Enterprise Implementation of Digital Pathology: Feasibility, Challenges, and Opportunities.

Digital pathology is becoming technically possible to implement for routine pathology work. At our institution, we have been using digital pathology for second opinion intraoperative consultations for over 10 years. Herein, we describe our experience in converting to a digital pathology platform for primary pathology diagnosis. We implemented an incremental rollout for digital pathology on subspecialty benches, beginning with cases that contained small amounts of tissue (biopsy specimens). We successfully scanned over 40,000 slides through our digital pathology system. Several lessons (both challenges and opportunities) were learned through this implementation. A successful conversion to digital pathology requires pre-imaging adjustments, integrated software and post-imaging evaluations.

Antimicrobial photodynamic therapy as an alternative to systemic antibiotics: results from a double-blind, randomized, placebo-controlled, clinical study on type 2 diabetics.

AIM: This double-blind, placebo-controlled clinical study compared multiple applications of the antimicrobial photodynamic therapy (aPDT) treatment protocol, to systemic doxycycline as adjuvant to scaling and root planing (SRP) on type 2 diabetic patients on clinical, systemic and immune-inflammatory outcomes. MATERIALS AND METHODS: Thirty patients with Hba1c >7% were allocated in two groups, SRP + Doxy (n = 15) using systemic doxycycline 100 mg/day (14 days) and SRP + aPDT (n = 15) with multiple applications (0, 3, 7 and 14 days). Primary outcome was glycated haemoglobin levels (HbA1c). Clinical parameters: plaque score (PS), bleeding on probe, probing depth, suppuration, gingival recession, and clinical attachment level, percentage of pockets with desired clinical endpoint were measured at baseline and 3 months after therapy. Cytokine profile was assessed at 0, 1 and 3 month to measure IL1-ß, TNF-α and TGF-ß on gingival crevicular fluid. RESULTS: No significant difference was detected on HbA1c, between treatments. The SRP + aPDT group showed advantage on reducing moderate pockets in single-rooted teeth at 3 months. SRP + aPDT presented better results at 3 months on IL1-ß levels. There were no significant differences between TNF-α and TGF-ß. CONCLUSIONS: Both treatments improved clinical and systemic outcomes (Hba1c). SRP + aPDT performed better in moderate probing pocket depth on single-rooted teeth, reduced favourably inflammation in short term, and may be an alternative to systemic antibiotics. (Clinicaltrials.org ID NCT01595594).

Safety and efficacy of low intensity shockwave (LISW) treatment in patients with erectile dysfunction.

UNLABELLED: The primary goal in the management strategy of a patient with ED would be to determine its etiology and cure it when possible, and not just to treat the symptoms alone. One of the new therapeutic strategies is the use of low intensity extracorporeal shockwave (LISW) therapy. The mechanism of shockwave therapy is not completely clear. It is suggested that LISW induces neovascularization and improvement of cavernosal arterial flow which can lead to an improvement of erectile function by releasing NO, VEGF and PCNA. MATERIALS AND METHODS: 31 patients between February and June 2013 with mild to severe ED and non-Phosphodiesterase 5 inhibitors responders were enrolled. Patients underwent four weekly treatment sessions. During each session 3600 shocks at 0.09mJ/ mm2 were given, 900 shocks at each anatomical area (right and left corpus cavernosum, right and left crus). Improvement of the erectile function was evaluated using the International Index of Erectile Function (IIEF-EF), the Sexual Encounter Profile (SEP) diaries (SEP-Questions 2 and 3) and Global Assessment Questions (GAQ-Q1 and GAQ-Q2). RESULTS: At 3-month follow-up IIEF-EF scores improved from 16.54±6.35 at baseline to 21.03±6.38. Patients answering 'yes' to the SEP-Q2 elevated from 61% to 89% and from 32% to 62% in the SEP-Q3. A statistically significant improvement was reported to the Global Assessment Questions (GAQ-Q1 and GAQ-Q2). CONCLUSION: In conclusion, we can affirm that LISW is a confirmed therapeutic approach to erectile dysfunction that definitely needs more long-term trials to be clarified and further verified.

Reranking docking poses using molecular simulations and approximate free energy methods.

Fast and accurate identification of active compounds is essential for effective use of virtual screening workflows. Here, we have compared the ligand-ranking efficiency of the linear interaction energy (LIE) method against standard docking approaches. Using a trypsin set of 1549 compounds, we performed 12,250 molecular dynamics simulations. The LIE method proved effective but did not yield results significantly better than those obtained with docking codes. The entire database of simulations is released.

