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OBJECTIVES: To test the performance of ex vivo fluorescence confocal microscopy (FCM; Vivascope 2500M-G4), as compared to intra-operative frozen section (IFS) analysis, to evaluate surgical margins during robot-assisted radical prostatectomy (RARP), with final pathology as the reference standard. METHODS: Overall, 54 margins in 45 patients treated with RARP were analysed with: (1) ex vivo FCM; (2) IFS analysis; and (3) final pathology. FCM margins were evaluated by two different pathologists (experienced [M.I.: 10 years] vs highly experienced [G.R.: >30 years]) as strongly negative, probably negative, doubtful, probably positive, or strongly positive. First, inter-observer agreement (Cohen's κ) between pathologists was tested. Second, we reported the sensitivity, specificity, positive predictive (PPV) and negative predictive value (NPV) of ex vivo FCM. Finally, agreement between ex vivo FCM and IFS analysis (Cohen's κ) was reported. For all analyses, four combinations of FCM results were evaluated. RESULTS: At ex vivo FCM, the inter-observer agreement between pathologists ranged from moderate (κ = 0.74) to almost perfect (κ = 0.90), according to the four categories of results. Indeed, at ex vivo FCM, the highly experienced pathologist reached the best balance between sensitivity (70.5%) specificity (91.8%), PPV (80.0%) and NPV (87.1%). Conversely, on IFS analysis, the sensitivity, specificity, PPV and NPV were, respectively, 88.2% vs 100% vs 100% vs 94.8%. The agreement between the ex vivo FCM and IFS analyses ranged from moderate (κ = 0.62) to strong (κ = 0.86), according to the four categories of results. CONCLUSION: Evaluation of prostate margins at ex vivo FCM appears to be feasible and reliable. The agreement between readers encourages its widespread use in daily practice. Nevertheless, as of today, the performance of FCM seems to be sub-par when compared to the established standard of care (IFS analysis).

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We designed a phase 3, prospective, randomized trial to evaluate the impact of augmented reality and augmented reality frozen section analysis in reducing the rates of positive surgical margins after robot-assisted radical prostatectomy.

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BACKGROUND: Currently, 13 Asian and European facilities deliver carbon ion radiotherapy (CIRT) for preclinical and clinical activity, and, to date, 55 clinical studies including CIRT for adult and paediatric solid neoplasms have been registered. The National Center for Oncological Hadrontherapy (CNAO) is the only Italian facility able to accelerate both protons and carbon ions for oncological treatment and research. METHODS: To summarise and critically evaluate state-of-the-art knowledge on the application of carbon ion radiotherapy in oncological settings, the authors conducted a literature search till December 2022 in the following electronic databases: PubMed, Web of Science, MEDLINE, Google Scholar, and Cochrane. The results of 68 studies are reported using a narrative approach, highlighting CNAO's clinical activity over the last 10 years of CIRT. RESULTS: The ballistic and radiobiological hallmarks of CIRT make it an effective option in several rare, radioresistant, and difficult-to-treat tumours. CNAO has made a significant contribution to the advancement of knowledge on CIRT delivery in selected tumour types. CONCLUSIONS: After an initial ramp-up period, CNAO has progressively honed its clinical, technical, and dosimetric skills. Growing engagement with national and international networks and research groups for complex cancers has led to increasingly targeted patient selection for CIRT and lowered barriers to facility access.

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The recent advancements in breast cancer precision medicine have highlighted the urgency for the precise and reproducible characterization of clinically actionable biomarkers. Despite numerous standardization efforts, biomarker testing by conventional methodologies is challenged by several issues such as high inter-observer variabilities, the spatial heterogeneity of biomarkers expression, and technological heterogeneity. In this respect, artificial intelligence-based digital pathology approaches are being increasingly recognized as promising methods for biomarker testing and subsequently improved clinical management. Here, we provide an overview on the most recent advances for artificial intelligence-assisted biomarkers testing in breast cancer, with a particular focus on tumor-infiltrating lymphocytes, programmed death-ligand 1, phosphatidylinositol-3 kinase catalytic alpha, and estrogen receptor 1. Challenges and solutions for this integrative analysis in pathology laboratories are also provided.

