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Surveillance of carbapenem-resistant Enterobacterales (CRE) carriers is the first measure of hospital infection control. Screening of CRE carriage can be assessed through culture and molecular techniques, each with specific properties of turnaround-times, sensitivity and specificity.This was a prospective study in a 1200-bed university hospital in Genoa, Italy, where CRE screening is performed analysing cultures from rectal swabs. Our 18-months intervention was to extend the incubation time of the corresponding plates from 48 to 288 h, after reporting negative tests, to evaluate the possible impact on the cultures.A total of 362 patients giving 19,278 swabs and corresponding plates were included. After baseline incubation, plate positivity was 3%, while after the overall lengthened times it was 3.7%. Extended incubation was associated with change in the relative frequency of the most represented species. In particular, we observed reduced presence of total and resistant Klebsiella pneumoniae strains (P0.001) and increased presence of Enterobacter cloacae complex, total and sensitive (P0.001). By extending incubation time, a reduced frequency of overall Enterobacterales strains with high resistance to ertapenem (MIC ≥4 mg/L) was also found (P=0.005), particularly that of K. pneumoniae (P0.001), while the presence of E. cloacae complex increased among organisms with low resistance levels to ertapenem (P0.001).Extending the incubation time of the cultures increased the number of CREs grown, and expanded the bacterial scenario of rectal colonization through the recovery of poorly resistant strains and otherwise undetected species.

Understanding brain mechanisms is a currently open challenge. There are several methods available to characterize the functional aspects. However, the information that one can retrieve about the organization of the wiring between the cells across the brain that could support the network organization and the circuit mechanism remains limited. This appears to be a general problem, even when the cell anatomy is well described and potentially accessible at scales up to the synaptic level across the whole brain. Typically, such high-resolution analyses focus on the reconstruction of a limited portion of the brain or of a specific circuit, while missing the general organization principles of the neuronal networks within the brain, the network components, and internetwork connection schemes. To extend the investigation on these aspects, we present a theoretical and computational framework using modularity analysis to identify from connectome datasets the brain-wide circuit organization and the underlying cell modules based on synaptic connectivity patterns. We applied this approach on an Electron Microscopy (EM) dataset with about 25-thousand annotated cell reconstructions that incorporate synaptic information from the hemibrain of adultDrosophila melanogaster. The analysis provided a representation of the connectome organized in a hierarchical structure and characterized by several cell modules with precise and topographically organized connection patterns. We then tested this approach on a “synthetic” connectome, one obtained from a light microscopy dataset of more than three thousand neuronal skeletonizations from the zebrafish larvae brain, where a cell-to-cell proximity rule was used to assign synaptic contacts. Despite this approximation, the developed approach revealed reported connectivity patterns and functional connection schemes. In conclusion, we show that our framework is scalable and amenable for different types of datasets, independently from the particular anatomical reconstruction method adopted, to reveal the neuronal architectures supporting the brain mechanisms.

Background Bone marrow-derived macrophages account for substantial GBM tumor volume and contribute to the local inflammatory tumor microenvironment, disease progression and treatment response. Material and Methods We have developed a genetically modified, autologous hematopoietic stem cell-based platform designed to deliver Interferon-alpha (IFNa), thanks to a transcriptional and post-transcriptional control mechanism mediated by miRNA target sequences, specifically into the tumor microenvironment via Tie-2 expressing monocytes (Temferon). Results As of Feb 2022, 3 escalating doses of Temferon (from 0.5 to 2.0x106/kg) were tested across 15 patients with newly diagnosed, unmethylated MGMT glioblastoma (GBM) assigned to 5 cohorts. The duration of follow-up from surgery is 6 - 28 mo (2 - 25 mo after Temferon). To date, no dose limiting toxicities have been identified. As expected, one month after the administration of the highest tested dose, the hematopoietic system of Temferon-treated patients was composed of up to 30% of CD14+ genetically modified cells, as determined by the presence of vector genomes in the DNA in peripheral blood and bone marrow cells. Temferon-derived progeny persisted, albeit at lower levels, up to 18 months (longest time of analysis). Despite the substantial proportion of engineered cells, very low median concentrations of IFNα were detected in the plasma (D+30, 5.9; D+90, 8.8pg/mL) and in the CSF (D+30, 1.5; D+90, 2.4pg/mL), indicating tight regulation of transgene expression. SAEs were mostly attributed to conditioning chemotherapy (e.g. infections) or disease progression (e.g. seizures). 1 SUSAR (persistent GGT elevation) has occurred. Median OS is 15 mo from surgery (range 6.1-28.4 mo; 10.8 mo post Temferon). Of the 15 pts treated so far, 4 pts belonging to low dose cohorts underwent 2nd surgery. Homing of transduced cells from BM to the tumor site was demonstrated by the presence of gene-marked cells in the specimens collected from 3 of the 4 analyzed pts. Single-cell RNA seq performed on CD45+ cells purified from the TME of Temferon-treated pts compared to recurrent tumors belonging to GBM pts treated as per the current standard of care, highlighted a Temferon signature defined by the induction of markers of IFNa responses and macrophage repolarization. Potential long-term benefit with Temferon was identified in a patient from cohort 3, who had disease progression at D+120 with two distant enhancing lesions, and increased tumor necrosis. One year following Temferon, with no 2nd line therapy added, there was approximately 40% reduction in enhancing tumor volume compared to D+180 with a stable clinical and imaging picture thereafter. Conclusion The results provide initial evidence of Temferon’s potential to modulate the TME of GBM patients, and anecdotal evidence for long lasting effects of Temferon in prevention of disease progression.