A Randomized comparative clinical study of two surgical procedures to improve root coverage with the acellular dermal matrix graft.

AIM: This randomized, controlled, clinical study compared two surgical techniques for root coverage with the acellular dermal matrix graft (ADMG) to evaluate which procedure could provide better root coverage and greater amounts of keratinized tissue. MATERIALS AND METHODS: Fifteen pairs of bilateral Miller Class I or II gingival recessions were treated and assigned randomly to the test group, and the contra-lateral recessions were assigned to the control group. The ADMG was used in both groups. In the control group, the graft and flap were positioned at the level of the cemento-enamel junction (CEJ), and in the test group, the graft was positioned 1 mm apical to the CEJ and the flap 1 mm coronal to the CEJ. The clinical parameters were taken before the surgeries and after 6 months. The gingival recession area, a new parameter, was measured in standardized photographs through a special device and software. RESULTS: There were statistically significant differences favouring the proposed technique for all parameters except for the amount of keratinized tissue at 6 months. CONCLUSIONS: The proposed test technique is more suitable for root coverage procedures with ADMG, and the new parameter evaluated appears valuable for root coverage analysis. (Clinicaltrials.gov Identifier: NCT01175720).

Testosterone deficiency causes penile fibrosis and organic erectile dysfunction in aging men. Evaluating association among Age, TDS and ED.

INTRODUCTION: We studied the possible correlation between age, testosterone deficiency, cavernosal fibrosis and erectile dysfunction (ED). METHODS: 47 patients with ED were enrolled between September 2010 and October 2011. IIEF-EF score, NPTR test using the Rigiscan method, total and free testosterone levels, and cavernosum biopsy were carried out on all patients. Patients aged 65 or over were defined as Old Age (OA) while patients under 65 were defined Young age (YA). The strength of the relationships found was estimated by Odds Ratio. RESULTS: 74% of patients with values of over 52% collagen fibers in the corpora cavernosa were found to have organic ED. A significant difference was found in age, percentage of collagen fibers, testosterone levels between patients with Positive Rigiscan (PR) and Negative Rigiscan (NR). Hypotestosteronaemia increased the risk of ED with PR (OR: 21.4, 95% CI: 20.2-22.6) and in both young age patients (OR: 4.3, 95% CI: 2.4-6.2) and old age patients (OR: 15.5, 95% CI: 13.4-17.6). Moreover cavernosal fibrosis increased the risk of ED with PR in both young age patients (OR: 8.2, 95% CI: 6.4-10.0 and old age patients (OR: 24.6, 95% CI: 20.8-28.4). CONCLUSIONS: This study demonstrates a strong association among age, testosterone deficiency, cavernosal fibrosis and ED with PR. Age, testosterone deficiency and cavernosal fibrosis are potentially correctable factors of cavernosal fibrosis and organic ED. Further, prospective studies are needed to evaluate if testosterone treatment, alone or in association with PDE5 inhibitors, may lower the risk of cavernosal fibrosis or decrease the severity the fibrosis in ED patients.

Management of systemic prostate cancer: current algorithm from castration sensitive to castration resistant setting.

In recent years, the advanced knowledge of clinical, biological and molecular features of prostate cancer have led to the introduction of new drugs and have allowed the relocation of old drugs in different settings. In this way, the new concepts of systemic disease arise: high risk or high volume vs. low risk and low volume disease castration sensitive prostate cancer (CSPC), diversifying the use of previously approved drugs (CRPC) and opening new scenarios for sequence therapy. The aim of this review is to integrate new developments into the medical management of systemic prostate cancer.

Zoledronic acid enhances Vδ2 T-lymphocyte antitumor response to human glioma cell lines.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain tumor in humans, responds modestly to treatment: most patients survive less than one year after diagnosis, despite both classical and innovative treatment approaches. A recent paper focused on γδ T-cell response in GBM patients, suggesting the application of an immunomodulating strategy based on γδ T-cells which is already in clinical trials for other tumors. Human Vγ2 T-cells recognize changes in the mevalonate metabolic pathway of transformed cells by activating cytotoxic response, and by cytokine and chemokine release. Interestingly, this activation may also be induced in vivo by drugs, such as zoledronic acid, that induce the accumulation of Vγ2 T-cell ligand Isopentenyl-pyrophosphate by blocking the farnesyl pyrophosphate synthase enzyme. The aim of our work is to confirm whether bisphosphonate treatment would make glioma cell lines more susceptible to lysis by in vitro expanded γδ T-cells, improving their antitumor activity. We expanded in vitro human Vγ2 T-cells by phosphoantigen stimulation and tested their activity against glioma cell lines. Co-culture with glioma cells induced Vγ2 T-cell differentiation in effector/memory cells, killing glioma cells by the release of perforin. Interestingly, glioma cells were directly affected by zoledronic acid; moreover, treatment increased their activating ability on Vγ2 T-cells, inducing an effective antitumor cytotoxic response. Taken together, our results show that aminobisphosphonate drugs may play a dual role against GBM, by directly affecting tumor cells, and by enhancing the antitumor response of Vγ2 T-cells. Our results confirm the practicability of this approach as a new immunotherapeutic strategy for GBM treatment.

