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Introduction and objectives: The impact of multiple subcutaneous (s.c.) esketamine injections on the blood pressure (BP) and heart rate (HR) of patients with unipolar and bipolar treatment-resistant depression (TRD) is poorly understood. This study aimed to assess the cardiovascular safety of multiple s.c. doses of esketamine in patients with TRD. Methods: Seventy TRD patients received 394 weekly s.c. esketamine injections in conjunction with oral antidepressant therapy for up to six weeks. Weekly esketamine doses were 0.5, 0.75 or 1.0 mg/kg according to each patient’s response to treatment. Participants were monitored before each treatment and every 15 minutes thereafter for 120 minutes. We assessed systolic blood pressure (SBP), diastolic blood pressure (DBP), and HR measurements for the entire treatment course. Results: BP increased after the first s.c. esketamine injection, reaching maximum mean SBP/DBP levels of 4.87/5.54 mmHg within 30–45 minutes. At the end of monitoring, 120 minutes post dose, vital signs returned to pretreatment levels. We did not detect significant differences in BP between doses of 0.5, 0.75, and 1 mg/kg esketamine. Mean HR did not differ significantly between doses or before and after s.c. esketamine injection. Conclusions: The BP changes observed with repeated s.c. esketamine injections were mild and well tolerated for doses up to 1 mg/kg. The s.c. route is a simple and safe method of esketamine administration, even for patients with clinical comorbidities, including obesity, hypertension, diabetes, and dyslipidemia. However, 14/70 patients experienced treatment-emergent transient hypertension (SBP >180 mmHg and/or a DBP >110 mmHg). Therefore, we strongly recommend monitoring BP for 90 minutes after esketamine dosing. Since s.c. esketamine is cheap, requires less frequent dosing (once a week), and is a simpler procedure compared to intravenous infusions, it might have an impact on public health.

Objective To systematically review current literature to determine the effectiveness of the ischemic compression (IC) technique on pain and function in individuals with shoulder pain. Methods This review was conducted according to recommendations of Preferred Reporting Items for Systematic Reviews and Meta-Analyses and the Cochrane Collaboration for Systematic Reviews; a search was performed in the electronic databases PubMed, Cumulative Index to Nursing and Allied Health Literature, SPORTDiscus, Physiotherapy Evidence Database, and Web of Science. Randomized clinical trials and quasi-randomized clinical trials were included, and the methodological quality was evaluated through the Physiotherapy Evidence Database scale. Results The search found 572 studies; of these, 71 were selected by title and, subsequently, 29 were selected through abstract analyses. After critical analyses, 5 studies were included. The methodological quality ranged from 4 (reasonable) to 9 (excellent) points. Pain was assessed by all studies using the visual analog scale, Global Perceived Effect scale, Numerical Rating Scale, pressure pain threshold, or Perceived Amelioration Numerical Scale. Function was evaluated by 3 studies through the Shoulder Pain and Disability Index; Neck Disability Index; American Shoulder and Elbow Surgeons Standardized Shoulder Assessment; and Disabilities of the Arm, Shoulder, and Hand questionnaires. The studies showed that the IC technique produces immediate and short-term positive effects for pain, and positive short-term effects for shoulder function in individuals with shoulder pain. Conclusion The IC technique seems to be beneficial for pain and shoulder function. However, caution is needed when considering this evidence owing to the limited quality of some studies, the few articles found, and the lack of standardization of the application parameters of the technique to facilitate its reproducibility.

Aims: ACC1 couples glucose metabolism with de novo lipogenesis in lipogenic tissues, by converting acetyl-CoA to malonyl-CoA. In β-cells, ACC1 plays a critical role in β-cell growth and insulin secretion. However, very little is known about ACC1 regulation in β-cell. The aims of this study were to 1) assess the role of ACC1S79 in β-cell function and whole body glucose homeostasis; and 2) screen for novel phosphosites that may regulate ACC1 activity in β-cell. Methods: 1) ACC1 single knock-in mouse model, with ACC1S79 mutated to alanine ACC1S79A, was obtained by crossing ACC1 double knock-in mice with wild type mice to isolate the ACC1S79A point mutation. 2) ACC1 protein was purified from INS1 β-cell following culture with different glucose concentrations. Quantitative phosphoproteomics was performed to characterise ACC1 phosphosites using a Q-Executive MS. Data analysed using MaxQuant and Perseus. Results: 1) Female ACC1S79A mice exhibited no alternations in glucose homeostasis. In male mice we found no differences in serum insulin, GTT or ITT, although a small but significant reduction in fasting blood glucose was recorded in ACC1S79A mice compared to littermate controls. There was no significant change in GSIS from isolated ACC1S79A islets at 2mM glucose or in response to 7.5 and 20mM glucose, suggesting that ACC1S79 phosphorylation does not play a role in insulin secretion under physiological conditions.2) Using phosphoproteomics, we identified twenty phosphosites on ACC1 protein in β-cells. ACC1S1215 was highly phosphorylated at 2mM glucose, and showed a significant reduction in phosphorylation in response to 15mM glucose. In contrast, phosphorylation of ACC1S25 was lower at basal glucose and increased upon glucose stimulation. Conclusions: Our data show that ACC1S79 phosphorylation does not play a major role as a glucose-regulated phosphosite in β-cells. However, other phosphosites show greater dynamic regulation by glucose and may play a role in regulating β-cell ACC1 activity. Disclosure R. Bany bakar: None. S. Liberatori: Employee; Self; LONZA. A. Veprik: None. N. N. Tebeka: None. B. E. Kemp: None. S. Mohammed: None. J. Cantley: Other Relationship; Self; Oxstem Beta.