Male infertility, a condition that has during the last decade raised significant concern, is a diagnostically demanding and socially sensitive topic. The number of unsolved issues on infertility etiology, especially potential environmental causes, in couples demonstrates the need for further investigations into infertility biomarkers. Semen parameters are often insufficient for reliable profiling of male infertility. Thus, this study aims to evaluate for the first time seminal plasma N-glycosylation as a biomarker of environmental exposure in semen samples from 82 normozoospermic men and 84 men with abnormal semen parameters and compare it with genome damage measured by DNA fragmentation. We obtained information about chronic exposure to environmental factors from the self-reported questionnaire, and determined sperm DNA fragmentation by sperm chromatin dispersion, while N-glycans were characterized with liquid chromatography-mass spectrometry (LC-MS). Based on previously published results, ten N-glycans were selected. Results show that the selected seminal plasma N-glycans were significantly associated with smoking, exposure to pesticides, air pollution, agents emitted during photocopying, alcohol consumption, and obesity. Some N-glycans showed a simultaneous association with DNA fragmentation, semen parameters, and environmental stressors. These subgroups of N- glycans are new potential candidates for biomonitoring of exposure to different environmental factors in men with semen abnormalities.

STUDY QUESTION Is it safe to perform controlled ovarian stimulation (COS) for fertility preservation before starting anticancer therapies or ART after treatments in young breast cancer patients? SUMMARY ANSWER Performing COS before, or ART following anticancer treatment in young women with breast cancer does not seem to be associated with detrimental prognostic effect in terms of breast cancer recurrence, mortality or event-free survival (EFS). WHAT IS KNOWN ALREADY COS for oocyte/embryo cryopreservation before starting chemotherapy is standard of care for young women with breast cancer wishing to preserve fertility. However, some oncologists remain concerned on the safety of COS, particularly in patients with hormone-sensitive tumors, even when associated with aromatase inhibitors. Moreover, limited evidence exists on the safety of ART in breast cancer survivors for achieving pregnancy after the completion of anticancer treatments. STUDY DESIGN, SIZE, DURATION The present systematic review and meta-analysis was carried out by three blinded investigators using the keywords ‘breast cancer’ and ‘fertility preservation’; keywords were combined with Boolean operators. Eligible studies were identified by a systematic literature search of Medline, Web of Science, Embase and Cochrane library with no language or date restriction up to 30 June 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS To be included in this meta-analysis, eligible studies had to be case-control or cohort studies comparing survival outcomes of women who underwent COS or ART before or after breast cancer treatments compared to breast cancer patients not exposed to these strategies. Survival outcomes of interest were cancer recurrence rate, relapse rate, overall survival and number of deaths. Adjusted relative risk (RR) and hazard ratio (HR) with 95% CI were extracted. When the number of events for each group were available but the above measures were not reported, HRs were estimated using the Watkins and Bennett method. We excluded case reports or case series with MAIN RESULTS AND THE ROLE OF CHANCE A total of 1835 records were retrieved. After excluding ineligible publications, 15 studies were finally included in the present meta-analysis (n = 4643). Among them, 11 reported the outcomes of breast cancer patients who underwent COS for fertility preservation before starting chemotherapy, and 4 the safety of ART following anticancer treatment completion. Compared to women who did not receive fertility preservation at diagnosis (n = 2386), those who underwent COS (n = 1594) had reduced risk of recurrence (RR 0.58, 95% CI 0.46–0.73) and mortality (RR 0.54, 95% CI 0.38–0.76). No detrimental effect of COS on EFS was observed (HR 0.76, 95% CI 0.55–1.06). A similar trend of better outcomes in terms of EFS was observed in women with hormone-receptor-positive disease who underwent COS (HR 0.36, 95% CI 0.20–0.65). A reduced risk of recurrence was also observed in patients undergoing COS before neoadjuvant chemotherapy (RR 0.22, 95% CI 0.06–0.80). Compared to women not exposed to ART following completion of anticancer treatments (n = 540), those exposed to ART (n = 123) showed a tendency for better outcomes in terms of recurrence ratio (RR 0.34, 95% CI 0.17–0.70) and EFS (HR 0.43, 95% CI 0.17–1.11). LIMITATIONS, REASONS FOR CAUTION This meta-analysis is based on abstracted data and most of the studies included are retrospective cohort studies. Not all studies had matching criteria between the study population and the controls, and these criteria often differed between the studies. Moreover, rate of recurrence is reported as a punctual event and it is not possible to establish when recurrences occurred and whether follow-up, which was shorter than 5 years in some of the included studies, is adequate to capture late recurrences. WIDER IMPLICATIONS OF THE FINDINGS Our results demonstrate that performing COS at diagnosis or ART following treatment completion does not seem to be associated with detrimental prognostic effect in young women with breast cancer, including among patients with hormone receptor-positive disease and those receiving neoadjuvant chemotherapy. STUDY FUNDING/COMPETING INTEREST(S) Partially supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC; grant number MFAG 2020 ID 24698) and the Italian Ministry of Health—5 × 1000 funds 2017 (no grant number). M.L. acted as consultant for Roche, Pfizer, Novartis, Lilly, AstraZeneca, MSD, Exact Sciences, Gilead, Seagen and received speaker honoraria from Roche, Pfizer, Novartis, Lilly, Ipsen, Takeda, Libbs, Knight, Sandoz outside the submitted work. F.S. acted as consultant for Novartis, MSD, Sun Pharma, Philogen and Pierre Fabre and received speaker honoraria from Roche, Novartis, BMS, MSD, Merck, Sun Pharma, Sanofi and Pierre Fabre outside the submitted work. I.D. has acted as a consultant for Roche, has received research grants from Roche and Ferring, has received reagents for academic clinical trial from Roche diagnostics, speaker’s fees from Novartis, and support for congresses from Theramex and Ferring outside the submitted work. L.D.M. reported honoraria from Roche, Novartis, Eli Lilly, MSD, Pfizer, Ipsen, Novartis and had an advisory role for Roche, Eli Lilly, Novartis, MSD, Genomic Health, Pierre Fabre, Daiichi Sankyo, Seagen, AstraZeneca, Eisai outside the submitted work. The other authors declare no conflict of interest. The funding organizations had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript and decision to submit the manuscript for publication. REGISTRATION NUMBER N/A.

