bacterial cultures of wound tissues, were performed. Results: Histological examination of wounds in the group treated with topical and intraperitoneal LL-37 showed increased re-epithelialization, formation of the granulation tissue, collagen organization, and angiogenesis. Conclusions: Based on the mode of action, LL-37 has a potential future role in the management of infected wounds.

An adequate placental vascularization allows the proper development of the fetus and it is crucial for the gestational success. A number of factors regulate angiogenesis, including vascular endothelial growth factor (VEGF), which induces the synthesis of nitric oxide (NO), a potent vasodilator produced by three different nitric oxide synthase (NOS) isoforms. NO is essential to maintain a low vascular resistance in the fetoplacental circulation, although at high concentrations, it may combine with excess superoxide to produce peroxynitrite, which reacts with proteins giving rise to nitrotyrosine. Since obesity, whose incidence is increasing worldwide, is characterized by a low-grade inflammatory state and increased levels of oxidative and nitrative stress, both affecting placental function, our aim was to evaluate the expression of VEGF, eNOS, and iNOS in full-term placentas obtained from normal weight and pre-pregnancy obese women by means of immunohistochemistry and real-time PCR. Moreover, we assessed the NO levels and the nitrotyrosine immunoexpression in the same sample groups. Our results show a significantly higher immunohistochemical expression of VEGF and eNOS in the endothelium of placentas from obese women than in controls, whereas the immunoexpression of iNOS was comparable in the two groups. These data agree with those of the gene expression analysis, thus suggesting the possible existence of a compensatory mechanism for changes in placental blood flow associated with obesity. As concerns nitrotyrosine and NO levels, we observed a significant increase in placental tissue from obese women which may contribute to the development of metabolic and cardiovascular diseases both in the mother and the offspring.

Background CEACAM1, a valuable biomarker for several cancers, have remained unexplored up to the present in laryngeal squamous cell carcinoma (LSCC). We aimed to examine CEACAM1 expression and evaluate its combinational clinical significance for the diagnosis or prognosis and treatment decision making in LSCC. Methods CEACAM1 expression was assessed by immunohistochemistry in 54 LSCCs and evaluate its correlation with clinical and histopathological features. Results CEACAM subtype 1 (CEACAM1) expression was positive in 50% of the cases. No significant difference was observed in relation to age, gender, tumor size, and tumor stage. CEACAM1 expression correlated with tumor grade, development of local recurrence, node and distant metastasis. Kaplan-Meier survival curves showed that CEACAM1 staining was inversely correlated with both overall and disease-specific 5-year survival. Conclusions Our study is the first to demonstrate that CEACAM1 expression is associated with an adverse prognosis in LSCC. CEACAM1 is a valuable biomarker and a promising therapeutic target in LSCC.

Aging, chronic oxidative stress, and inflammation are major pathogenic factors in the development and progression of age-related macular degeneration (AMD) with the loss of retinal pigment epithelium (RPE). The human RPE contains a subpopulation of progenitors (i.e., RPE stem cells—RPESCs) whose role in the RPE homeostasis is under investigation. We evaluated the paracrine effects of mature RPE cells exposed to oxidative stress (H2O2) on RPESCs behavior through co-cultural, morphofunctional, and bioinformatic approaches. RPESCs showed a decline in proliferation, an increase of the senescence-associated β-galactosidase activity, the acquisition of a senescent-like secretory phenotype (SASP), and the reduction of their stemness and differentiation competencies. IL-6 and Superoxide Dismutase 2 (SOD2) seem to be key molecules in RPESCs response to oxidative stress. Our results get insight into stress-induced senescent-associated molecular mechanisms implicated in AMD pathogenesis. The presence of chronic oxidative stress in the microenvironment reduces the RPESCs abilities, inducing and/or maintaining a pro-inflammatory retinal milieu that in turn could affect AMD onset and progression.

Background Cutaneous neoplasms include melanoma and non-melanoma skin cancers (NMSCs). Among NMSCs, basal cell carcinoma (BCC) represents the most common lesion. On the contrary, although accounting for less than 5% of all skin cancers, melanoma is responsible for most of cutaneous malignancy-related deaths. Paraoxonase-2 (PON2) is an intracellular enzyme exerting a protective role against production of reactive oxygen species within mitochondrial respiratory chain. Recently, a growing attention has been focused on exploring the role of PON2 in cancer. The aim of this study was to investigate the diagnostic and prognostic role of PON2 in skin neoplasms. Materials and methods 36 cases of BCC, distinguished between nodular and infiltrative lesions, as well as 29 melanoma samples were analysed by immunohistochemistry to evaluate PON2 protein expression. Subsequent statistical analyses were carried out to explore the existence of correlations between intratumour enzyme levels and clinicopathological features. Results Results obtained showed PON2 overexpression in BCCs compared with controls. In particular, distinguishing between less and more aggressive tumour forms, we found no significant differences in enzyme levels between nodular BCCs and controls. Conversely, PON2 expression was significantly higher in infiltrative BCCs compared with controls. Moreover, the enzyme was strongly upregulated in melanoma samples with respect to controls. Interestingly, PON2 levels were positively correlated with Breslow thickness, Clark level, regression, mitoses, lymph node metastases, primary tumour (pT) parameter and pathological stage. Conclusions Reported findings seem to suggest that PON2 expression levels could be positively related with tumour aggressiveness of both BCC and melanoma.