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D6 is an atypical chemokine receptor acting as a decoy and scavenger for inflammatory CC chemokines expressed in lymphatic endothelial cells. Here, we report that D6 is expressed in Kaposi sarcoma (KS), a tumor ontogenetically related to the lymphatic endothelium. Both in human tumors and in an experimental model, D6 expression levels were inversely correlated with tumor aggressiveness and increased infiltration of proangiogenic macrophages. Inhibition of monocyte recruitment reduced the growth of tumors, while adoptive transfer of wild-type, but not CCR2(-/-) macrophages, increased the growth rate of D6-competent neoplasms. In the KS model with the B-Raf V600E-activating mutation, inhibition of B-Raf or the downstream ERK pathway induced D6 expression; in progressing human KS tumors, the activation of ERK correlates with reduced levels of D6 expression. These results indicate that activation of the K-Ras-B-Raf-ERK pathway during KS progression downregulates D6 expression, which unleashes chemokine-mediated macrophage recruitment and their acquisition of an M2-like phenotype supporting angiogenesis and tumor growth. Combined targeting of CCR2 and the ERK pathway should be considered as a therapeutic option for patients with KS.

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We evaluated histologic results for surgical left maze with an high-intensity focused ultrasound (HIFU) energy source. Two patients came to our attention 6 and 48 months, respectively, after ablation concomitant to a valve procedure. Tissue specimens, obtained from the lesion site on the mitral isthmus and from the "box lesion" around the pulmonary veins were analyzed histologically. A complete transmural lesion was found in all specimens. Chronic lesions exhibited replacement of the muscular band with connective tissue. The atrial wall maintained normal thickness and vascularization. HIFU ablation represents an acceptable energy source to create transmural lesions on the beating human left atrium.

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BACKGROUND: Autologous haematopoietic stem cells transplantation (HSCT) has been shown to be effective in refractory Crohn's disease. AIM: We analysed the effects of HSCT on the immune response of patients treated for moderate-severe Crohn's disease, refractory or intolerant to multiple drugs. METHODS: Unselected peripheral blood stem cells were collected after mobilisation with cyclophosphamide (CTX) and G-CSF. The conditioning regimen included CTX and rabbit antithymocyte globulin. Blood samples for immunological analyses were collected at baseline, after mobilisation, and 3, 6 and 12 months after transplantation. Immunological analyses evaluated: (1) CD4(+)/CD25(high+)/FoxP3(+) regulatory T cells (T-regs); (2) Toll-like receptor 2-(TLR2) and TRL4-expressing monocytes (CD14(+) cells); (3) IL-12, IL-10, TNF-alpha-production by mitogen-stimulated CD14(+) cells and IFN-gamma production by CD4(+) T cells. Immunological results were compared with healthy donors and associated with clinical and endoscopic response during 12 months of follow-up. RESULTS: Overall, T-regs increased, whilst TLR4-expressing cells, as well as TNF-alpha and IL-10, all higher than healthy donors at baseline, significantly decreased after transplantation. Full responders at T(3) had higher T-regs and lower IFN-gamma and IL12. T-regs decreased and IL12 and TLR2 increased in the only relapsed patient. CONCLUSIONS: HSCT can induce and maintain clinical and endoscopic remission in refractory Crohn's disease, which is associated with immunomodulation.

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Vagal activity has protective effects in ischemic heart disease. We tested whether vagal stimulation (VS) could modulate the inflammatory reaction, a major determinant of cardiac injury after ischemia/reperfusion. Four groups of male rats underwent myocardial ischemia (30 minutes) and reperfusion (24 hours). One group underwent VS (40 minutes), 1 VS plus atrial pacing (VS + Pacing), and 1 VS plus nicotinic inhibition by mecamylamine (VS + MEC). After 24 hours, the area at risk, infarct size, inflammation parameters, and apoptosis were quantified. Infarct size was reduced in all VS-treated rats (controls, 53 ± 18%; VS, 6.5 ± 3%; VS + Pacing, 23 ± 6%; VS + MEC, 33 ± 9%; P < 0.005 vs. controls). The infarct size in the VS + MEC group was larger than that in VS-treated animals, despite similar heart rate, suggesting partial loss of protection. The number of macrophages, neutrophils, and apoptotic cells in the area at risk and the plasma cytokines levels were significantly reduced in all VS-treated animals. In conclusion, VS decreases infarct size and inflammatory markers during ischemia/reperfusion independent of the heart rate. The anti-inflammatory and antiapoptotic properties of the nicotinic pathway are the primary underlying mechanism. The vagally mediated modulation of inflammatory responses may prove valuable in the clinical management of acute coronary syndromes and of heart failure.