Background and aim COVID-19 (COronaVIrus Disease 19) is an acute respiratory disease caused by SARS CoV 2 virus. The correlation between SARS-CoV2 infection and comorbidities is complex; patients with multiple comorbidities present often with the most severe symptoms that could potentially lead to death. Patients undergoing hemodialysis are generally frail and immunodeficient. This leads to a greater risk of contracting infectious diseases. In the literature, the estimated incidence of SARS-CoV2 infection is 3.24% in chronic hemodialysis patients. Method Fondazione Policlinico A. Gemelli is a COVID hospital. During the pandemic patients from several dialysis centers converged in our hospital. FPG has two dialysis centers, one for outpatients and one for inpatients. Patients admitted for COVID-19 infection have been treated in three different settings: 1. isolation room within the dialysis center; 2. Bedside; 3. In a COVID-19 dialysis center. We retrospectively collected data of patients treated from March 2020 to January 2021 and analyzed the SARS-CoV2 incidence in our center’s chronic hemodialysis patients. Results 66 hemodialysis patients affected by COVID-19 have been treated in our hospital from March 2020 to January 2021, 60 patients undergoing chronic dialysis and 6 patients diagnosed with acute kidney injury (AKI) stage III non-intensive care unit. Among chronic patients, 64 underwent chronic hemodialysis and 2 patients underwent peritoneal dialysis. Median age was 68.19 (46 males, 20 females), all patients had multiple comorbidities: 37.8% of patients had diabetes mellitus; 72.7% cardiovascular diseases and 16.6% a positive clinical history for cancer. Among the 6 AKI cases, 3 patients regained total kidney function; the other 3 had to continue renal replacement therapy. The mean hospital stay length was 18.5 days with a mean time of COVID-19 infection of 21.23 days. The overall mean Charlson Comorbidty Index was 6.21. Among the 66 treated patients, 43 were diagnosed with COVID-19-related pneumonia, 14 had the infection, no pulmonary involvement, but presented with other complications, and 5 patients resulted positive although asymptomatic. Among the 116 hemodialysis outpatients, only 4 presented with SARS-CoV2 infection, 3 were contacts of a positive family member and 1 resulted positive during a hospital stay for Clostridium Difficile infection. All patients required hospitalization. 14 (21%) patients died. Among the deceased patients, the mean age was 76.90 years (9 males, 3 females), mean Charlson Comorbidity Index was 7.3, mean hospital stay length was 9 days. Among patients who survived the disease the mean age was 76.92 years (34 males, 14 females), mean Charlson Comorbidty Index was 5.87 and mean hospital stay length was 19.47 days. Statistical significance was reached for age (p value 0.005) and Charlson Comorbidty Index (p value 0.39), but not for mean hospital stay length (p value 0.13). All COVID-19 patients were treated with bicarbonate hemodialysis and a Theranova 400 Baxter® filter. This filter was chosen for its efficiency on medium-size molecules removal (between 25 kDa and 60 kDa) that may be associated with inflammation. Bedside treatments were performed using the Genius© Fresenius system. Each treatment lasted 180 minutes, in order to reduce the time of exposure to COVID-19 of medical staff and the risk of virus spread on one hand, but still ensuring an optimal and complication-free treatment to patients. Conclusion Our experience seems to confirm the national data collected so far, both in terms of patients’ outcomes and mortality rate. Our study confirms that age is a risk factor for mortality. How to properly manage chronic hemodialysis patients affected by COVID-19 remains a challenging and burdensome question. However, there is the need of new flexible solutions that guarantee the patients and the medical staff’s safety on one hand and a personalized management on the other.