The plant diterpene epoxysiderol targets Hsp70 in cancer cells, affecting its ATPase activity and reducing its translocation to plasma membrane.

The ATP-dependent molecular chaperone Hsp70 is over-expressed in cancer cells where it plays pivotal roles in stabilization of onco-proteins, promoting cell proliferation and protecting cells from apoptosis and necrosis. Moreover, a relationship between the ability of cancer cells to migrate and the abundance of membrane-associated Hsp70 was shown. However, although Hsp70 is a promising target for cancer therapy, there is a still unsatisfied requirement of inhibitors possibly blocking its cancer-associated activities. Moving from the evidence that the plant diterpene oridonin efficiently targets Hsp70 1A in cancer cells, we set up a small kaurane diterpenoids collection and subjected it to a Surface Plasmon Resonance-screening, to identify new putative inhibitors of this chaperone. The results obtained suggested epoxysiderol as an effective Hsp70 1A interactor; therefore, using a combination of bioanalytical, biochemical and bioinformatics approaches, this compound was shown to bind the nucleotide-binding-domain of the chaperone, thus affecting its ATPase activity. The interaction between epoxysiderol and Hsp70 1A was also demonstrated to actually occur inside cancer cells, significantly reduced the translocation of the chaperone to the cell membrane, thus suggesting a possible role of epoxysiderol as an anti-metastasis agent.

Transcritpional effects of S100B on neuroblastoma cells: perturbation of cholesterol homeostasis and interference on the cell cycle.

S100B is a Ca2+ binding protein mainly secreted by astrocytes in the vertebrate brain that is considered a multifunctional cytokine and/or a damage-associated molecular pattern (DAMP) protein and a marker of brain injury and neurodegeneration when measured in different body fluids. It has been widely shown that this protein can exert diverse effects in neural cultures depending on its concentration, having detrimental effects at micromolar concentrations. The molecular mechanisms underlying this effect are still largely unknown. This study attempts to delineate the genome-wide gene expression analysis of the events associated with exposure to micromolar concentration of S100B in a human neuroblastoma cell line. In this experimental condition cells undergo a severe perturbation of lipid homeostasis along with cell cycle arrest. These mechanisms might reasonably mediate some aspects of the S100B-related detrimental effects of S100B, although obvious differences between mature neurons and neuroblastoma cells have to be considered.

Advanced/metastatic bladder cancer: current status and future directions.

In 2015 bladder cancer was the fourth most frequent malignancy and the eighth cause of death for cancer. At diagnosis, about 30% of bladder cancer (BC) patients present a muscle-invasive bladder cancer (MIBC) and 5% a metastatic bladder carcinoma (MBC). For fit MBC patients, combination chemotherapy (CC) is the standard of care for first-line treatment. CC includes both the treatment with methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) either the classical or the dose-dense MVAC regimen, and the doublet therapy with cisplatin and gemcitabine (CG). Median progression free survival (PFS) was 7 months and median overall survival (OS) was 15 months. The present review provides an update on the management of MBC, with focus on target therapies, immune checkpoint inhibition, looking for prognostic and predictive factors.

Penile cancer: prognostic and predictive factors in clinical decision-making.

Penile cancer (PC) is a typical tumor of non-industrialized countries. The incidence is 20-30 times higher in Africa and South America, considering the elevated prevalence of sexually transmitted diseases. Histologically, PC includes squamous cell carcinoma (SCPC), the most frequent, and nonsquamous carcinoma (NSCPC). Early diagnosis is the goal, whereas later diagnosis relates to poor functional outcomes and worse prognosis. The 5-year survival rate is 85% for patients with histologically regional negative lymph nodes, compared to 29%-40% for those with histologically regional positive lymph nodes. To date no new drugs are approved, and there are few new data about molecular mechanisms underlying tumorigenesis. The SCPC remains a rare tumor and the current therapeutic algorithm is based principally on retrospective analysis and less on prospective trials. In this review article, biomarkers of prognosis and efficacy of current treatments are summarized with a focus on those that have the potential to affect treatment decision-making in SCPC.