This study evaluated single intra-articular injections of Hymovis MO.RE., a hyaluronic acid hexadecyl derivative (HYADD4-G), to manage post-traumatic or degenerative knee or ankle chondropathy in professional soccer players.Twenty-five players affected by knee (n = 12) or ankle (n = 13) chondropathy were prospectively enrolled and treated by two single Hymovis MO.RE. (32 mg/4 ml) injections at the beginning of the football season (V0, baseline) and at mid-season (V1, 19-20 weeks thereafter), and were followed-up until the end of the season (V2, after further 19-20 weeks). Knee cases were evaluated using the 2000 IKDC knee subjective examination form and the modified Lysholm scoring system. Ankle cases were evaluated using the American Orthopaedic Foot Ankle Society (AOFAS) ankle-hindfoot score. Patients were also evaluated using a VAS Likert scale and a four-category scale recording both the patient's and the doctor's assessment on joint mobility in degrees and overall treatment efficacy. Adverse events, patient withdrawals and local reaction to injections were also assessed.In knee patients, the 2000 IKDC subjective score improved from 46.8 ± 11.4 at V0 to 83.1 ± 12.5 at V2. Their modified Lysholm score improved from 58.8 ± 8.9 at V0 to 90.6 ± 8.3 at V2. In the ankle patients, the AOFAS score improved from 52.2 ± 5.6 at V0 to 96.4 ± 4.5 at V2. VAS Likert values and subjective evaluations improved at V1 and were maintained at V2. No side effects were recorded.A single Hymovis MO.RE. (32 mg/4 ml) intra-articular injection, repeated after 19-20 weeks, may be a viable option to improve symptoms and function in professional soccer players suffering from knee and ankle chondropathy.

Brain metastases (BMs) are frequent events in patients with HER2-positive metastatic breast cancer (MBC) and are associated with poor prognosis. Small-molecule anti-HER2 tyrosine kinase inhibitors (TKIs) are promising agents for the treatment of BM. In this study, we assess the clinical outcomes of patients with HER2-positive MBC and BM treated with TKI-containing regimens compared with those treated with non-TKI-containing regimens.PubMed, Embase, Cochrane Library, and conference proceedings (ASCO, SABCS, ESMO, and ESMO Breast) were searched up to June 2021. The primary endpoint was progression-free survival (PFS) in patients with BM. Secondary endpoints included PFS in patients without BM and overall survival (OS). The study was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Overall effects were pooled using random-effects models.This systematic review and meta-analysis included data from 2437 patients (490 with and 1947 without BM at baseline) enrolled in five trials assessing tucatinib-, lapatinib-, pyrotinib-, or afatinib-based combinations. A nonstatistically significant PFS benefit favoring TKI-containing regimens was observed in both patients with BM [hazard ratio (HR) 0.67, 95% confidence interval (CI) 0.41-1.12; P = 0.13] and without BM (HR 0.55, 95% CI 0.24-1.26; P = 0.16). Sensitivity analysis, excluding each study singly, demonstrated a significant PFS benefit favoring TKI-containing regimens in patients with BM after the exclusion of afatinib from the analysis (HR 0.56, 95% CI 0.35-0.90; P = 0.016). No statistically significant differences in OS were observed between the comparison groups.A trend in PFS favoring TKI-containing regimens was observed in patients with BM. Sensitivity analysis including only trials that evaluated regimens containing tucatinib, lapatinib, or pyrotinib demonstrated a significant PFS benefit favoring TKI-containing regimens in patients with BM.

Background The negative prognostic effect of chronic kidney disease (CKD), anaemia and diabetes in patients with acute coronary syndromes is well known. However, data about the prevalence and the prognostic importance of these comorbidities in unselected, contemporary STEMI patients treated with primary PCI are limited. Purpose We sought to investigate the prevalence of CKD, anaemia and diabetes mellitus in this patient population, as well as possible interactions between these comorbidities. Methods Between January 2006 and December 2018, 3395 consecutive patients with STEMI underwent primary PCI in two centres. Hb and creatinine were determined on a blood test obtained immediately upon the arrival at the Hospital and eGFR was estimated with the CKD-EPI equation. Renal impairment (RI) was defined as stage 3B or worse CKD (eGFR ≤44 ml/min/1.73m2). Anaemia was defined as Hb Results The age of patients was (mean±SD) 67.2±12.9 years and 27.3% of them were females. Diabetes was present in 22.1%, anaemia in 18.1%, and RI in 9.8% of patients. The presence of diabetes, anemia or RI, individually or in various combination, was associated with higher mortality (see figure). Interestingly, these comorbidities presented an addictive, but non synergistic, effect (P for interaction = NS for all combinations). The covariates associated with mortality are shown in the Table. Notably, female gender was independently associated with lower mortality. Conclusions In contemporary patients treated with primary PCI, diabetes, anaemia and RI are frequently present, individually or in combination. All these comorbidities are strong independent predictors of mortality, and the coexistence of more conditions has addictive, but not synergistic, effect. The identification of patients at higher risk could promote a closer follow-up and more stringent measures of secondary prevention. Funding Acknowledgement Type of funding source: None

The objective of this study was to evaluate the efficacy of the endothelin receptor antagonist, bosentan, in patients with Raynaud’s phenomenon secondary to systemic sclerosis never treated with prostanoids and without digital ulcers. The study design is a preliminary, prospective open label trial. The patients recruited took one 62.5 mg dose of bosentan twice daily for 4 weeks, followed by 125 mg twice daily for 24 weeks. Of the 10 patients recruited, all completed the study. The reduction in Raynaud’s phenomenon attacks at week 24 from the baseline was statistically significant (Δ-1.3, P=0.0126). The Raynaud’s condition score showed a statistically significant improvement (Δ-1.4, P=0.0279), as did the visual analog pain scale (Δ-1.5, P=0.0016) at the 24th week. Bosentan appears to be effective and may be a valid alternative for the treatment of severe secondary Raynaud’s phenomenon for patients where prostanoids therapy is contraindicated or refused.

Aims:High-resolution melting (HRM) screening and scanning for single-nucleotide polymorphisms (SNPs) afford the advantages of a quicker, less expensive, and less demanding option compared to other methods for sequence analysis. The evaluation of large populations of patients for multiple SNPs in a high-throughput manner is the next phase in individualized medicine. Results: We demonstrated that Tm profiles can be generated from gDNA samples that clearly differentiate homozygous ancestral, homozygous SNP, and heterozygous genotypes, while identifying samples of unique outcome without the cumbersome processes of normalization, temperature shifting, and difference plot generation. Conclusions: Through expanded primer selection criterion and inclusion of a cloning fragment length double-stranded DNA sequence-specific control template, we are now able to generate additional data via HRM melt domains that are greatly simplified, while considering both the peak melt temperature and profile.