Background: A large number of studies indicate that subanesthetic doses of ketamine induce a fast antidepressant effect. Limited studies have investigated the subcutaneous (SC) route, and it remains unclear for whom this treatment is most suitable. Aims: The aim of this study was to examine the effect on depressive symptoms of repeated subanesthetic doses of SC esketamine in unipolar and bipolar treatment-resistant depression (TRD) and clinical predictors of response. Methods: A retrospective analysis of 70 patients who received six SC esketamine doses weekly as an adjunctive treatment was carried out. Doses started at 0.5 mg/kg and it could be titrated up to 1 mg/kg, according to response. The primary outcome was reduction in depressive symptoms. Statistical analysis to investigate clinical predictors of effectiveness included logistic regression analysis using a dependent variable of a 50% reduction in rating scale scores at the end of treatment. Comparisons between groups were made through analysis of variance and treatment effects. Results: At baseline, our sample presented with severe treatment resistance in 65.7%, as assessed by the Maudsley Staging Method (MSM), and 47.1% had anxiety disorder comorbidity. The response rate was 50%. A better outcome was predicted by mild and moderate MSM scores (OR = 3.162, p = 0.041) and anxiety disorder comorbidity (OR = 3.149, p = 0.028). Conclusions: Our results suggest that higher levels of treatment resistance may be associated with a poor response to SC esketamine. Unlike traditional pharmacotherapies, it might benefit those with poor prognosis such as patients with depression and comorbid anxiety. Therefore, future research could investigate whether esketamine should receive a more prominent place in the treatment algorithm for TRD.

Background Handheld dynamometers (HHD) provide quick and low-cost assessments of muscle strength and their use has been increasing in clinical practice. There is no available data related to the validity of HHD for this measurement. Objective To verify the concurrent validity of scapular protraction measurements using an HHD. Methods Individuals with traumatic anterior glenohumeral instability were allocated in Instability Group (n = 20), healthy swimmers were allocated in Athletes Group (n = 19) and healthy subjects were allocated in Sedentary Group (n = 21). Concurrent validity was verified by the Pearson correlation test between HHD and isokinetic measurements. The agreement between instruments was verified by Bland–Altman plots, for each of the two HHD positions. Results A moderate correlation was observed between seated (r = 0.59) and lying supine HHD (r = 0.54) and isokinetic dynamometer measurements for the all groups. Separated group analysis exhibited a strong correlation between seated HHD and isokinetic dynamometer measurements in the Instability Group (r = 0.80), Sedentary Group (r = 0.79) and Athletes Group (r = 0.76). The Bland–Altman plot showed greater agreement in the seated position than the lying supine position when comparing measurements with the HHD and isokinetic in both the general sample and separated groups. Conclusion The HHD may be considered a valid tool for assessing scapular protraction muscle strength among healthy athletes, non-athletes and subjects with shoulder instability. We recommend to assess subjects in the seated position and to be aware that the HHD tends to overestimate the peak force, compared with the gold-standard isokinetic dynamometer.

Aim Data on the outcome of chronic kidney disease (CKD) patients who are hospitalized and start unplanned urgent haemodialysis (HD) are lacking. This prospective, longitudinal, observational study aimed to define the hospital mortality rate and associated factors in CKD patients who start unplanned urgent HD. Methods Between January 2003 and December 2009, all patients with CKD who were hospitalized, diagnosed with ESRD and started unplanned urgent haemodialysis at Haemodialysis Service of the Catholic University of Rome, Italy were recruited. Exclusion criteria were: acute renal failure, prior history of dialysis, multiple organ failure, coma, and dementia. Hospital mortality rate was the primary outcome. Results Three and hundred sixteen patients were studied: 99 died after 19.5 ± 27.3 days and 217 survived until discharge. Of these, 154 were prescribed chronic HD and 63 restored renal function. Patients who died were significantly older and had a higher Charlson Comorbidity Index score. The mortality rates were 51.1% in patients with 81–90 years, 37.8% with 71–80 years, 34.1% with 61–70 years and 13.9% with age ≤60 years. Logistic regression analysis showed that age only was an independent risk factor for all-cause mortality. Conclusions In CKD patients who need hospitalization and start unplanned urgent haemodialysis the mortality is very high and significantly related to age.