Breslow thickness and Clark level are still important factors for cutaneous melanoma, but do not provide a precise prognosis in all cases. It is necessary to find new factors capable of a more accurate prediction of the tumor course. Angiogenesis is essential for tumor development and progression and is regulated by vascular endothelial growth factor A (VEGF-A) and semaphorins (SEMA), in particular, SEMA3A inhibits angiogenesis by affecting VEGF signaling. However, the prognostic role of angiogenetic factors remains unclear. To date, no information is available on SEMA3A in human melanoma. Microvessel density, immunohistochemical and mRNA VEGF and SEMA3A expression level in 60 thin (Breslow thickness ≤ 1.0 mm), 60 intermediate (1.1-4.0 mm) and 50 thick (>4.0 mm) primary human cutaneous melanomas were investigated and related to clinical/pathological parameters and disease-specific survival. No positive association between Breslow thickness, Clark level, metastasis presence and survival was identified; Clark level was poorly related to survival. VEGF and microvessel density were significantly higher in intermediate and thick melanomas and related to Breslow thickness and Clark level but not to metastasis status and survival. On the contrary, SEMA3A was significantly reduced in intermediate and thick melanomas and associated to metastasis and poor survival. VEGF/SEMA3A ratio was higher in the worst prognosis, resulting the most closely related factor with metastasis and survival. SEMA3A expression and VEGF/SEMA3A ratio turned out to be valuable prognostic biomarkers in patients affected by cutaneous melanoma, in particular with Breslow thickness >1 mm. SEMA3A might serve as a candidate tumor suppressor in cutaneous melanoma therapy.

Dalbavancin is an effective antibiotic that is widely used to treat skin infection. Our aim was to determine the effect of dalbavancin administration on wound healing compared to that of vancomycin and to elucidate if epidermal growth factor receptor (EGFR), matrix metalloproteinase 1 (MMP-1), MMP-9, and vascular endothelial growth factor (VEGF) could be involved in its therapeutic mechanism. A mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection was established. Mice were treated daily with vancomycin (10 mg/kg) and weekly with dalbavancin at day 1 (20 mg/kg) and day 8 (10 mg/kg). After 14 days, wounds were excised, and bacterial counts were performed. Wound healing was assessed by histological and immunohistochemical staining, followed by protein extraction and immunoblotting. Our microbiological results confirmed that both dalbavancin and vancomycin are effective in reducing the bacterial load in wounds. The dalbavancin group showed a strong effect compared with infected untreated animals and the vancomycin-treated group. The wounds treated with dalbavancin showed robust epidermal coverage with reconstitution of the regular and keratinized epidermal lining and well-organized granulation tissue with numerous blood vessels, although slightly less than that in the uninfected group. While in the vancomycin-treated group the epithelium appeared, in general, still hypertrophic, the granulation tissue appeared even less organized. We observed elevated EGFR and VEGF expression in both treated groups, although it was higher in dalbavancin-treated mice. MMP-1 and MMP-9 were decreased in uninfected tissue and in both treated tissues compared with untreated infected wounds. This study showed faster healing with dalbavancin treatment that might be associated with higher EGFR and VEGF levels.

This corrects the article "Anastomotic healing in a rat model of peritonitis after non-steroidal anti-inflammatory drug administration " in volume 64(1):3085 In the published article “Anastomotic healing in a rat model of peritonitis after non-steroidal anti-inflammatory drug administration” Eur J Histochem 2020;64(1):3085, https://doi.org/10.4081/ejh.2020.3085,” one affiliation was published incorrectly. The authors apologize for any inconvenience that it may have caused. Roberto Ghiselli,1 Guendalina Lucarini,2 Monica Ortenzi,1 Eleonora Salvolini,3 Stefania Saccomanno,2 Fiorenza Orlando,4 Mauro Provinciali,4 Fabio Casciani,1 Mario Guerrieri1 1Clinic of Surgery, Marche Polytechnic University, Ancona2Department of Clinical and Molecular Sciences, Marche Polytechnic University, Ancona3Department of Odontostomatologic and Specialized Clinical Sciences, Marche Polytechnic University, Ancona4Experimental Animal Models for Aging Units, Research Department, Italian National Institute on Aging (INRCA) IRCCS, Ancona, Italy The affiliation should be corrected as follows: 4Experimental Animal Models for Aging Units, Scientific Technological Area, IRCCS INRCA, Ancona, Italy

The tissue inflammatory response can influence the outcome of anastomotic healing. Anastomotic leakage represents a dreadful complication after gastrointestinal surgery, in particular sepsis and intra-abdominal infections impair the restorative process of colic anastomoses. It has been debated whether the administration of non-steroidal anti-inflammatory drugs (NSAIDs) is a risk factor for dehiscence, since many patients receive NSAIDs in the early postoperative period. Our aim was, for the first time, to analyze the morpho-functional effects of postoperative administration of two commonly used NSAIDs, Diclofenac and Ketorolac, on the healing process of colo-colic anastomoses constructed under condition of fecal peritonitis in a rat model. Sixty adult male rats underwent two surgical procedures: peritonitis induction and colo-colic anastomosis, and were divided into three groups: 20 rats received saline; 20 rats 4 mg/kg Diclofenac and 20 rats 5 mg/kg Ketorolac. We assessed anastomosis strength, morphological features of tissue wound healing, immunohistochemical metalloproteinase 9 (MMP9) expression and collagen deposition and content by Sirius red staining and hydroxyproline level. We found no significant difference in bursting pressure, collagen content and organization and morphological features between the groups, except a significantly reduced presence of inflammatory cells and MMP9 expression in the groups treated with NSAIDs. Our findings showed that Diclofenac and Ketorolac administration did not affect post-surgical healing and did not increase the leakage risk of colo-colic anastomoses during peritonitis.