Increases in heme oxygenase-1 (HO-1) and administration of heme degradation products CO and biliverdin inhibit vascular inflammation and vasoocclusion in mouse models of sickle cell disease (SCD). In this study, an albumin (alb) promoter-driven Sleeping Beauty (SB) transposase plasmid with a wild-type rat hmox-1 (wt-HO-1) transposable element was delivered by hydrodynamic tail vein injections to SCD mice. Eight weeks after injection, SCD mice had three- to five-fold increases in HO-1 activity and protein expression in liver, similar to hemin-treated mice. Immunohistochemistry demonstrated increased perinuclear HO-1 staining in hepatocytes. Messenger RNA transcription of the hmox-1 transgene in liver was confirmed by quantitative real-time polymerase chain reaction restriction fragment length polymorphism (qRT-PCR RFLP) with no detectible transgene expression in other organs. The livers of all HO-1 overexpressing mice had activation of nuclear phospho-p38 mitogen-activated protein kinase (MAPK) and phospho-Akt, decreased nuclear expression of nuclear factor-kappa B (NF-kappaB) p65, and decreased soluble vascular cell adhesion molecule-1 (sVCAM-1) in serum. Hypoxia-induced stasis, a characteristic of SCD, but not normal mice, was inhibited in dorsal skin fold chambers in wt-HO-1 SCD mice despite the absence of hmox-1 transgene expression in the skin suggesting distal effects of HO activity on the vasculature. No protective effects were seen in SCD mice injected with nonsense (ns-) rat hmox-1 that encodes carboxy-truncated HO-1 with little or no enzyme activity. We speculate that HO-1 gene delivery to the liver is beneficial in SCD mice by degrading pro-oxidative heme, releasing anti-inflammatory heme degradation products CO and biliverdin/bilirubin into circulation, activating cytoprotective pathways and inhibiting vascular stasis at sites distal to transgene expression.

The results obtained in the stereospecific copolymerization of 1,3-butadiene with 2-phenylbutadiene are reported. The copolymers were generally obtained using catalysts prepared from substituted aluminum hydrides, AlI3 and TiCl4. Homopolymerization of 2-phenylbutadiene with such catalysts gives an amorphous, low M.W. polymer having prevailing 1,4-cis structure. In the copolymers the 2-phenylbutadiene units still have a substantially cis structure; also the butadiene units are mainly 1,4-cis up to 90-95%. The 1,2 content in the copolymers is always very low. The content of 2-phenylbutadiene units in the copolymers varies in a broad range from 10 to 80% and the reaction rate and the average molecular weight diminish as the phenylbutadiene increases. The monomer reactivity ratios were calculated and the distribution of sequence lengths in the copolymer chains was made by statistical analysis. The copolymer composition was determined by IR and NMR analysis and the results compared with spectra obtained from mechanical mixtures of the homopolymers. The formation of a true copolymer was confirmed by extractions, fractionations, and glass transition temperature determinations. Mechanical properties of the copolymers are given and compared with those of other polymers, in particular with those of 1,4-cis polybutadiene.

The technology of Single Nucleotide Polymorphism (SNP) detection has evolved steadily through mobility shift studies, mass cleavage product evaluations, heterodimer differences in chemical, conformational, and enzymatic properties, mass spectroscopy and sequencing, to allele-specific hybridization probe methods. Each method presented challenges of labor intensity, unreliable efficiencies, complicated optimizations, and issues of sample quantity and quality. Concurrently the value of SNP detection in basic research and personalized medicine has continued to grow. Accessing the secrets of genetic individuality is the next frontier in moving medicine from the description of very low frequency and highly deleterious nucleotide changes to the study of very low frequency polymorphisms, lower penetrance polymorphisms, and polymorphisms with public health importance. High-Resolution Melting (HRM) analysis of SNP status became an option for high throughput settings with the development of double-stranded dyes that do not interfere with PCR amplification in saturation, eliminate dye jumping, and nearest neighbor sequence changes influence melt temperature via amplicon strand locking chemistry. This method is able to distinguish transitions, transversions, and identify novel changes at or near the SNP of interest rapidly, inexpensively, and without post-amplification assay techniques or extensive technical interpretation of data. For probe or solid matrix based assays, the investigator initially defines a set of target sequences for binding. These assays are not only difficult due to the optimization of binding conditions but are unable to detect sequences that were not included in the design, often have marginalized binding due to a "one size fits all" reaction, and are not distinct in the case of heterozygotes.

The technology of Single Nucleotide Polymorphism (SNP) detection has evolved steadily through mobility shift studies, mass cleavage product evaluations, heterodimer differences in chemical, conformational, and enzymatic properties, mass spectroscopy and sequencing, to allele-specific hybridization probe methods. Each method presented challenges of labor intensity, unreliable efficiencies, complicated optimizations, and issues of sample quantity and quality. Concurrently the value of SNP detection in basic research and personalized medicine has continued to grow. Accessing the secrets of genetic individuality is the next frontier in moving medicine from the description of very low frequency and highly deleterious nucleotide changes to the study of very low frequency polymorphisms, lower penetrance polymorphisms, and polymorphisms with public health importance. High-Resolution Melting (HRM) analysis of SNP status became an option for high throughput settings with the development of double-stranded dyes that do not interfere with PCR amplification in saturation, eliminate dye jumping, and nearest neighbor sequence changes influence melt temperature via amplicon strand locking chemistry. This method is able to distinguish transitions, transversions, and identify novel changes at or near the SNP of interest rapidly, inexpensively, and without post-amplification assay techniques or extensive technical interpretation of data. For probe or solid matrix based assays, the investigator initially defines a set of target sequences for binding. These assays are not only difficult due to the optimization of binding conditions but are unable to detect sequences that were not included in the design, often have marginalized binding due to a "one size fits all" reaction, and are not distinct in the case of heterozygotes.