Protection of mice against syngeneic C1300 neuroblastoma challenge by immunization with membranes of C1300 neuroblastoma cells.

Male A/J mice 2-3 months old were inoculated sc with membranes from syngeneic C1300 neuroblastoma cells (clone NB6R) in complete Freund's adjuvant. Significant immunoprophylaxis was noted in the sensitized mice upon sc challenge with viable NB6R cells. During the experiment (60 days from viable cell challenge), each control mouse developed a palpable tumor and died within 50 days. Complete protection was obtained with a program of 4 inoculations of NB6R cell membranes. Each mouse given only 1 inoculation of NB6R cell membranes developed a palpable tumor, but afer 60 days only 1 mouse in 7 had died, which indicated a significant degree of protection. With in vitro tests of lymphocyte proliferation, rosette formation, and complement fixation, it was shown that these mice had mounted both cellular and humoral immune response against the tumor cells.

Interferon gamma up-regulates alpha 2 macroglobulin expression in human astrocytoma cells.

An established human astrocytoma cell line (T67) was shown to constitutively produce the proteinase inhibitor alpha 2 macroglobulin (alpha 2M). Interferon gamma (IFN gamma), a potent immunoregulatory lymphokine, was able to increase the synthesis of alpha 2M by these cells, as measured by ELISA on cell supernatants. The alpha 2M induction was also observed in other human glioma cell lines (T70 and ADF) and in human fetal astrocyte cultures following IFN gamma treatment. In T67 cells this effect was dose-dependent and the maximum (2.7-fold increase) was obtained with 2000 U/ml of IFN gamma. A corresponding enhanced alpha 2M mRNA accumulation was demonstrated by PCR and Northern blot techniques. Our results suggest an important role of alpha 2M during inflammatory and immune processes in the CNS. An increased release of alpha 2M following IFN gamma stimulation may allow the removal of the bulk of proteases released at the site of inflammation, strengthening at the same time the antigen presentation processes.

Modulation of fas-ligand (Fas-L) on human microglial cells: an in vitro study.

The expression of Fas-Ligand (Fas-L) on microglia could be relevant in multiple sclerosis immunopathology. The present study was performed to evaluate in vitro the expression of Fas-L in human microglial cells both unstimulated and after stimulation with IFN-gamma, beta-IFN-1b and beta-IFN-1b+IFN-gamma. Cells were stimulated for 6,12, 24 and 48 h. Surface Fas-L was evaluated by flow cytometry, total Fas-L by Western blot, whereas mRNA for Fas-L was measured by RT-PCR. We also evaluated the capacity of microglial cells to induce, in vitro, apoptosis on Fas-positive T leukemia Jurkat cells. Our results showed a constitutive expression of Fas-L on microglia. IFN-gamma downregulated the expression of the molecule, while beta-IFN-1b and beta-IFN-1b+IFN- gamma did not. The amount of surface Fas-L was related to the ability of microglial cells to induce apoptosis in Fas-positive target cells, which was partly inhibited by blockade of the Fas-Fas-L pathway.

Modulation of the alpha 2 macroglobulin receptor/low density lipoprotein receptor related protein by interferon-gamma in human astroglial cells.

Alpha 2 macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 Mr/LRP) is a multi-functional cell surface receptor that has been implicated in important processes, such as atherogenesis, cellular migration, immune response and degenerative diseases. Its expression increases in human brain during Alzheimer's disease, tissue injury and neoplastic transformation. In the present paper we studied the regulation of alpha 2 Mr expression by interferon-gamma (IFN gamma) in human astrocytoma cell lines and in fetal astrocytes. Western blots demonstrated an increase of the alpha 2 Mr expression after 24 h of IFN gamma treatment. This effect paralleled the up-regulation of alpha 2 Mr mRNA, as detected by PCR. By prolonging incubation with IFN gamma, we observed a decrement of alpha 2 Mr in IFN gamma treated cells, both by western blot and cytometric analysis. Since in the same cells IFN gamma also up-regulates alpha 2 macroglobulin, this effect may be due to an augmented degradation of the receptor during its recycling.

The effect of nitric oxide on cytokine-induced release of PGE2 by human cultured astroglial cells.