Histological studies born in Ancona as morphological ultrastructural investigations. The ability to correlate structure and function concomitantly with the development of bio-molecular technologies was an important step forward for the scientific growth of the histological group. Starting from studies on tissue regeneration, which focused on cells, engineered material biocompatibility and appropriate biochemical factors interrelationship, researches were directed both to the study of wound healing and to deepen our knowledge on adult mesenchymal stem cells behaviour in normal and in pathological conditions. Knowledges of the group were important starting point for fruitful collaborations with clinicians.

OBJECTIVE Sacral neuromodulation (SNM) is proposed to treat different anorectal dysfunctions but its mechanism of action is not yet known. Our previous study demonstrated how SNM can significantly increase neuronal nitric oxide synthase NOS (n-NOS) and inducible NOS (i-NOS) expression in the anus and rectum of rats. There are no reports regarding the relation between SNM and NOS in colonic cells: our aim was to assess NOS expression in colonic rat model after SNM. MATERIALS AND METHODS Twenty-six female Sprangue-Dawley rats were considered: group I, normal control rats; group II, sham treatment rats, in whom electrodes for electrical stimulation were placed in S1 foramen bilaterally and left in place, without performing neuromodulation; group III, rats in whom SNM was performed. After 14 days, the rats were sacrificed and we evaluated n-NOS and i-NOS in colonic specimens by immunohistochemistry and Western Blot analysis. RESULTS Western Blot analysis showed that levels of n-NOS and i-NOS were higher in colon of the III group rats respect to the others; in particular, immunohistochemistry revealed that, after neuromodulation, n-NOS expression in the muscle cells and i-NOS expression in glandular epithelium and nervous cells were highly represented (p

Malignant mesothelioma (MM) is a very aggressive, lethal cancer, and its incidence is increasing worldwide. Development of multi-drug resistance, therapy related side-effects, and disease recurrence after therapy are the major problems for the successful treatment of MM. Emerging evidence indicates that dietary phytochemicals can exert anti-cancer activities by regulating microRNA expression. Until now, only one dietary phytochemical (ursolic acid) has been reported to have MM microRNA regulatory ability. A large number of dietary phytochemicals still remain to be tested. In this paper, we have introduced some dietary phytochemicals (curcumin, epigallocatechin gallate, quercetin, genistein, pterostilbene, resveratrol, capsaicin, ellagic acid, benzyl isothiocyanate, phenethyl isothiocyanate, sulforaphane, indole-3-carbinol, 3,3'-diindolylmethane, diallyl disulphide, betulinic acid, and oleanolic acid) which have shown microRNA regulatory activities in various cancers and could regulate MM microRNAs. In addition to microRNA regulatory activities, curcumin, epigallocatechin gallate, quercetin, genistein, resveratrol, phenethyl isothiocyanate, and sulforaphane have anti-mesothelioma potentials, and pterostilbene, capsaicin, ellagic acid, benzyl isothiocyanate, indole-3-carbinol, 3,3'-diindolylmethane, diallyl disulphide, betulinic acid, and oleanolic acid have potentials to inhibit cancer by regulating the expression of various genes which are also known to be aberrant in MM.

Squamous cell carcinoma of the oral cavity represents the sixth most common cancer worldwide and it is often preceded by pre-neoplastic lesions. Sometimes it is still difficult for pathologists to make objective differential diagnoses only on histological characteristics. Tumorigenesis is accompanied by altered expression of cell adhesion molecules, like carcinoembryonic antigen cell adhesion molecule (CEACAM)1. We wanted to investigative CEACAM1 in oral dysplastic lesions, carcinoma in situ (CIS) and oral squamous cell carcinoma (OSCC). We examined immunohistochemical CEACAM1 expression in 50 OSCC, 30 oral CIS and 40 pre-neoplastic lesions and assessed its correlation with clinical and pathological parameters. CEACAM1 was not expressed in normal mucosa, significantly expressed in CIS while it was negative in all the dysplastic lesions. In OSCC, high CEACAM1 expression was associated with tumor grade and inversely correlated with both overall and disease-specific 5-year survival. We showed that CEACAM1 expression is very dynamic: absent in dysplastic lesions, up-regulated in CIS and OSCC. We suggest that CEACAM1 could be a prognostic marker of OSCC and oral CIS. Our most important finding was that it could help pathologists diagnosing oral carcinoma in situ.

Breast cancer is one of the most common forms of cancers in females. Traditional therapies are associated with adverse side effects, and drug resistance, recurrence, and lack of treatment in metastasis are the major problems. Accumulating evidence indicates that dietary phytochemicals may exert therapeutic effects by regulating miRNA expression. A number of dietary phytochemicals have been tested as miRNA regulatory agents against breast cancer, and some other numbers of dietary phytochemicals have not yet been tested. Our aim is to introduce some of the potential dietary phytochemicals (benzyl isothiocyanate, capsaicin, epigallocatechin gallate, oleanolic acid, phenethyl isothiocyanate, and ursolic acid) that have shown miRNA regulatory activities, and have not yet been tested against breast cancer miRNAs. We also discuss the effects of curcumin, diallyl disulphide, 3,3′-diindolylmethane, ellagic acid, genistein, indole-3-carbinol, quercetin, resveratrol, and sulforaphane on regulation of expression of breast cancer miRNAs in various in vitro and in vivo models.

