Immunomodulatory effects of metronomic vinorelbine (mVRL), with or without metronomic capecitabine (mCAPE), in hormone receptor positive (HR+)/HER2-negative metastatic breast cancer (MBC) patients: final results of the exploratory phase 2 Victor-5 study.

Tregs are able of suppressing tumor-specific effector cells, such as lymphocytes CD8+, CD4+ and Natural Killer cells. Different drugs, especially different schedules of administration, like metronomic chemotherapy (mCHT), seem to be able to increase anticancer immunity, by acting on downregulation of Tregs. Most of the data available regarding the immunomodulating effect of mCHT have been obtained with Cyclophosphamide (CTX). Aim of the present study was to explore the effects of mVRL and mCAPE administration, alone or in combination, on T cells. Observation of 13 metastatic breast cancer patients lasted controlling for 56 days, where Treg frequencies and function, spontaneous anti-tumor T-cell responses were monitored, as well as the clinical outcome. No depletion in Treg absolute numbers, or percentage of T lymphocytes, was observed. Only in 5 patients, a modest and transient depletion of Tregs was observed during the first 14 days of treatment. To better describe the effect on Tregs, we subsequently looked at the variations in Memory, Naïve and Activated Treg subpopulations: we observed a trend in reduction for memory Treg (Treg MEM) and an increase for Treg Naïve (Treg NAIVE) and Treg Activated (Treg ACT) components. We finally analyzed the average trend of Treg in the Treg depleted patients and non-depleted ones, without fiding any significant differences. The trend of the Treg MEM appeared different, showing a reduction during the first 14 days, followed by an increase at the levels before treatment at Day 56 in the group of depleted patients and a progressive substantial reduction in the group of non-depleted patients along the entire course of treatment. Opposed to the data known, treatment with mVRL w/o mCAPE did not show any effect on Tregs.

Translational and transdisciplinary approach to the human papilloma virus - Preliminary evidence from the Italian "HPV board: a future without papilloma virus" project.

Human papillomavirus (HPV) is considered to be one of the viral infections associated with cancers and other diseases. HPV is detected asymptomatically in the oral mucosa. The presence of human papillomavirus in the oral mucosa appears to be closely associated with a series of benign and malign oral lesions. The aim of this paper is to report the Italian experience in applying translational protocols, using new technologies and multidisciplinary strategies in Human Papilloma virus detection and treatment. The "HPV board: a future without papilloma virus" project was born, promoted by CNEL (Italian Council of Economics and Labor) with the collaboration of numerous scientific societies to commonly approach to public knowledge of HPV-related oral lesions and their clinical management. The preliminary results are related to the assessment of the proof-of-concept of this new project. More in details, "HPV Board" is a project that plans the presence of a working group, made up of otolaryngologists, dentists, oral and maxillofacial surgeons, in close contact with gynecologists, oncologists and pediatricians; this working group manages to combine very transversal skills, in order to promote primary prevention projects, early diagnosis and adequate therapies. The "HPV BOARD" project will give the opportunity to increase the attention of patients and doctors on the early diagnosis of oncological diseases dependent on infection by the infectious agent HPV. In this panorama, dentists will have the role of "first sentinel" of public health because oral health is an indicator, too often overlooked, for the prevention of numerous diseases.

Aerosol-generating procedures in head and neck surgery - can we improve practice after COVID-19?

The COVID-19 pandemic has had a dramatic impact on international medicine practice. The propensity for head and neck surgery to generate aerosols needs special consideration over and above simply adopting personal protective equipment. This study sought to interrogate the literature and evaluate whether which additional measures might provide benefit if routinely adopted in minimising viral transmission.

Staphylococcus aureus intra-mammary infection affects the expression pattern of IL-R8 in goat.

IL-1R8 is a member of Interleukin-1 receptor family acting as a negative regulator of inflammation reliant on ILRs and TLRs activation. IL-1R8 role has never been evaluated in acute bacterial mastitis. We first investigated IL-1R8 sequence conservation among different species and its pattern of expression in a wide panel of organs from healthy goats. Then, modulation of IL-1R8 during natural and experimental mammary infection was evaluated and compared in blood, milk and mammary tissues from healthy and Staphylococcus aureus infected goats. IL-1R8 has a highly conserved sequence among vertebrates. Goat IL-1R8 was ubiquitously expressed in epithelial and lymphoid tissues with highest levels in pancreas. IL-1R8 was down-regulated in epithelial mammary cells following S. aureus infection. Interestingly it was up-regulated in leukocytes infiltrating the infected mammary tissues suggesting that it could represent a target of S. aureus immune evasion.