Hemolysis, oxidative stress, inflammation, vaso-occlusion, and organ infarction are hallmarks of sickle cell disease (SCD). We have previously shown that increases in heme oxygenase-1 (HO-1) activity detoxify heme and inhibit vaso-occlusion in transgenic mouse models of SCD. HO-1 releases Fe(2+) from heme, and the ferritin heavy chain (FHC) ferroxidase oxidizes Fe(2+) to catalytically inactive Fe(3+) inside ferritin. FHC overexpression has been shown to be cytoprotective. In this study, we hypothesized that overexpression of FHC and its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic SCD mice in response to plasma hemoglobin. We utilized a Sleeping Beauty (SB) transposase plasmid to deliver a human wild-type-ferritin heavy chain (wt-hFHC) transposable element by hydrodynamic tail vein injections into NY1DD SCD mice. Control SCD mice were infused with the same volume of lactated Ringer's solution (LRS) or a human triple missense FHC (ms-hFHC) plasmid with no ferroxidase activity. 8 weeks later, LRS-injected mice had ~40% microvascular stasis (% non-flowing venules) 1 h after infusion of stroma-free hemoglobin, while mice overexpressing wt-hFHC had only 5% stasis (p0.05), and ms-hFHC mice had 33% stasis suggesting vascular protection by ferroxidase active wt-hFHC. The wt-hFHC SCD mice had marked increases in splenic hFHC mRNA and hepatic hFHC protein, ferritin light chain (FLC), 5-aminolevulinic acid synthase (ALAS), heme content, ferroportin, nuclear factor erythroid 2-related factor 2 (Nrf2), and HO-1 activity and protein. There was also a decrease in hepatic activated nuclear factor-kappa B (NF-κB) phospho-p65 and vascular cell adhesion molecule-1 (VCAM-1). Inhibition of HO-1 activity with tin protoporphyrin demonstrated HO-1 was not essential for the protection by wt-hFHC. We conclude that wt-hFHC ferroxidase activity enhances cytoprotective Nrf2-regulated proteins including HO-1, thereby resulting in decreased NF-κB-activation, adhesion molecules, and microvascular stasis in transgenic SCD mice.

In the framework of research on generation-IV reactors, it is very important to have infrastructures specifically dedicated to the study of fundamental parameters in dynamics and kinetics of future fast-neutron reactors. Among various options pursued by international groups, Italy focused on lead-cooled reactors, which guarantee minimal neutron slowdown and capture and efficient cooling. In this paper it is described the design of a the low-power prototype generator, LEADS, that could be used within research facilities such as the National Laboratory of Legnaro of the INFN. The LEADS has a high safety standard in order to be used as a training facility, but it has also a good flexibility so as to allow a wide range of measurements and experiments. A high safety standard is achieved by limiting the reactor power to less than few hundred kW and the neutron multiplication factor keff to less than 0.95 (a limiting value for spent fuel pool), by using a pure-uranium fuel (no plutonium) and by using solid lead as a diffuser. The proposed core is therefore intrinsically subcritical and has to be driven by an external neutron source generated by a proton beam impinging in a target. Preliminary simulations, performed with the MCNPX code indicated, for a 0.75mA continuous proton beam current at 70MeV proton energy, a reactor power of about 190kW when using a beryllium converter. The enriched-uranium fuel elements are immersed in a solid-lead matrix and contained within a steel vessel. The system is cooled by helium gas, which is transparent to neutrons and does not undergo activation. The gas is pumped by a compressor through specific holes at the entrance of the active volume with a temperature which varies according to the operating conditions and a pressure of about 1.1MPa. The hot gas coming out of the vessel is cooled by an external helium-water heat exchanger. The beryllium converter is cooled by its dedicated helium gas cooling system. After shutdown, the decay is completely dissipated by conduction through the lead reflector and steel vessel, and then evacuated by irradiation from the vessel surface to the external ambient air.

Protoporphyria is a genetic disorder in which a deficiency of mitochondrial ferrochelatase activity causes accumulation of protoporphyrin that produces severe liver damage in some patients. In this study, mutations of the ferrochelatase gene were examined in eight unrelated patients who had liver transplantation. RNA was prepared from liver and/ or lymphoblasts, and specific reverse transcriptase-nested polymerase chain reactions amplified and sequenced ferrochelatase cDNAs. Products shorter than normal resulted from an exon 3 deletion in three patients, exon 10 deletion in two, exon 2 deletion in one, and deletion of five nucleotides in exon 5 in one. Sequence of normal-size products revealed no other mutations. Western blot showed a reduced quantity of normal-size ferrochelatase protein in protoporphyria liver compared with normal liver (19-51%, mean 32% of normal). Levels of the mitochondrial protein F1-ATPase beta-subunit were not decreased to a similar degree. Liver ferrochelatase activity was reduced more than could be explained by the decrease in ferrochelatase protein (4-20%, mean 9% of normal). These results establish genetic heterogeneity in the most severe phenotype of protoporphyria. However, the gene mutations found share the property of causing a major structural alteration in the ferrochelatase protein.

A careful and precise examination of the knee is mandatory for correct diagnosis, and fundamental for decision-making. It must be preceded by a complete patient history in order to address the clinical examination, even if symptoms are often non-specific. In addition, the clinical examination must be bilateral and well standardized. Every surgeon must use those tests he or she is most confident with, variably combined to improve sensitivity. Imaging studies are used only to confirm the diagnosis formulated on the basis of history and clinical examination. The patellofemoral joint is frequently injured but meniscal lesions, chondral lesions, and instability are common as well. Although these injuries often coexist, they are discussed separately here in order to present a comprehensive and reproducible method of clinical assessment.