To date, there are evidence-based guidelines available for cervical dysplasia diagnosed in pregnancy. Certain functional biomarkers have proven useful in the prediction of regressing and non-regressing cervical intraepithelial neoplasia (CIN) lesions in non-pregnant women. In the present study, Ki-67 and p16 immunostaining were evaluated in different grades of CIN lesions diagnosed in pregnant or non-pregnant women with the aim to identify any differences in order to better understand the behavior of CIN in pregnancy. The current retrospective case-control study included 17 pregnant patients that conceived naturally with first-time onset of CIN occurring at no later than 16 gestational weeks. The control group included 17 non-pregnant patients matched for age, parity and number of previous sexual partners. Exclusion criteria included previous cervical treatment, immunocompromised status, chronic hepatitis B and/or C and cigarette smoking. p16 and Ki-67 protein expression were respectively detected using the CINtec Histology kit and monoclonal antibodies against Ki-67. p16 and Ki-67 staining were analyzed using a classification system based on the distribution of positivity on a semi-quantitative three point-scale. p16 and Ki-67 immune reactivity correlated positively with the grade of epithelial dysplasia in the total cohort of pregnant and non-pregnant patients; expression increased linearly from CIN1 to CIN3. Furthermore, the association between p16 immunostaining and CIN grade was significant in non-pregnant patients but not in pregnant patients. In pregnant patients, positivity for Ki-67 was less intense than in non-pregnant patients. These results appear to suggest that pregnancy status interferes with the expression of cellular proteins involved in cell-cycle regulation and the carcinogenic process induced by high-risk human papilloma virus, exhibiting increased variability in their staining.

BACKGROUND Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) represent the most common forms of nonmelanoma skin cancers (NMSCs). Although successful treatment of these neoplasms is based on surgical excision, an increasing number of BCCs relapses and many SCCs display high rates of recurrence and metastasis. Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme, which was found to be upregulated in different solid tumours. However, there are no data regarding enzyme expression in NMSCs. The aim of this study was therefore to evaluate the potential involvement of NNMT in BCCs and SCCs. MATERIALS AND METHODS Immunohistochemical analyses were carried out on 40 BCC cases and 39 SCC cases, to evaluate enzyme expression in tumour and surrounding healthy margins. Moreover, the relationship between NNMT intratumour levels and clinico-pathological parameters were explored. RESULTS Nicotinamide N-methyltransferase was found to be overexpressed in BCCs compared with control tissues, while a significant enzyme downregulation was detected in SCCs with respect to corresponding healthy margins. In addition, NNMT levels were negatively related to aggressiveness of both BCCs (distinguishing between infiltrative and nodular tumours) and SCCs (considering head and neck forms and tumours of the extremities and trunk). CONCLUSIONS These evidences seem to demonstrate that the different NNMT dysregulation detected in BCC and SCC may be the result of important biological traits distinctively characterizing these two forms within NMSCs. In addition, enzyme levels seem to be inversely correlated with tumour aggressiveness, thus suggesting the potential suitability of the enzyme as a prognostic biomarker for both neoplasms.

Periosteum derived progenitor cells (PDPCs) represent promising mesenchymal stem cells (MSCs) for skeletal regeneration and to test bone cell based tissue engineering strategies. Most of regenerative medicine approaches based on MSCs require a noteworthy amount of cells that must be expanded in vitro prior to their use. As culture expansion method may impact on cell behaviour, we assessed the replicative and metabolic capacity (nitric oxide production and glucose consumption), senescence hallmarks of PDPCs serially passaged as well as the expression of selected genes specifically related to early osteoblastic differentiation, bone remodelling and stemness during PDPC sequential passaging. We also scouted a Systems Biology approach to examine and elucidate the experimental results through mathematical modelling and in silico simulations. PDPC subculture led to a progressive proliferative decline but, despite this, PDPCs maintained almost constant their metabolic activity. In vitro, senescent PDPCs displayed the typical "replicative senescence" features, involving p16 and not p53 in the regulation of this phenomenon. Gene expression analysis evidenced the tendency of sub-cultured PDPCs to increase the expression of genes involved in bone resorption. The mathematical analysis of the experimental results showed a strict similarity between replicative senescence and age-related changes, enabling the definition of an in silico model mimicking PDPC behaviour in terms of nitric oxide (NO) production. The relationship between NO production and subculture passages could represent a cutting edge "replicative senescence index". Overall, our findings suggest the possibility to use early-passage PDPCs for bone regenerative approaches based on the local recruitment of stem cells, whilst the later cell passages could be a suitable in vitro tool to validate scaffolds intended for bone regeneration in elderly subjects.