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BACKGROUND: Chronic hepatitis C is more aggressive during HIV infection. Available data about risk factors of liver fibrosis in HIV/HCV co-infected patients derive from studies based on a single liver biopsy. OBJECTIVES: To evaluate the risk factors of liver fibrosis progression (LFP) and to investigate the role of antiretroviral therapy (ARV) in HIV/HCV patients who underwent paired liver biopsy. PATIENTS AND METHODS: We retrospectively studied 58 patients followed at two Infectious Diseases Departments in Northern Italy during the period 1988-2005. All specimens were double-blinded and centrally examined by two pathologists. LFP was defined when an increase of at least one stage occurred in the second biopsy, according to the Ishak-Knodell classification. RESULTS: In a univariate analysis, serum levels of alanine aminotransferase (ALT) > 150 IU/L at the first biopsy (P = 0.02), and a > 20% decrease in CD4+ cell count between the two biopsies (P = 0.007), were significantly associated with LFP. In multivariate analysis, a > 20% decrease in CD4+ cell count remained independently associated to LFP (Odds Ratio, 3.99; 95% confidence interval, 1.25-12.76; P < 0.02). Analysis of life survival curves confirmed the correlation between CD4+ cell count and LFP. CONCLUSIONS: Our findings highlight that in HIV/HCV coinfected patients, an effective antiretroviral therapy that assures a good immune-virological profile contributes to reducing the risk of LFP.

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We retrospectively evaluated autopsy-proven invasive fungal infections (IFIs) in patients with AIDS who died between 1984 and 2002. IFIs were identified in 297 (18.2%) of 1,630 autopsies. Their prevalence significantly decreased over time (from 25.0% in 1984-1988 to 15% in 1998-2002; P = .004), mainly owing to a significant decrease in pneumocystosis (P = .017) and cryptococcosis (P = .003), whereas the prevalence of aspergillosis and histoplasmosis remained relatively stable and of candidiasis and zygomycosis tended to increase in the last years (P = .028 and P = .042, respectively). IFIs were suspected or confirmed during life in only 46.8% of the cases; this proportion did not vary significantly over time (P = .320). The infections contributed to the deaths of 103 patients (34.7%), and their global impact on mortality was 6.3%. Of fatal cases, 38 (36.9%) were characterized by missed antemortem diagnoses, 17 (45%) of which met Goldman criteria for class I errors. The epidemiology of IFIs in patients with AIDS is evolving and not completely mirrored by clinical diagnoses or current diagnostic methods. Our results confirm the valuable role of autopsy data, even with highly effective therapies and advanced technologies.

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BACKGROUND AND AIMS: The National UK IBD audit tool is an electronic database created to improve the quality and safety of care for IBD patients by auditing individual patient care, service resources and organisation against national standards. We used the National UK IBD audit tool to compare the organisation and process of IBD care between services in Oxford (UK) and Milan (Italy), as a pilot study to evaluate its application outside national boundaries. METHODS: Clinical and demographic data of patients with CD and UC, consecutively admitted during a 2month period, were collected and compared between the centres, to each other and to the UK IBD standards obtained by previous audit analyses performed in Oxford in 2006. RESULTS: 20 and 26 patients with UC were admitted in Oxford and Milan, as well as 21 and 20 patients with CD, respectively. Most admissions in Milan were planned admissions for moderately active treatment-refractory disease. No patient died. Oxford had a higher surgery rate. Endoscopy for UC consisted mainly of colonoscopy in Milan (92%) and flexible sigmoidoscopy in Oxford (64%). In CD, Oxford data revealed a higher use of immununomodulators and CT scan, compared with higher use of bowel ultrasound in Milan. CRP was the preferred biomarker of disease activity. The following areas did not reach the standards set for the 2006 UK IBD Audit: the lack in Milan of IBD specialist nurses and few dietitian visits, as well as little attention to heparin prophylaxis and abdominal radiography in UC. Both sites paid little attention to stool cultures and revealed a high rate of active smokers in CD and little attention to bone protection in steroids users. Since the 2006 audit in Oxford, improvements include IBD specialist nurse visits, dietitian visits, number of active smokers, stool samples, prophylactic heparin, bone protection and nutritional assessment. CONCLUSIONS: Consistent procedural differences between Oxford and Milan identified by audits of both UC and CD could be resolved by organisational change, with an improvement in the service. The UK IBD audit tool is an easy instrument to assess the processes and outcomes of care delivery in IBD and can be applied also outside UK.