Evaluation of the transfer efficiency of a rat heme oxygenase-1 (HO-1) transgene into mice requires differentiation of rat and mouse HO-1. However, rat and mouse HO-1 have 94% homology; antibodies and enzyme activity cannot adequately distinguish HO-1. We designed a quantitative real-time polymerase chain reaction (qRT-PCR) method to monitor HO-1 transcription relative to a housekeeping gene, GAPDH. The ratio of rat and mouse HO-1 mRNA could be estimated through restriction fragment length polymorphism (RFLP) analysis of the PCR products. In vitro, murine AML12 hepatocytes were transfected with rat HO-1. After 40 h, the total HO-1 mRNA was enriched 2-fold relative to control cells, and rat HO-1 comprised 84% of HO-1 cDNA. In vivo, the rat HO-1 transgene was cloned into a Sleeping Beauty transposase (SB-Tn) construct and was injected hydrodynamically into a mouse model of sickle cell disease (SCD). After 21 days, there was a 32% enrichment of HO-1 mRNA relative to control mice and the rat transgene comprised 88% of HO-1 cDNA. After 21 days, HO-1 protein expression in liver was increased 2.5-fold. In summary, qRT-PCR RFLP is a useful and reliable method to differentiate the transgene from host gene transcription, especially when the host and transgene protein are identical or highly homologous. This method has translational applications to the design, delivery, and monitoring of gene-therapy vectors.

Objective: Hypereosinophilic syndromes are a heterogeneous group of uncommon disorders characterized by the presence of marked peripheral blood eosinophilia, tissue eosinophilia, or both, resulting in a wide variety of clinical manifestations, often without an identifiable cause. Churg-Strauss syndrome is a systemic vasculitis characterized by prominent peripheral eosinophilia, asthma and systemic involvement. The presence of mild to severe eosinophilia and systemic involvement raise the search of many trigger factor that need to be ruled out. Distinguishing CSS from idiopathic hypereosinophilic syndrome may be particularly challenging, especially in ANCA negative patients. Methods: The aim of the present study was to present a small case series of patients referred to a Rheumatology Unit for mild to severe eosinophilia and signs and symptoms of systemic involvement and to outline the clinical significance of molecular biology in the work-up of hypereosinophilia. Results: Eleven patients with moderate to severe peripheral eosinophylia, were referred to our Unit from 1996 to 2007. Female to male ratio was 7/4, mean age 40.54 (range 22-75). Three out of eleven patients resulted positive for molecular biology. The diagnosis of idiopathic hypereosinophylia was confirmed in one out of three on the basis of the clinical picture and bone marrow biopsy. Conclusions: Molecular biology may be useful in the screening and in the follow-up of a new hypereosinophylic patient.

After standard management of stage III–IV ovarian cancer patients by surgical reduction of tumor mass and subsequent cisplatin-based combination chemotherapy, eradication of residual intraabdominal disease remains a major clinical problem. In an effort to increase response to therapy without adding marrow toxicity, after laparotomy, 21 stage III–IV ovarian cancer patients were treated with systemic chemotherapy comprising cisplatin, doxorubicin, and cyclophosphamide (PAC) on Day 1 followed by intraperitoneal (ip) cytosine arabinoside (Ara-C) on either Day 8 or 14, every 28 days. Ara-C, an S-phase-specific drug, was administered ip to exploit the pharmacologic advantage of an ip regimen at a time when a possible PAC-induced recruitment of cells into the proliferative pool could further maximize cell kill. Kinetic features of ovarian neoplastic cells recovered from peritoneal washings were monitored during treatment by measurement of the thymidine labeling index (TLI): data from four patients indicate that there is an increase in proliferating cells on Days 8 and 14 after PAC treatment. Toxicity of treatment was acceptable. Although 95% of evaluable patients had more than 2 cm of residual disease, response was observed in 47% of patients. The therapeutic potential of this regimen should be tested in patients with small-volume disease after debulking surgery.

Frequently we hear and read that drinking water will be the most scarce resource in the coming years, and that, unlike oil, have no substitutes, therefore, whoever controls it will be controlling the universal economy and the life on the planet in the near future. It is also known that 3 percent of the earth’s drinking water is found on the planet surface. The rest is below it and it has been stored there for more than 10,000 years.

While ovarian cancer is one of the most sensitive cancers to cytotoxic drugs, with objective response rates of 60-80% routinely being reported in previously untreated patients, the majority of individuals with advanced disease ultimately relapse. Paclitaxel, a new and novel antimicrotubule agent, has shown activity as a salvage therapy in epithelial ovarian cancer. More importantly, in a prior study, it has been shown to be active in tumors that have displayed resistance to platinum compounds, with a reported response rate of 20%. Ifosfamide has shown activity in the treatment of patients who previously demonstrated clinical resistance to a platinum-cyclophosphamide combination. Recently, a synergistic activity of Taxol combined with ifosfamide has been reported in ovarian cell lines. Based on these data, a phase I/II study of a combination treatment with paclitaxel and ifosfamide was performed.Thirty-one patients with recurrent ovarian cancer or ovarian cancer refractory to cisplatin (CDDP)-containing regimens were treated with paclitaxel at a dose of 135 mg/m2 on day 1; ifosfamide was administered at 1 g/m2 on days 2 and 3 for the first cycle and 1.5 and 2 g/m2 with the same schedule in cycles 2 and 3, respectively. In the absence of toxicity, the dose of ifosfamide was maintained at 2 g/m2 for the last three cycles. Cytotoxic therapy was repeated every 3 weeks.A 30% overall objective response rate was achieved in the 30 patients assessable for response. Among 21 platinum-resistant patients, 4 partial responses (19%) were observed, while in the 9 platinum-sensitive patients 2 complete responses and 3 partial responses (55%) were observed. Myelosuppression was the predominant toxicity. Leukopenia (WHO grade 3-4) occurred in 10% of patients who received ifosfamide at a dose of 1 g/m2 and in 18% of patients treated with ifosfamide at 1.5 g/m2.Our results confirmed a low activity of paclitaxel in platinum-resistant patients. The results of this combination treatment with paclitaxel-ifosfamide in our platinum-sensitive patients support further investigations in a randomized study of the combination regimen against paclitaxel alone or retreatment with organoplatinum compounds.