In this study, we investigate the relation between vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS) in periodontal disease of diabetic and non-diabetic individuals. We evaluated immunohistochemical VEGF and iNOS expressions in gingival biopsies from healthy individuals (no chronic periodontitis (CP)), patients with periodontitis alone (CP), patients with diabetes alone (DM) and diabetic patients with chronic periodontitis (DM + CP). We found a significant positive correlation between VEGF and iNOS expression in non-diabetic groups, but not in diabetic ones. Periodontal clinical parameters were not found to be significantly correlated with the inflammatory markers in no-CP, CP, and DM groups, whereas in DM + CP, positive and significant correlations were found between all the considered periodontal parameters and epithelial VEGF and endothelial iNOS. The uncoupling of VEGF and iNOS expression in diabetic individuals could allow a greater involvement of the markers in the inflammatory process of periodontitis.

Uncomplicated treatments for pulpitis and periodontitis continues to be challenging and regenerative approaches could meet this contingency. Dental pulp stem cells (DPSCs) represent a good candidate for oral recovering therapies. Here, we investigated changes in morphology, proliferation, and in vitro differentiation toward mesenchymal and neuronal phenotypes of human DPSCs harvested from differently aged donors. Aging is a physiologic phenomenon occurring with time that hamper body's capability to maintain homeostasis also affecting the functional reserve. Cytofluorimetric, immunohistochemical, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and western blot analyses were performed to gain insight for successful regenerative strategies in elderly. We observed a decline in DPSCs proliferation and differentiation potential with age. Interestingly, these cells behaved differently under osteogenic or odontogenic stimuli, showing different age-related mineralization capabilities. Similarly, neurogenic differentiation decreased with age. In conclusion, our observations represent a valid tool for the development of tailored regenerative strategies in an aging society.

Although the etiology of leiomyoma is unclear, a progenitor/undifferentiated cell population has been described whose dysregulation may be involved in the onset of uterine conditions. Moreover, inflammation is involved in the development of several tumors. The aim of this work was to understand if progenitor cells sustain a chronic inflammatory microenvironment that enhances leiomyoma development. Cells from 12 human leiomyoma and 12 normal myometrium samples of the same patients were in vitro isolated and exhaustively characterized (morphology, proliferation, cytofluorometry, differentiation, RT-PCR, immunofluorescence, immunohistochemistry, and Western blotting assays). Selected cytokines (ELISA) and inflammation-related genes (RT-PCR) were analyzed to identify healthy myometrium progenitor cells (MPCs) and leiomyoma progenitor cells (LPCs). Results show that (i) MPCs and LPCs share stemness features, such as immunophenotype and multidifferentiation assay, (ii) LPCs have a significantly shorter doubling time and a significantly higher expression of stemness genes (p<0.05), and (iii) LPCs secreted significantly higher levels (p<0.05) of cytokines related to chronic inflammation and significantly lower amounts (p<0.05) of cytokines related to acute inflammation. Despite the limited sample size, comparisons between leiomyoma and normal myometrium tissue from each patient allowed normalization of patient-specific differences. The evidenced cytokine expression pattern related to chronic inflammation in LPCs may play a role in the increased risk of adverse obstetric outcomes (infertility, spontaneous miscarriage, and preterm birth) in women affected by leiomyomas. These women should be recognized as “high risk” and subjected to specialized management both before and during pregnancy.

It was reported recently that allyl isothiocyanate (AITC) could inhibit various types of cancer cell growth. In the present study, we further investigated whether AITC could inhibit the growth of human breast cancer cells. Unexpectedly, we found that AITC did not inhibit, rather slightly promoted, the proliferation of MDA-MB-231 breast cancer cells, although it did have inhibitory effect on MCF-7 breast cancer cells. Cytofluorimetric analysis revealed that AITC (10 µM) did not induce apoptosis and cell cycle arrest in MDA-MB-231 cells. In addition, AITC significantly (p < 0.05) increased the expression of BCL-2 and mTOR genes and Beclin-1 protein in MDA-MB-231 cells. No significant changes in expression of PRKAA1 and PER2 genes, Caspase-8, Caspase-9, PARP, p-mTOR, and NF-κB p65 proteins were observed in these AITC-treated cells. Importantly, AITC displayed cytotoxic effect on MCF-10A human breast epithelial cell line. These observations suggest that AITC may not have inhibitory activity in MDA-MB-231 breast cancer cells. This in vitro study warrants more preclinical and clinical studies on the beneficial and harmful effects of AITC in healthy and cancer cells.

Prolonged hospitalization and antibiotic therapy are risk factors for the development of methicillin-resistant Staphylococcus aureus (MRSA) infections in thermal burn patients. We used a rat model to study the in vivo efficacy of daptomycin in the treatment of burn wound infections by S. aureus , and we evaluated the wound healing process through morphological and immunohistochemical analysis. A copper bar heated in boiling water was applied on a paraspinal site of each rat, resulting in two full-thickness burns. A small gauze was placed over each burn and inoculated with 5 × 10 7 CFU of S. aureus ATCC 43300. The study included two uninfected control groups with and without daptomycin treatment, an infected control group that did not receive any treatment, and two infected groups treated, respectively, with intraperitoneal daptomycin and teicoplanin. The main outcome measures were quantitative culture, histological evaluation of tissue repair, and immunohistochemical expression of wound healing markers: epidermal growth factor receptor (EGFR) and fibroblast growth factor 2 (FGF-2). The highest inhibition of infection was achieved in the group that received daptomycin, which reduced the bacterial load from 10 7 CFU/ml to about 10 3 CFU/g ( P < 0.01). The groups treated with daptomycin showed better overall healing with epithelialization and significantly higher collagen scores than the other groups, and these findings were also confirmed by immunohistochemical data. In conclusion, our results support the hypothesis that daptomycin is an important modulator of wound repair by possibly reducing hypertrophic burn scar formation.