1. The role of the L-arginine-nitric oxide (NO) pathway on the formation of prostaglandin E2 (PGE2) by human cultured astroglial cells incubated with interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) was investigated. 2. Incubation of T 67 astroglial cell line with IL-beta (10 ng ml(-1)) and TNF-alpha (500 u ml(-1)) produced a significant (P<0.05) increase of both nitrite (the breakdown product of NO), cyclic GMP and PGE2 levels in cell supernatants. N omega-nitro-L-arginine methyl ester (L-NAME; 20-300 microM), an inhibitor of NO synthase (NOS), inhibited the increase of cyclic GMP and nitrite levels found in supernatants of cytokine-treated astroglial cells and reduced the release of PGE2. The latter effect showed that the enhanced arachidonic acid (AA) metabolism subsequent to stimulation of astroglial cells with IL-1beta and TNF-alpha was, at least in part, induced by NO. This occurred also when sodium nitroprusside (SNP; 120 microM), an NO donor, was incubated with astroglial cells, an effect antagonized by oxyhaemoglobin (OxyHb; 10 microM). 3. The inhibition elicited by L-NAME on PGE2-release by cytokine-treated astroglial cells was reversed by adding AA (40 microM), showing that the effect of NO on cytokine-dependent PGE2 release occurred at the cyclo-oxygenase (COX) level. Furthermore, the release of PGE2 in cytokine-treated astroglial cells was inhibited by indomethacin (10 microM), a COX inhibitor as well as by preincubating cells with dexamethasone (20 microM), an inhibitor of inducible enzymes, showing that the inducible isoform of COX (COX-2) was involved. 4. On the other hand, pretreating astroglial cells with methylene blue (MB; 10 microM), an inhibitor of NO biological activity acting at the guanylate cyclase level, failed to affect PGE2 release in cytokine-treated astroglial cells, leading to the conclusion that cyclic GMP changes related to NO formation are not involved in the generation of AA metabolites. 5. The present experiments demonstrated that the release of PGE2 by astroglial cells pretreated with IL-1beta and TNF-alpha is due to enhanced COX-2 activity via activation of the L-arginine-NO pathway, and this may be relevant to the understanding of the pathophysiological mechanisms underlying neuroimmune disorders.

Mycobacterium tuberculosis enhances iNOS mRNA expression and HIV replication in human astrocytoma cells.

The aim of this study was to investigate whether Mycobacterium tuberculosis (MTB) infection affects human immunodeficiency virus (HIV) replication in T67 human astrocytoma cells and whether HIV and MTB infections induce iNOS mRNA expression in T67 cells. T67 cells were susceptible to both HIVLai and MTB infections, and MTB was able to increase HIV infection in T67 cells, as demonstrated by a marked increase in p24 release. Furthermore, both HIV and MTB infections strongly induced inducible nitric oxide synthase (iNOS) mRNA expression, as verified by RT-PCR. These findings suggest that HIV and MTB-induced iNOS expression of astroglial cells may be involved in the neuronal damage associated with HIV infection, particularly in the presence of opportunistic infections such as tuberculosis.

Basic fibroblast growth factor modulates in vitro differentiation of human fetal microglia.

The effects of basic fibroblast growth factor (bFGF) on differentiation of human fetal microglial cells were investigated. Human ramified microglial cells treated with human recombinant bFGF underwent a morphological change which resulted in a round-shape phenotype. bFGF was also able to induce a dose- and time-dependent increase in cell proliferation and enhanced phagocytic and non-specific esterase activity. These results indicate that ramified microglia, when properly stimulated, are regulated in their physiological and proliferative activities and are transformed into amoeboid forms. Growth factors, such as bFGF, are likely to play a key role in microglial transformation in both normal developing brain and in central nervous system injury.

Differential expression of fibroblast growth factor receptors by human neurones, astrocytes and microglia.

Expression of fibroblast-growth factor receptors (FGFRs) was studied in human fetal neurones, astrocytes and microglia in culture. Northern blot analysis showed that neurones and microglia expressed the mRNAs for FGFR-1, FGFR-2, FGFR-3, FGFR-4 at different levels, whereas astrocytes expressed only FGFR-1 and FGFR-4 mRNAs. Immunocytochemical localization of FGFR-1 revealed that this receptor was predominantly localized on the axon hillock membrane in neurones, and was associated with the plasma membrane of ameboid, activated microglia and of glial-fibrillar acidic protein positive astrocytes. The expression of various members of the FGFR family in all the cell types investigated implicates FGFs in human brain development and functions.