Pentraxin 3 is up-regulated in epithelial mammary cells during Staphylococcus aureus intra-mammary infection in goat.

Pentraxin 3 is the prototypic long pentraxin and is produced by different cell populations (dendritic cells, monocytes/macrophages, endothelial cells, and fibroblasts) after pro-inflammatory stimulation. Different studies demonstrated the up-regulation of PTX3 during mastitis in ruminants, but its role is still unknown. We first investigated the conservation of PTX3 sequence among different species and its pattern of expression in a wide panel of organs from healthy goats. We studied the modulation of PTX3 during natural and experimental mammary infection, comparing its expression in blood, milk and mammary tissues from healthy and Staphylococcus aureus infected animals. We confirmed the high conservation of the molecule among different species. Goat PTX3 was expressed at high levels in bone marrow, mammary gland, aorta, rectum, pancreas, skin and lungs. PTX3 was up-regulated in epithelial mammary cells and in milk cells after S. aureus infection, suggesting that it represents a first line of defense in goat udder.

Alpha tocopherol loaded chitosan oleate nanoemulsions for wound healing. Evaluation on cell lines and ex vivo human biopsies, and stabilization in spray dried Trojan microparticles.

An amphiphilic chitosan salt, chitosan oleate (CS-OA), was previously proposed for the physical stabilization of lemongrass antimicrobial nanoemulsions (NE) through a mild spontaneous emulsification process. As both chitosan and oleic acid are described in the literature for their positive effects in wound healing, in the present study CS-OA has been proposed to encapsulate alpha tocopherol (αTph) in NEs aimed to skin wounds. A NE formulation was developed showing about 220 nm dimensions, 36% drug loading, and αTph concentration up to 1 mg/ml. Both CS-OA and αTph NE stimulated cell proliferation on keratinocytes and fibroblast cell cultures, and in ex vivo skin biopsies, suggesting the suitability of CS-OA and of the antioxidant agent for topical application in wound healing. αTph stability was further improved with respect of encapsulation, by spray drying the NE into a powder (up to about 90% αTph residual after 3 months). The spray drying process was optimized, to improve powder yield and αTph recovery, by a design of experiments approach. The powder obtained was easily re-suspended to deliver the NE and resulted able to completely release αTph.

Circulating tumor DNA changes for early monitoring of anti-PD1 immunotherapy: a proof-of-concept study.

BACKGROUND: Recent clinical results support the use of new immune checkpoint blockers (ICB), such as anti-PD-1 (e.g. nivolumab and pembrolizumab) and anti-PD-L1 antibodies. Radiological evaluation of ICB efficacy during therapy is challenging due to tumor immune infiltration. Changes of circulating tumor DNA (ctDNA) levels during therapy could be a promising tool for very accurate monitoring of treatment efficacy, but data are lacking with ICB. PATIENTS AND METHODS: This prospective pilot study was conducted in patients with nonsmall cell lung cancer, uveal melanoma, or microsatellite-instable colorectal cancer treated by nivolumab or pembrolizumab monotherapy at Institut Curie. ctDNA levels were assessed at baseline and after 8 weeks (w8) by bidirectional pyrophosphorolysis-activated polymerization, droplet digital PCR or next-generation sequencing depending on the mutation type. Radiological evaluation of efficacy of treatment was carried out by using immune-related response criteria. RESULTS: ctDNA was detected at baseline in 10 out of 15 patients. At w8, a significant correlation (r = 0.86; P = 0.002) was observed between synchronous changes in ctDNA levels and tumor size. Patients in whom ctDNA levels became undetectable at w8 presented a marked and lasting response to therapy. ctDNA detection at w8 was also a significant prognostic factor in terms of progression-free survival (hazard ratio = 10.2; 95% confidence interval 2.5-41, P < 0.001) and overall survival (hazard ratio = 15; 95% confidence interval 2.5-94.9, P = 0.004). CONCLUSION: This proof-of-principle study is the first to demonstrate that quantitative ctDNA monitoring is a valuable tool to assess tumor response in patients treated with anti-PD-1 drugs.

A dynamic distention protocol for whole-organ bladder decellularization: histological and biomechanical characterization of the acellular matrix.