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JC virus (JCV) is a human polyomavirus that causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease that mainly affects immunocompromised subjects. Since its discovery, PML has been considered a rapidly progressing fatal disease; however, amino acid substitutions in the capsid viral protein have recently been tentatively associated with changes in PML clinical course. In order to provide more insight to PML pathogenesis and identify potential prognostic markers, seven cerebrospinal fluid (CSF) samples and four brain autopsy samples were collected from patients afflicted with PML with different clinical courses (fast- and slow-progressing), and the JCV VP1 coding region was amplified, cloned, and sequenced. In addition, urine samples were collected and analyzed from nine patients with PML or other neurological diseases (ONDs) as a control group. Sequencing analysis of the genomic region encoding the VP1 outer loops revealed polymorphic residues restricted to four positions (74, 75, 117, and 128) in patients with slow PML progression, whereas no significant mutation was found in JCV isolated from urine. Collectively, these data show that JCV VP1 loop mutations are associated with a favorable prognosis for PML. It is therefore possible that slower progression of PML may be related to the emergence of a less virulent JCV strain with a lower replication rate.

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Cerebral aspergillosis is a mostly lethal infection of the central nervous system. Former results identified low cerebral complement levels as one cause for insufficient immune reaction. Therefore we studied cerebral complement expression after fungal invasion and investigated putative mechanisms of Aspergillus spp to cope with the complement-induced selection pressure. Brain tissue derived from patients with cerebral aspergillosis or non-infected individuals was analyzed immunohistochemically for complement synthesis. Correlations between expression levels were determined statistically. Increased complement synthesis, a prerequisite for strengthened antifungal potency, was visible in resident astrocytes, neurons, oligodendrocytes as well as in infiltrating macrophages after fungal infection. Surprisingly, microglia, although regarded as major immune cells, only marginally participated in synthesis of most complement proteins. Several evasion mechanisms were found that help the fungus to establish a cerebral infection even in the presence of complement: Fungal hyphae limit the surface deposition of C3 and thus interfere with complement-dependent phagocytosis. Furthermore, the "sealing off" in brain abscesses not only inhibits fungal spreading but also forms protection shields against complement attack. Complement indeed seems to represent an important selection pressure and evokes the development of fungal evasion mechanisms. Counteractions for these evasion processes might represent interesting therapeutic approaches.

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The chemokine system includes at least three "silent" receptors, DARC, D6 and CCX CKR, with distinct specificity and tissue distribution. D6 binds most inflammatory, but not homeostatic, CC chemokines and shuttles in a ligand-independent way from the plasma membrane to endocytic compartments where chemokines are targeted to degradation. In vitro and in vivo evidence, including results with gene-targeted mice, is consistent with the view that D6 acts as a decoy and scavenger for inflammatory CC chemokines. Thus, D6 has unique functional and structural features, which make it ideally adapted to act as a chemokine decoy and scavenger receptor, strategically located on lymphatic endothelium to dampen inflammation in tissues and draining lymph nodes.