Rhabdomyolysis is a syndrome due to a damage of skeletal muscle and the leakage of intracellular contents into the extracellular fluid and the circulation. Several causes may induce rhabdomyolysis and the major one is the crush syndrome. Most cases of non-traumatic rhabdomyolysis are related to drugs. Many molecules are subject to hepatic metabolism and the concomitant use of drugs, as statins, with other medications acting as substrates of the same isoenzymes can interact and increase the risk of myopathy. Subclinical rise of creatine kinase may be the expression of rhabdomyolysis that can present as a medical emergency such as acute kidney injury (AKI), compartment syndrome, cardiac dysrhythmias and disseminated intravascular coagulopathy. The main pathophysiological mechanisms of myoglobinuric-related AKI are renal vasoconstriction, formation of intraluminal casts and direct cytotoxicity promoted by heme-protein. The aim of this review is to analyze the pathophysiology of myolysis, the causes of rhabdomyolysis and especially the link between the liver and the kidney, which can represent the connecting element for the development of the syndrome.

Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7-10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17-secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.

During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis.

Aim Autonomic dysfunction (AD) is an early feature of systemic sclerosis (SSc). A regular endothelial function is a prerequisite for normal response of the myocardial blood flow (MBF) to cold pressure test (CPT). The aim of the study was to evaluate the relation between MBF and AD at rest and after CPT in asymptomatic SSc patients. Methods Twenty SSc patients and 10 age-, sex- and body mass index-matched healthy controls underwent cardiac magnetic resonance at rest and after CPT. All subjects underwent 24 hours ambulatory 3-channel electrocardiogram Holter to evaluate AD by heart rate variability. Results We did not observe any significant difference in MBF (mL/g/min) at rest and after CPT between SSc patients and healthy controls. Delta of MBF (difference between MBF after CPT and rest MBF) was lower (P = 0.039) in SSc patients than healthy controls (0.28 [0.04-0.40] vs 0.33 [0.24-0.54]). The low frequency/high frequency (LF/HF) was higher (P = 0.002) in SSc patients than healthy controls (3 [1.7-6] vs 1.8 [1.1-2.8]). The high frequencies (HF), modulated mainly by paraympathetic system, was lower (P = 0.003) in SSc patients than healthy controls (30 [16-42] vs 36.5 [24-44]). Sympathetic hyperactivity, due to reduction of parasympathetic activity (HF), is present in SSc patients. A negative correlation was observed between Delta of MBF and LF/HF (r = -0.572, P = 0.0031). Conclusion AD, characterized by sympathovagal imbalance due to a reduced parasympathetic tone with high LF/HF ratio, could be responsible for the reduced myocardial vasodilatory response after CPT.

Objectives Obese children are often taller than age-matched subjects. Reports on GH and IGF-I levels in obese individuals are controversial, with normal and reduced GH-IGF-I levels having been reported in this group of patients. Thus, the aim of this study was to analyse insulin-like growth factor type 1 receptor (IGF-IR) mRNA expression in obese children. Methods Forty-seven pre-pubertal children were included in this study: 29 were obese and taller than their target height, and 18 were normal eutrophic controls. Fasting blood samples were collected for IGF-IR mRNA expression in isolated lymphocytes and serum IGF-I, ALS, IGFBP-3, and IGFBP-1 concentration analysis. Results Relative IGF-IR gene expression (2− ΔΔCT) was significantly (P = 0.025) higher in obese children (median 1.87) than in controls (1.15). Fourteen of the 29 obese subjects showed 2− ΔΔCT values greater than or equal to 2, while only 2 individuals in the control group showed values above 2 (P = 0.01). Obese children showed significantly (P = 0.01) higher IGF-I concentrations than the control group (237 ng/ml and 144 ng/ml, respectively). Among obese patients, 65.5% had IGF-I values above the 75 percentile of the control group (P = 0.02). ALS concentration was significantly (P = 0.04) higher in the obese group, while IGFBP-3 levels were similar in obese and control children. IGFBP-1 concentration was lower in obese children, while insulin levels and HOMA-IR index were higher than in controls. Conclusions The higher IGF-IR mRNA expression observed in obese children, associated with the higher IGF-I and ALS and the lower IGFBP-1 levels, suggest that the higher stature observed in these children may be due to increased IGF-I bioactivity.