A combined physical-chemical protocol for whole full-thickness bladder decellularization is proposed, based on organ cyclic distention through repeated infusion/withdrawal of the decellularization agents through the urethra. The dynamic decellularization was intended to enhance cell removal efficiency, facilitating the delivery of detergents within the inner layers of the tissue and the removal of cell debris. The use of mild chemical detergents (hypotonic solution and non-ionic detergent) was employed to limit adverse effects upon matrix 3D ultrastructure. Inspection of the presence of residual DNA and RNA was carried out on decellularized matrices to verify effective cell removal. Histological investigation was focused on assessing the retention of adequate structural and functional components that regulate the biomechanical behaviour of the acellular tissue. Biomechanical properties were evaluated through uniaxial tensile loading tests of tissue strips and through ex vivo filling cystometry to evaluate the whole-organ mechanical response to a physiological-like loading state. According to our results, a dynamic decellularization protocol of 17 h duration with a 5 ml/min detergent infusion flow rate revealed higher DNA removal efficiency than standard static decellularization, resulting in residual DNA content < 50 ng/mg dry tissue weight. Furthermore, the collagen network and elastic fibres distribution were preserved in the acellular ECM, which exhibited suitable biomechanical properties in the perspective of its future use as an implant for bladder augmentation.

Efficacy and Safety of the All-Oral Schedule of Metronomic Vinorelbine and Capecitabine in Locally Advanced or Metastatic Breast Cancer Patients: The Phase I-II VICTOR-1 Study.

Background. Vinorelbine (VRB) and capecitabine (CAPE) are demonstrated to be active in pretreated metastatic breast cancer patients. Different studies have demonstrated that the metronomic treatment is active with an acceptable toxicity profile. We designed a Phases I-II study to define the MTD of oral metronomic, VRB, and CAPE. Patients and Methods. Phase I: fixed dose of CAPE was 500 mg thrice a day, continuously. Level I of VRB was 20 mg/tot thrice a week for 3 weeks (1 cycle). Subsequent levels were 30 mg/tot and 40 mg/tot (Level III), respectively, if no Grades 3-4 toxicity were observed in the previous level. Phase II: further 32 patients received the MTD of VRB plus CAPE for a total of 187 cycles to confirm toxicity profile. Results. 12 patients were enrolled in Phase I and 22 in Phase II. Phase I: the MTD of VRB was 40 mg. Phase II: 187 cycles were delivered, observing 5.9% of Grades 3-4 toxicity. 31 patients are evaluable for efficacy, obtaining a clinical benefit rate of 58.1%. Conclusion. MTD of VRB with fixed dose of CAPE was 40 mg thrice a week and was the recommended dose for the ongoing Phase II multicenter study.

Models of epithelial histogenesis.

Epithelial tissues emerge from coordinated sequences of cell renewal, specialization and assembly. Like corresponding immature tissues, adult epithelial tissues are provided by stem cells which are responsible for tissue homeostasis. Advances in epithelial histogenesis has permitted to clarify several aspects related to stem cell identification and dynamics and to understand how stem cells interact with their environment, the so-called stem cell niche. The development and maintenance of epithelial tissues involves epithelial-mesenchymal signalling pathways and cell-matrix interactions which control target nuclear factors and genes. The tooth germ is a prototype for such inductive tissue interactions and provides a powerful experimental system for the study of genetic pathways during development. Clonogenic epithelial cells isolated from developing as well mature epithelial tissues has been used to engineer epithelial tissue-equivalents, e.g. epidermal constructs, that are used in clinical practise and biomedical research. Information on molecular mechanisms which regulate epithelial histogenesis, including the role of specific growth/differentiation factors and cognate receptors, is essential to improve epithelial tissue engineering.

Stimulation of osteoblast growth by an electromagnetic field in a model of bone-like construct.

The histogenesis of bone tissue is strongly influenced by physical forces, including magnetic fields. Recent advances in tissue engineering has permitted the generation of three dimensional bone-like constructs. We have investigated the effects of electromagnetic stimulation on human osteoblast cells grown in a hydrophobic polyurethane scaffold. Bone-like constructs were stimulated by pulsed electromagnetic fields in a bioreactor. Proliferation, bone protein expression and calcified matrix production by osteoblasts were measured using histochemical methods. In stimulated cultures, the number of cells was significantly higher compared to static (control) cultures. In both stimulated and control cultures, cells were immunoreactive to osteoblast markers, including type-I collagen, osteocalcin and osteopontin, thus suggesting that the expression of bone-related markers was maintained throughout the in vitro experiments. Morphometric analysis of von Kossa-stained sections revealed that stimulation with electromagnetic field significantly increased matrix calcification. The data lend support to the view that the application of a magnetic field can be used to stimulate cell growth in bone-like constructs in vitro. This finding may be of interest for the production of biomaterials designed for clinical applications.