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To understand the modulation of dendritic cell (DC) function by IL-10, gene expression profiling was performed by using Affymetrix technology (Santa Clara, CA) in human monocyte-derived DC treated with IL-10, alone or in combination with LPS. The modulation of selected genes was validated by real-time PCR, Northern blot, and protein production. IL-10 regulated in DC the expression of a limited number of genes, including IL-7, the receptors for transferrin and vitamin D(3), structural matrix proteins, and signal transduction elements. The combined treatment with LPS plus IL-10 modulated a number of genes comparable to LPS alone, but the expression profiles were distinct. As expected, IL-10 suppressed the expression of several LPS-inducible proinflammatory molecules. Among genes uniquely modulated by the concomitant treatment with LPS plus IL-10, phosphatidylinositol 3-kinase gamma was down-regulated while the suppressor of cytokine signaling 3, signaling lymphocytic activation molecule, regulator of G protein signaling 16, and the chemokine, CXC chemokine ligand (CXCL) 13, were up-regulated. Overall, four distinct transcriptional programs were identified, related to: 1) control of immunity and inflammation; 2) tuning of cytokine receptor and G protein-coupled receptor signaling; 3) remodeling of extracellular matrix; and 4) B cell function and lymphoid tissue neogenesis. Among the latter genes, we further demonstrate that IL-10 synergizes with TLR ligands for the production of functionally active B cell-attracting chemokine, CXCL13, in both myeloid and plasmacytoid DC. This novel finding reveals that IL-10 sustains humoral immunity by inducing the production in APCs of the chemokine, CXCL13, which amplifies B cell recruitment and promotes lymphoid tissue neogenesis.

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The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in extracellular matrix degradation and cell migration in the central nervous system (CNS). To investigate the role of the uPA/uPAR system in the pathophysiology of acquired immunodeficiency syndrome dementia complex (ADC), we measured soluble uPAR (suPAR) levels in cerebrospinal fluid (CSF) and plasma from human immunodeficiency virus (HIV)-1-infected patients and controls. CSF suPAR levels were significantly higher in HIV-1-infected patients than in controls and in patients with ADC or opportunistic CNS infections (CNS-OIs) than in neurologically asymptomatic patients, irrespective of HIV-1 disease stage. The highest levels of suPAR were found in patients with ADC, and among those with CNS-OIs in patients with cytomegalovirus encephalitis or cryptococcosis. Plasma suPAR levels were higher in HIV-1-infected patients than in controls and increased with HIV-1 disease stage regardless of the presence of CNS disease. In patients with ADC or CNS-OIs, CSF suPAR levels correlated with CSF HIV-1 RNA, but not with plasma suPAR concentrations. Highly active antiretroviral therapy was associated with a significant and parallel decrease of both CSF suPAR and HIV-1 RNA. In brain tissue from patients with HIV-1 encephalitis, uPAR was highly expressed by microglial and multinucleated giant cells staining positively for HIV-1. The overexpression of uPAR in the CNS of patients with ADC suggests that the uPA/uPAR system may contribute to the tissue injury and neuronal damage in this disease.

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Anderson-Fabry disease (AFd) is a rare X-linked lisosomal storage disorder of glycosphingolipid (GL) metabolism, caused by a deficiency of the activity of alpha-galactosidase A (alpha-gal A). The progressive accumulation of GL in tissues results in the clinical manifestations of the disease, that are more evident in hemizygous males, and include characteristic skin lesions (angiokeratomas), neurological symptoms (acroparesthesia), ocular features (cornea verticillata), cardiac involvement (left ventricular enlargement, conduction abnormalities), cerebrovascular manifestations (thromboses, hemorrhage, etc.), and kidney involvement with progression to end-stage renal failure (ESRF). ESRF is a common manifestation in hemizygous males (3rd-5th decade) and death occurs around the 5th decade of life because of severe cardiac and/or cerebrovascular complications. Heterozygous females have an attenuated form of this systemic disease. In the kidney, accumulation of GL occurs in the endothelial cells of every vessel, in the epithelial cells of every tubular segment, and in all kinds of glomerular cells. The broad spectrum of renal lesions is a pathophysiological continuum with progressive impairment in the renal function related to continuous intracellular deposition of GL. Electron microscopic study of renal biopsies shows typical osmiophilic inclusion bodies in the cytoplasm of all kind of renal cells, characterized by concentric lamellation of clear and dark layers (35-50 A of periodicity). ESRF is treated by dialysis and kidney transplantation: neither treatment modifies the progression of the cardiovascular and cerebrovascular lesions due to progressive GL deposition. The outcome of kidney transplantation seems to be similar to that found in other non-diabetic patients, but the survival rate on dialysis is lower than in patients with other causes of ESRF. Nowadays, treatment with enzyme replacement infusion with purified alpha-Gal A, produced by a genetically engineered human cell line or Chinese hamster ovocytes, seems to be effective and safe.

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