From June 1990 to October 1994, 111 advanced ovarian cancer patients with minimal (less than 2 cm) residual disease after platinum-based front-line chemotherapy and second-look laparotomy entered a cooperative randomized study aimed at evaluating the effectiveness and the toxicity of the addition of interferon-alpha2 to carboplatin, both intraperitoneally (ip) administered. Patients were randomized to receive either 3 courses of ip Carboplatin 400 mg/m2 Day 1 q 28 days (54 pts) (CBDCA) or ip interferon-alpha 25 x 10(6) U Day 1 + ip carboplatin 400 mg/m2 Day 2 q 28 days (57 pts) (CBDCA + IFN). Patients treated with interferon experienced more severe (WHO grade 3-4) leukopenia (28% vs 17.1%) and anemia (14% vs 4.2%). Fever (P = 0.000) and flu-like syndrome (P = 0.02) were significantly more frequent in the combination arm. No difference in gastroenteric, neurologic, or renal toxicity was observed. At a median follow-up time of 13 months (range 1-72) 71 patients showed a disease progression (31 CBDCA, 40 CBDCA + IFN) and 44 patients died (21 CBDCA, 23 CBDCA + IFN). Median progression-free survival was 11 months in the CBDCA group and 10 months in the CBDCA + IFN arm. Median survival was 22 and 29 months in CBDCA and CBDCA + IFN arm, respectively. In conclusion, intraperitoneal interferon-alpha does not seem to improve the results achievable with intraperitoneal carboplatin in this subset of patients, while the toxicity and the costs of the combination are consistently higher than with chemotherapy alone.

Background Systemic sclerosis (SSc) is a chronic autoimmune inflammatory pathogenic disease of the connective tissue, characterized by progressive fibrosis thickening of skin and internal organs. SSc is caused by the accumulation of collagen (gastrointestinal system, heart, lung, kidney, joints and muscles) and widespread of vascular disease damage. The first vascular event is Raynaud’s phenomenon(RP). Objectives In our study, we evaluated the efficacy of Aminaftone in the treatment of RP in 19 patients with SSc consecutively recruited and divided in two groups. Methods Group A:9 Patients that not tolerated endothelin receptor antagonist (ERA) due to side effects (eg increased> 3 times the liver enzymes, headache, edema of lower limbs), in treatment with ACE inhibitors, prostanoids and Aminaftone. Group B:10 Patients in treatment with ACE inhibitors, prostanoids and ERA All Patients were aged between 40 and 68 years (Group A 51.6±10.2;Group B 52.1±10.5), with SSc according to ACR criteria, presenting RP and DUs with an onset between 6 and 9 months.11 of the 19 patients had the limited form of SSc withpositive anticentromere antibody value and 8 of them had a diffuse form withpositive SCL-70 antibody value. The mid duration of SSc was 6.4 years with 10.8 years of average duration of onset of RP. The A group tooks three tablets of Aminaftone 75mg/day while the B group tooks two tablets of Bosentan 125mg/day. Follow-up was performed every 4 weeks for 6 months and each patient was given a diary to report on each check, which indicate: -Date of onset of Raynaud’s Phenomenon -Duration: minutes -Raynaud’s Condition Score(RCS): Limitation of daily activity on a scale of 1 to 10 (meaning 10 as a total inability to do the activity) -Pain VAS (1-10): 1 meaning the least pain and 10 as the maximum pain -Number of daily attacks -Ulcer onset (date) -Location ulcer/s Results The number of RP attacks in both groups of patients decreased from baseline to Week 24, obtaining statistically significance (ΔA-2,80 p-0,02;ΔB-3.10 p-0,01). The duration of the RP was decreased, but did not obtainstatistically significance (ΔA-10,90 ΔB-12,30). The RCS has shown a statistically significant improvement (ΔA-2,10 p-0,04;ΔB-2.3 p-0,03), as well as the pain VAS (ΔA-3,20 p-0,04;ΔB-3,5 p-0,02) at week 24. The number of DUs decreased in both group of patients and in most cases (especially in Group B), the lesions met complete resolution. Furthermore, in patients treated with Aminaftone, no extras DUs appeared. Our data showed a statistically significant improvement of baseline in the number of attacks of RP, VAS pain and RCS. Conclusions In conclusion, the use of Aminaftone for the treatment of RP in SSc patients, seems to have been effective, with good tolerability and no adverse events. Although the ERA ( Bosentan ) is more effective, we believe that, at least for those patients whose therapy with ERA is not tolerated, Aminaftone may be a viable alternative treatment for RP. Long term studies with a wider coverage must be performed to assess the effects of Aminaftone . References Kahaleh MB. Raynaud phenomenon and the vascular disease in scleroderma. Curr Opin Rheumatol 2004;16: 718–22. Arefiev Kait et al. Endothelin Receptor Antagonists for the Treatment of Raynaud’s Phenomenon and Digital Ulcers in Systemic Sclerosis. International Journal of Rheumatology Volume 2011, Article ID 201787,7 Disclosure of Interest None Declared

Background Spondyloarthritis (SpA) is characterised by the inflammatory involvment of the enthesis. In the last few years, several studies have provided relevant data on the advantages and validity of using ultrasound (US) to study enthesis in SpA. Objectives To evaluate with ultrasound the presence of erosions in Achilles tendons enthesis and their recovery capacity, in spondyloarthritis (SpA). Methods A prospective study of Achilles enthesis erosions in SpA was undertaken. US and X-Ray examinations were performed at baseline and at 6 and 12 months of follow-up. Clinical outcomes measures were collected. Results Bilateral Achilles enthesis of 45 patients (30 men, 15 women) were investigated. The mean C reactive protein (CRP) levels were 9,9±6,9 mg/L. US visualised 14 erosions (6,3%) in 12 patients. X-Ray 11 erosions in 11 patients (4,9%). At 6 and 12 months of follow-up, 20% and 35% of basal erosion had disappeared, respectively and of the new erosions that appeared at 6 months, 30% had disappeared 6 months later. A stastically significant association between erosion and CRP levels, entheseal Doppler signals, MASES and SPARCC was found. Conclusions The recovery capacity of Achilles tendons in SpA seems reasonably related to formation of new bone in the enthesis area. Conclusions: US is reliable and sensitive to evaluate the presence of enthesis erosions and their changes in time, and more sensitive than X-Ray. An association was found between Achilles enthesis erosions and objective activity-based measurements of SpA outcomes. Disclosure of Interest None Declared