[Internal carotid artery aneurisms: two cases]. / Anévrismes de l'artère carotide interne extra-crânienne: à propos de 2 cas.

INTRODUCTION: Aneurysms of the extracranial portion of the internal carotid artery (ICA) are rare (accounting for only 0.1-2% of all surgical procedures affecting the ICA, 0.4-1% of all arterial aneurysms, and 4% of all aneurysms involving peripheral arteries), but they are nonetheless clinically significant because of the high related risk of cerebral thromboembolism. Given the rarity of these lesions, it seems worthwhile to report on two extracranial ICA aneurysms, one of atherosclerotic, the other of fibrodysplastic etiology that came under our observation. PATIENTS AND METHODS: Our experience concerns just two cases, treated at the Department of Surgical and Gastroenterological Sciences of the Policlinico G.B. Rossi in Verona, presenting with very different clinical and instrumental findings, and requiring a different surgical treatment. The former underwent resection of the aneurysm and end-to-end reconstruction; in the latter, we performed a carotid transposition with internalization of the external carotid artery. RESULTS: Neither patient suffered from any major or minor neurological complications during or after surgery, and the follow-up confirmed a normal extracranial carotid patency. CONCLUSIONS: Based on our, albeit limited experience and an analysis of the literature, we make a few points concerning the diagnostic approach (which differs from the case of stenosing carotid lesions), the indications and type of treatment for extracranial ICA aneurysms.

Mid-term experience with the ALN retrievable inferior vena cava filter.

OBJECTIVE: To report the mid-term results of 63 patients who received a new commercially-available retrievable vena cava filter, ALN. METHODS: Between January 2001 and October 2005, 63 patients (mean age 65 +/- 15 years) underwent placement of ALN filters. Filter removal was performed when anti-thrombotic prophylaxis was considered unnecessary or when the patient could safely resume full anticoagulant therapy. RESULTS: Thirty-five patients (55%) had ilio-femoral venous thrombosis and 28 patients (45%) had ilio-caval thrombosis. Overall, 49% had pulmonary embolism. Technical success for filter insertion was 100%, without any complications. None of the procedures aborted or was converted due to technical difficulties. After a median follow-up of 21-months (range 1-48, median 18), there were no cases of pulmonary embolism or vena cava thrombosis. Two patients died of a cause unrelated to deep venous thrombosis during the follow-up period, without clinical evidence of pulmonary embolism or filter-associated complications. No device migration was observed. There were 20 (31.7%) retrieval attempts: in 16 cases filters were retrieved successfully, but 4 cases were aborted. The mean implantation period of the retrieved filter was 179 days (range 53-370). CONCLUSION: Our results confirm the clinical efficacy of the ALN filter for preventing potentially fatal pulmonary embolism whilst implanted and in absence of post-insertion complications, even when left in place indefinitely.

Morphological alterations induced by cytochalasin D on serous cells of human submandibular gland in basal and stimulated conditions.

Cytochalasin D (CD) is a fungal toxin which binds to the faster growing end of actin microfilament and inhibits actin polymerization. By an in vitro incubation system of slices of human submandibular glands obtained at surgery, we investigated by light microscope (LM), transmission electron microscope (TEM), and high resolution scanning electron microscope (HRSEM) the morphological changes caused by CD on serous cells. We studied the effects of the drug on secretory events induced by isoproterenol (I) and carbachol (C). With LM, following CD incubation, canaliculi were enlarged and prominent vacuoles were seen throughout the cytoplasm. By TEM, the vacuoles, which in many cases were in continuity with the lumen, represented the distinctive feature of secretory cells. With HRSEM, intercellular canaliculi, seen from their cytoplasmic side, exhibited many small spherical bulges, corresponding to the coated pits seen with TEM and indicating that the retrieval of plasma membrane was arrested at an early phase by the disruption of the actin cytoskeleton. In specimens treated with secretagogues and CD, a consequence reported here for the first time was the presence of dense granules within the vacuoles. The protrusions seen by HRSEM on the cytoplasmic side of intercellular canaliculi, following secretagogues stimulations, appeared peculiar to each stimulants, even if combined with CD, suggesting that besides actin filaments, other components, unaffected by CD, also are involved in the process of exocytosis and related phenomena.

Managing anomalous splenic artery aneurysm: a review of the literature and report of two cases.