BAP1 is a gene situated on chromosome 3p in a region that can be modified in renal cell carcinomas (RCCs). Mutations that cause loss of expression of BAP1 frequently occur in primary clear cell renal carcinoma (ccRCC). In a previous work, we observed that loss of nuclear BAP1 expression was crucial in ccRCC progression; in the current study, we investigated BAP1 expression in a large series of small conventional ccRCCs treated with partial nephrectomy, to assess a possible role as biomarker and the prognostic value in terms of patients’ survival at long-term follow-up. One hundred sixty-two patients with single pT1 ccRCC were selected from those who underwent surgery at our Institute of Urology between 1987 and 2000. The features considered in this study were gender, age, tumor size, grade, incidence of metastasis, and patient-specific survival; they were correlated with immunohistochemical BAP1 nuclear expression in tumoral tissue. Median follow-up was 197.24 months (range 19 to 274); median survival was 125.34 months (range 5 to 274 months). None of our pT1 ccRCCs showed total loss of nuclear BAP1 staining; we found a significant negative correlation between nuclear BAP1 expression and tumor size and between nuclear BAP1 expression and grade. In small ccRCCs, nuclear BAP1 staining was not correlated with disease-specific 5-year survival. Our data confirm the established role of BAP1 as a tumor suppressor protein. This is the first report where BAP1 has been studied in pT1 ccRCC tumors, but nuclear BAP1 expression cannot help identify patients having high-risk disease in these patients.

Basal cell carcinoma (BCC) is the most common type of skin cancer in older persons and is a rapidly rising incidence. E-cadherin-mediated cell-cell adhesion activates Cdc42, a Rho GTPase essential for cell polarity in numerous settings. No study has yet addressed a biological significance of Cdc42 alterations in BCC pathogenesis. Our aim was to investigate E-cadherin-dependent cell-cell contacts and Cdc42 activity in BCC formation. We evaluated E-cadherin and Cdc42 expression by immunohistochemistry and Western blot analysis in samples of 15 normal skin (NS) and 30 BCC (10 superficial, 9 nodular and 11 infiltrative subtypes). Low E-cadherin and high Cdc42 immunohistochemical expression were found in BCC samples compared with NS. E-cadherin staining was significantly reduced in infiltrative BCC compared with superficial and nodular. A significantly greater Cdc42 expression was observed in BCC compared with NS; moreover, superficial BCC had a significantly lower Cdc42 expression in respect to the other subtypes. Western blot analysis confirmed the significantly decreased E-cadherin expression in infiltrative BCC as well as Cdc42 reduction in superficial BCC in respect to the other subtypes. In BCC the increased Cdc42 in association with reduced E-cadherin might contribute to the disruption of adhesion mechanisms and to the loss of cell polarity, thus explaining a mechanism by which cancer cells can escape from the control of adjacent normal keratinocytes. Our study also showed that Cdc42 and E-cadherin expression differed according to aggressive behaviour of BCC subtypes and suggested important functions of these molecules in regulating tumour demarcation and progression.

SummaryBackground Atopic dermatitis (AD) is a chronic and inflammatory disease characterized by a marked imbalance of T helper (Th)2 vs. Th1/Th17 cells in the early phase of AD, whereas a mixed Th1/Th2 pattern of inflammation is usually found at the chronic stage. These features have not been extensively evaluated in undifferentiated skin cells of patients affected by AD. Objectives To evaluate the relative expression of 22 genes encoding Th1, Th2 and Th17 cytokines and the secretion of the corresponding proteins in cutaneous mesenchymal stem cells (MSCs) isolated from skin of patients with AD (AD-MSCs) and their role in AD onset. Methods AD-MSCs were isolated, characterized and profiled by polymerase chain reaction array and enzyme-linked immunosorbent assay for the relative expression and secretion of cytokines involved in the Th1, Th2 and Th17 pathways. MSCs isolated from the skin of healthy people were used as controls (C-MSCs). Results AD-MSCs showed an upregulation of many Th1/Th17 cytokines [interleukin (IL)-6, IL-8, IL-12, IL-13, IL-17A, IL-17F, transforming growth factor-β, interferon-γ], while Th2 chemokines (IL-2, IL-4, IL-5, IL-23A) were downregulated in AD-MSCs. Finally, some genes/proteins (CCL1, IL-17C, tumour necrosis factor-α) did not show variations between C-MSCs and AD-MSCs. Conclusions The profile of MSCs obtained from patients with chronic AD retraces the Th1/Th17 cell environment observed in differentiated cells of chronic AD. This evidence could open a new scenario in the pathogenesis of AD, according to which the inflammatory process may involve MSCs early on.