From 1982 to 1984, 131 patients with FIGO stage Ic-IV epithelial ovarian cancer were included in a randomized clinical trial comparing cisplatin 50 mg m-2 plus cyclophosphamide 600 mg m-2 (PC regimen) with PC plus doxorubicin 45 mg m-2 (PAC regimen). Chemotherapy was repeated every 4 weeks for six cycles. The criteria for entry, the characteristics of the eligible patients, the response rates and the toxicities have been previously reported. The study was updated in August 1994 with a median follow-up of 10.5 years (range 10-12 years). In the whole series, the median time to progression is 13 months and the 12-year progression-free survival(PFS) is 18%, whereas the median time to survival is 21 months and the 12-year survival is 21%. By log-rank test survival is significantly related to residual disease after first surgery (P 2 cm 12-year PFS is 12.5% for PAC arm, while all patients of PC arm progressed by the sixth year. Conversely, the PFS curves are superimposable in the two treatment arms among patients with residual disease

Authors describe 1 case of rhabdomyolytic acute renal failure as a complication of Cardiopulmonary bypass.Case report.Department of Cardiac Surgery.Patient affected by rhabdomyolytic acute renal failure was treated with pharmacological therapy, plasmapheresis and continuous arteriovenous hemofiltration (CAVH).Anuric acute renal failure was diagnosed in 2nd post surgical day; serum myoglobin values was 16000 micrograms/l (normal values in non surgical patients88 micrograms/l), creatine phosphokinase serum values was 8790 UI/L (with Mb fraction8%). Patient underwent CAVH and two a day sessions of plasmapheresis; hematochemical and hemodynamic parameters improved progressively with resolution of acute renal failure and resumption of the diuresis on the 10th day. The CAVH was suspended on the 11th day and plasmapheresis on the 5th. Rhabdomyolytic syndrome should be correlated to the direct cannulation of the femoral artery for cardiopulmonary bypass and to prolonged time of extracorporeal circulation, with consequent leg ischemia/reperfusion induced injury which occurs during the extracorporeal circulation and continued during postoperative period; contributory causes should be the low cardiac output syndrome and alpha vasoconstrictor effect of high dose continuous intravenous administration of epinephrine.Rhabdomyolytic acute renal failure is a severe complication of cardiopulmonary bypass; plasmapheresis and continuous arteriovenous hemofiltration seems to be suitable treatments.

From 1987 to 1992 at our Institute, 253 patients with non palpable breast lesions (NBPL) underwent a surgical excision. Fifty-one lesions (19.7%) were localized by an injection of a sterile 3% charcoal suspension under sonographic guide. The sonographic features of NBPL were classified as follows: positive in one case (2%), doubtful in 31 cases (60%) and negative in 19 cases (38%). According to our data the preoperative sonographic localization is a useful alternative procedure to stereotactis mammography in case of breast masses and/or areas of increased tissue density or distorted breast architecture.

Abstract 378 Hemolysis, oxidative stress, inflammation, vaso-occlusion and organ infarction are hallmarks of sickle cell disease (SCD). We hypothesize that intravascular heme, liberated from hemoglobin S derived from hemolyzed sickle red blood cells, is fundamental to inflammation and vaso-occlusion in SCD. Heme can be degraded by heme-oxygenase-1 (HO-1), releasing Fe2+, carbon monoxide (CO), and biliverdin/bilirubin. We have previously shown that increases in HO-1 activity inhibit vascular inflammation and vaso-occlusion in transgenic mouse models of SCD. HO-1 products, CO and biliverdin/bilirubin, also are protective in SCD, but Fe2+ released from the heme ring requires further processing. The released Fe2+ from heme is oxidized by ferritin heavy chain (FHC) ferroxidase activity and safely stored as catalytically-inactive Fe3+ inside ferritin clusters. FHC overexpression has been shown to be cytoprotective in response to inflammation and oxidative stress in vivo and in vitro. In this study, we hypothesize that overexpression of FHC with its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic sickle mice in response to stroma-free hemoglobin. We utilized a Sleeping Beauty transposase plasmid to deliver a human wt-ferritin heavy chain (wt-FHC) transposable element by hydrodynamic tail vein injections to NY1DD SCD mice. Control mice were infused with the same volume of lactated Ringer's solution LRS) or a triple missense (ms-) human FHC plasmid encoding no ferroxidase enzyme activity. Eight weeks after injection, the mice were implanted with dorsal skin-fold chambers to access microvascular blood flow. LRS-treated mice had 40% microvascular stasis (% non-flowing venules) when infused with stroma-free hemoglobin, while wt-FHC overexpressing mice had only 5% stasis (p Disclosures: Vercellotti: Sangart Inc: Consultancy, Research Funding. Chen:Sangart Inc: Research Funding. Hebbel:Sangart Inc: Consultancy, Research Funding. Belcher:Sangart Inc: Research Funding.

Between January 1989 and December 1991, 64 patients with advanced cervical carcinoma FIGO stage IIb-III were randomized to receive radiotherapy (RT) alone or the sequential combination of chemotherapy (CT) and RT. RT consisted of external RT (40 Gy fractionated over 4 weeks)+brachy-therapy (40 Gy to point A)+an additional boost to the parameters (15-20 Gy) in arm RT; CT consisted of cisplatin 60 mg/m2 i.v. day 1 q 15 days administered for 2 cycles before the start of RT and for 4 cycles after the end of radiation treatment in CT+RT arm. Among the 58 evaluable patients objective response rate was as follows: in RT arm, CR in 40.7% of patients, PR in 40.7%, and SD in 18.6%; in CT+RT arm, CR in 42% of patients, PR in 35.5%, and SD in 22.5%. The median duration of response was 12 months (range: 3-38 + months). At a median follow-up of 36 months survival (S) and progression-free survival (PFS) were 83% and 72.4% in RT arm, 72% and 59.3% in CT+RT arm, respectively. No significant difference was observed between the 2 treatment arms, neither in terms of objective response nor in terms of S and PFS. Both treatments were generally well tolerated. In our experience the addition of chemotherapy to standard radiotherapy does not enhance morbidity and does not interfere with the correct delivery of the planned treatment. However, results of this combined modality regimen remain unsatisfactory, since no improvement in pelvic control and survival of patients with advanced cervical carcinoma was observed.