The splenic artery originates from the superior mesenteric artery in approximately 1% of cases, which may explain the extreme rarity of aneurysms involving this anomalous branch, with only five cases reported in the international literature to date. We report our experience of managing two patients with aneurysms involving splenic arteries arising from the superior mesenteric artery, one treated surgically and the other percutaneously. From a diagnostic point of view, the first approach is ultrasound, while computed tomographic (CT) scan and angiography enable a better definition of the lesion and of the anatomical anomaly; CT angiography is currently the method of choice for the preoperative workup. Finding these two anomalies in association is so rare that it is impossible to draw any final conclusions as to the best type of treatment. In the authors' experience, both surgery and percutaneous treatment can prove useful.

Serous and mucous cells of human submandibular salivary gland stimulated in vitro by isoproterenol, carbachol and clozapine: an LM, TEM, and HRSEM Study.

We have investigated by LM, TEM, and HRSEM the effects of D,L-isoproterenol (beta-adrenergic agent), carbachol (muscarinic agent) and clozapine on biopsy specimens of human submandibular gland stimulated in vitro in an inorganic oxygenated medium. Clozapine is a dibenzodiazepine derivative used in psychotic patients that provokes hypersalivation, a displeasing side effect that often causes discontinuance of therapy. Our findings demonstrate that clozapine acts on salivary mucous and seromucous (serous) cells of the gland as a muscarinic agonist. However, the induced secretory response seems to differ qualitatively and quantitatively from that resulting from carbachol. Thus, in agreement with published data resulting from therapeutic treatments and from experimental studies on rats, the mechanism of clozapine induced hypersialorrhea remains open to further investigation.

UV-induced DNA incision and proliferating cell nuclear antigen recruitment to repair sites occur independently of p53-replication protein A interaction in p53 wild type and mutant ovarian carcinoma cells.

The tumour suppressor gene TP53 plays an important role in the regulation of DNA repair, and particularly of nucleotide excision repair. The influence of p53 status on the efficiency of the principal steps of this repair pathway was investigated after UV-C irradiation in the human ovarian carcinoma cell line IGROV-1 (expressing wild-type p53) and in the derived clone IGROV-1/Pt1 (with p53 mutations at codons 270 and 282). Clonogenic survival after UV-C irradiation showed that IGROV-1/Pt1 cells were approximately 2-fold more resistant to DNA damage than parental cells. Modulation of p53 protein levels, cell cycle arrest and apoptosis were induced in UV-irradiated IGROV-1 cells, but not in the p53-mutant cell line. Exposure to UV or cisplatin induced down-regulation of p53-replication protein A (RPA) interaction in parental, but not in IGROV-1/Pt1 cells. However, persistent binding of p53 to RPA did not affect the early steps of DNA repair. In fact, both UV-induced DNA incision and the recruitment of proliferating cell nuclear antigen (PCNA) to DNA repair sites occurred to a comparable extent in p53-wild type and -mutant cell lines, although PCNA remained associated with chromatin for a longer period of time in IGROV-1/Pt1 cells. Global genome repair, as detected by immunoblot analysis of cyclobutane pyrimidine dimers, was not significantly different in the two cell lines at 3 h after UV irradiation. In contrast, lesion removal at 24 h was markedly reduced in IGROV-1/Pt1 cells, being approximately 25% of the initial amount of damage, as compared with approximately 50% repair in parental cells. These results indicate that the presence of mutant p53 protein and its persistent interaction with RPA do not affect the early steps of nucleotide excision repair in IGROV-1/Pt1 cells. Thus, repair defects in p53-mutant ovarian carcinoma cells may be attributed to late events, possibly related to a reduced removal/recycling of PCNA at repair sites.

Effects of topoisomerase II inhibitors on gastric cancer cells characterized by different genetic lesions.

Gastric cancer is poorly-responsive to widely used antitumour drugs, the efficacy of which is thought to be related to the capacity of triggering apoptosis. This process requires a series of gene products including a functional p53 protein. We tested the effects of two DNA topoisomerase II poisons, etoposide and doxorubicin, on gastric cancer cell lines with different genetic lesions. We characterised MKN74 and MKN28 cells for p53 gene status and for the expression of p53 and p21 proteins, as well as of topoisomerase II alpha and beta isoforms. After drug treatments, the cells were analysed for drug cytotoxicity, colony forming ability, cell cycle distribution and presence of apoptotic features. Our findings demonstrated that both etoposide and doxorubicin have a potent anti-proliferative effect on gastric cancer cells. Cell death kinetics was different in the two cell lines, MKN74 cells being more sensitive than MKN28 to the drugs. MKN74 cells, although harboring a wt p53 gene, were unable to undergo a massive apoptosis following etoposide treatment. The response of this cell line might be related to the topoisomerase II beta isozyme, the expression of which proved to be undetectable.