In staphylococci, quorum sensing regulates both biofilm formation and toxin production, moreover it has been demonstrated to be inhibited by RNAIII inhibiting peptide (RIP). Aim our study was to evaluate the in vitro activity and its in vivo efficacy of the combined administration of FS10, a novel RIP derivative, and tigecycline in an animal model of methicillin-resistant (MR) and methicillin-sensitive (MS) Staphylococcus aureus wound infection. Using a 1.x2 cm template, one full thickness wound was established through the panniculus carnosus on the back subcutaneous tissue of each animal. Infection was determined by inoculation of 5x107 CFU/ml of bacteria, that produced an abscess within 24 h, after this, treatment was initiated. The study included, for each strain, a control group without infection, a control infected group that did not receive any treatment and a control infected group with drug-free foam dressing, and three infected groups treated, respectively, with: FS10-soaked foam dressing (containing 20 μg FS10), daily intraperitoneal tigecycline (7 mg/Kg), FS10-soaked foam dressing (containing 20 μg FS10) and daily intraperitoneal injections of tigecycline (7 mg/Kg). The main outcome measures were quantitative culture and histological examination of tissue repair. The highest inhibition of infection was achieved in the group that received FS10-soaked and parenteral tigecycline reducing the bacterial load from 107 CFU/ml to about 103 CFU/g for MSSA and to about 104 CFU/g for MRSA. The group treated with FS10-soaked foam dressing associated with parenteral tigecycline showed, histologically, better overall healing with epithelialization and collagen scores significantly higher than those of the other groups in both strains. In conclusion, the combined use of topical FS10 with i.p. tigecycline induced positive interaction in vivo, resulting in an enhanced therapeutic benefit versus staphylococcal infections in murine wound models.

Background and Objective The accumulation of advanced glycation end-products (AGEs) seems to play an important role in the development of diabetes mellitus (DM)-associated periodontitis; however, some aspects of this issue are still scarcely known, such as the expression of AGEs in type 1 DM-associated periodontitis and the clinical factors able to affect their accumulation. This study aimed to clarify these points by evaluating the expression of AGEs in DM-associated periodontitis. Material and Methods Sixteen systemically and periodontally healthy subjects and 48 subjects suffering from generalized, severe, chronic periodontitis (16 with type 1 DM, 16 with type 2 DM and 16 systemically healthy subjects) were studied clinically, periodontally and metabolically. The immunohistochemical expression of AGEs in gingival tissues was also evaluated. Results Subjects affected with type 1 DM presented a significantly higher percentage of AGE-positive cells than did subjects affected with type 2 DM, not only in the epithelium, but also in vessels and fibroblasts. A positive and significant correlation was found between gingival expression of AGEs and length of time affected with DM both in type 1 and type 2 DM; glycated hemoglobin, lipid profile, body mass index and age did not correlate significantly with gingival AGEs in any of the classes of subjects studied. Conclusions Gingival AGEs are increased in both type 1 and type 2 DM-associated periodontitis; however, the clinical parameter that determines their accumulation, and therefore their degree of influence on the development of DM-associated periodontitis, may be the duration of DM.

Several studies have reported on the prognostic value of molecular markers for metastasis risk and survival in penile squamous cell carcinoma (SCC) patients. The usefulness of CD44 expression as such a marker has been studied in different tumors, but not in penile SCC. Our aim was to determine whether CD44 expression may serve as a prognostic marker for lymph node metastasis and survival in penile SCC patients. CD44 immunoistochemical expression was investigated in tissue specimens from 39 patients with penile SCC. CD44 cell positivity, staining intensity and distribution were analyzed and correlated with tumor stage, grade, lymph node status and disease-specific survival. CD44 expression was detected in epithelial cells of both intratumoral and normal tissues with different intensities and staining distributions. In normal tissues CD44 protein was mainly detected in cell membranes, whereas in the tumor compartments it was found in both the cell membranes and the cytoplasm. The intensities and percentages of CD44 expressing cells did not correlate with tumor stage and/or grade. Seventy-three percent of the patients with lymph node metastasis showed high intensities of CD44 staining, as compared to 44% of the patients without lymph node metastasis (P = 0.03). Lymph node-positive patients showed both cytoplasmic and membranous CD44 expression. High CD44 expression was found to be significantly correlated with a decreased 5 year overall survival (P = 0.01). CD44 levels and patterns of expression can be considered as markers for penile SCC aggressiveness and, in addition, may serve as predictive markers for lymph node metastasis, also in patients with clinically negative lymph nodes. CD44 expression may provide prognostic information for penile SCC patients, next to classical clinical-pathological factors.

Background Sacral neuromodulation is becoming established as a valid treatment option for patients with anorectal disorders. Nevertheless, despite its efficacy, little is known regarding its mechanism of action. The purpose of this study was to evaluate whether chronic sacral neuromodulation is able to influence the expression of nitric oxide synthetase (NOS) in the anorectum of rats. Materials and methods Twenty-six female Sprague-Dawley rats were divided into three groups; normal control rats (n = 6); sham treatment (n =10) and group in whom, electrical sacral neuromodulation was performed (n = 10). Bilateral electrode wires were placed in the S1 and electrical stimulation was performed for 14 d. At the end of the procedures the rats were sacrificed, proctectomy was performed, and anorectal specimens were sent to the laboratory for immunostaining with n-NOS and i-NOS. Results In the anal and rectal specimens, n-NOS and i-NOS expression was significantly increased in epithelial and muscle cells after neuromodulation of the anus and rectum of the animals. Conclusion Our results showed that this model can be applied in further experimental studies to better understand the mechanism of action of sacral neuromodulation in anorectal disorders.

We aimed to evaluate glycodelin immunostaining in pregnant women with a first diagnosis of cervical intraephitelial neoplasia (CIN) and to correlate the expression of CIN with Ki-67 and glycodelin immunostaining. A retrospective case-control study was performed including 20 patients with natural pregnancy and with first time onset of CIN occurring not later than 16 gestational weeks. The control group included 20 non-pregnant patients matched for age, parity, smoking status and number of previous sexual partners. Exclusion criteria included previous cervical treatment, immunocompromised status and chronic hepatitis B and/or C. Staining for Glycodelin and for Ki-67 was expressed using a classification based on the distribution of positivity on a semi-quantitative three-point scale. An inverse relationship was observed between glycodelin immunostaining and CIN grade in pregnant patients (p = 0.01), with a significantly higher expression in CIN1 than in CIN2 and CIN3, but not in non-pregnant patients (p = 0.81). Positivity for Ki-67 was less intense in pregnant than in non-pregnant patients. A significant inverse relationship was observed between glycodelin immunostaining and Ki-67 expression (p = 0.02). We suggest that the higher expression of glycodelin in pregnancy is related to a lower proliferative activity in CIN, which is probably associated to hormonal status of pregnancy. Further clinical studies are needed to support these findings.

Summary Background The expression of inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) and the level of total oxyradical scavenging capacity have been evaluated extensively in the cutaneous cells of patients with psoriasis. As yet, no indications are available about the undifferentiated cells, the mesenchymal stem cells (MSCs), isolated from skin. Objectives To isolate MSCs in patients with psoriasis and to compare them with those obtained from atopic and healthy subjects, in order to analyse whether MSCs show some typical psoriatic profiles and to understand whether pathophysiological events leading to psoriasis start early at the stem cell level. Methods MSCs isolated from seven patients with psoriasis, seven patients with acute atopic dermatitis and seven healthy subjects were characterized by fluorescence-activated cell sorting analysis. VEGF and nitric oxide (NO) content was measured in conditioned medium, the expression of VEGF and iNOS was analysed by immunohistochemistry, and the total oxyradical scavenging capacity towards peroxynitrite was tested. Results VEGF content was highest in the medium conditioned by psoriatic perilesional MSCs, whereas NO concentration was maximally increased in medium conditioned by MSCs isolated from lesional psoriatic skin. The ability to neutralize the oxidizing effects of peroxynitrite was lower for MSCs isolated from lesional psoriatic skin compared with other MSCs, except for MSCs of lesional atopic skin. Conclusions The microenvironment in psoriasis differs from those of atopic dermatitis and healthy skin; it could induce resident MSCs to produce angiogenic and proinflammatory mediators which lead to a reduction in the antioxidant capacity of these cells, contributing to the development of skin lesions in psoriasis.

Summary Background Chronic leg ulceration is a common health problem. It is well known that a clinically relevant bacterial load in chronic cutaneous wounds interferes significantly with the normal process of healing. Staphylococcus aureus is the most important representative of the staphylococcal group which causes clinically relevant infections within immunocompetent patients. Objectives To investigate the efficacy of a single treatment of antimicrobial photodynamic therapy (APDT) with RLP068/Cl in a mouse model of a surgical wound infection induced with a methicillin-resistant strain of S. aureus (MRSA). Methods Wounds, established through the panniculus carnosus of BALB/c and CD1 mice, were inoculated with 5 × 107 c.f.u. of MRSA. Mice were randomized into four groups respectively receiving no treatment, APDT with placebo, APDT with a new phthalocyanine derivative (RLP068/Cl) and intraperitoneal teicoplanin. Results On day 2 from infection, a strong reduction of bacterial counts (≈ 3 logs) was observed in mice treated with RLP068/Cl in comparison with infected untreated mice. On day 9 from infection, a comparable and significant (≈ 2 logs) reduction of bacterial counts was found in mice treated with RLP068/Cl or with teicoplanin. At this time, histological examinations revealed that wounds treated with RLP068/Cl showed a complete re-epithelialization with a continuous epithelial lining. Conclusions The results of the in vivo study demonstrated that APDT with RLP068/Cl may be useful in the management of chronic infected wounds, accelerating the repair process through a significant bacterial inhibition.

Asbestos fibres can be transformed into potentially non-hazardous silicates by high-temperature treatment via complete solid-state transformation. A549 cells were exposed to standard concentrations of raw cement asbestos (RCA), chrysotile and cement asbestos subjected to an industrial process at 1200 degrees C (Cry_1200 and KRY.AS, respectively), raw commercial grey cement (GC). Cell growth rate and viability (MTT test) were detected in vitro. RCA and KRY.AS subjected to comprehensive microstructural study by electron microscopy were further in vitro assayed to compare their cytotoxic potential by morphostructural studies, proliferation index (Ki-67 antigen), apoptosis induction (AO/EB staining) assays and detection of intracellular reactive oxygen species (ROS) with the fluorescent DCFA dye. More severe cytotoxic damage was induced by RCA than by KRY.AS after each incubation period. Exposure to KRY.AS and GC resulted in comparable cell growth rates and cytotoxic effects. Cells incubated with RCA showed greater apoptotic induction and ROS production and a lower cell proliferation index than those exposed to KRY.AS. Chrysotile asbestos and RCA subjected to heat treatment underwent complete microstructure transformation. The final product of heat treatment of cement asbestos, KRY.AS, was considerably more inert and had lower cytotoxic potential than the original asbestos material in all in vitro tests.