Background: Many patients admitted to a Department of Internal Medicine have different degrees of heart and kidney dysfunction. Mortality, morbidity and cost of care greatly increase when cardiac and renal diseases coexist. Methods: A retrospective cohort study was conducted on 1,087 patients admitted from December 2009 to December 2012 to evaluate the prevalence of the cardiorenal syndrome (CRS) and clinical features. Results: Out of 1,087 patients discharged from our unit during the study period, 190 (17.5%) were diagnosed as having CRS and classified into five types. CRS was more common in males (68.9%). CRS type 1 was associated with higher age (79.9 ± 8.9 years) and accounted for 61.5% of all deaths (p < 0.001), representing a risk factor for mortality (OR 4.23, 95% CI 1.8-10). Congestive heart failure was significantly different among the five CRS types (p < 0.0001) with a greater frequency in type 1 patients. Infectious diseases were more frequent in CRS types 1, 3 and 5 (p < 0.05). Pneumonia presented a statistically higher frequency in CRS types 1 and 5 compared to other classes (p < 0.01), and community-acquired infections were statistically more frequent in CRS types 1 and 5 (p < 0.05). The distribution of community-acquired pneumonia was different among the classes (p < 0.01) with a higher frequency in CRS types 1, 3 and 5. Conclusion: CRS is a condition that is more frequently observed in the clinical practice. The identification of predisposing trigger factors, such as infectious diseases, particularly in the elderly, plays a key role in reducing morbidity and mortality. An early recognition can be useful to optimize therapy, encourage a multidisciplinary approach and prevent complications.

The hypercoagulability of patients with nephrotic syndrome could be an important trigger for arterial and venous thrombotic events. Arterial thromboses are less frequent than venous thromboses and the most common locations are femoral arteries. The association of stroke and nephrotic syndrome is extremely rare. Here we report the case of a patient with stroke as first manifestation of nephrotic syndrome. Ischemic stroke can be the first manifestation of nephrotic syndrome and should be considered as a possible complication of the syndrome, when the commonest causes of ischemic stroke are excluded and especially in presence of pre-existing glomerular disease.

Patients with small vessel vasculitis present fluctuating antineutrophil cytoplasmic antibodies (ANCA) levels to the point that positive ANCA may be missed even if only up to 10% of patients with microscopic polyangiitis (MPA) are ANCA-negative. The first-line treatment of MPA is the association of steroids and cyclophosphamide, especially in the presence of a rapidly progressive glomerulonephritis. Plasmapheresis, intravenous immunoglobulins, and tumor necrosis factor inhibitors have been proposed as alternative to standard therapy. Disseminated intravascular coagulation (DIC) is a possible event in the course of small vessel vasculitis. Gabexate mesylate is a protease inhibitor able to suppress endothelial cell injury, and it may be administered to treat DIC related to different diseases. In ANCA-associated vasculitis, cytokines play a key role in promoting endothelial damage. DIC-related thrombocytopenia may be misinterpreted as drug-induced because of the immunosuppressive properties of cyclophosphamide. Two cases of ANCA-positive MPA associated with DIC and treated with gabexate are reported in the literature with improvement of both hematological disorder and renal function. Our patient presented a rapidly progressive glomerulonephritis, and the renal biopsy showed MPA, in the absence of ANCA. After two weeks of steroid treatment, our patient developed a DIC. This case represents the first report of ANCA-negative MPA managed with gabexate, which showed improvement of coagulation disorders and kidney function. In conclusion, the anti-inflammatory properties of gabexate could be helpful in MPA at increased bleeding risk when immunosuppressive treatment is contraindicated, even in ANCA-negative vasculitis.

OBJETIVOS: Avaliar a morfologia dos cardiomiocitos e quantificar o colageno presente no miocardio de ratas tratadas com extrato concentrado de soja ou 17β-estradiol (E2). METODOS: Vinte e oito ratas foram divididas em quatro grupos: GCtrl - fase de estro; GOvx - ovariectomizadas (Ovx); GIso - Ovx tratadas com extrato de soja (150 mg/kg, por dia); GE2 - Ovx tratadas com E2 (10 µg/kg, por dia). As drogas e o veiculo (0,2 mL de propilenoglicol) foram administrados apos 30 dias da realizacao da ovariectomia, por 30 dias consecutivos. No ultimo dia os animais foram anestesiados, o coracao retirado, mergulhado em formaldeido a 10%, e fragmentos dos ventriculos submetidos a processamento histologico, sendo os cortes corados pela hematoxilina e eosina ou pelo picrosirius-red. As analises histomorfometricas (contagem, volume nuclear e quantificacao do colageno) foram realizadas em microscopio de luz e software AxioVision Rel. 4.2, sendo o colageno determinado pelo programa Imagelab 2000. Os dados foram submetidos ao teste de ANOVA complementado pelo teste de Tukey (p GE2=70,5±14,8=GCtrl=66,3±9,6; p GIso=26,5±4,5=GOvx=22,4±2,9; p GCtrl=4,0±0,1=GIso=4,4±0,08=GE2=4,3±0,5; p

Iron availability plays an essential role in staphylococcal pathogenesis. We selected FhuD2, a lipoprotein involved in iron-hydroxamate uptake, as a novel vaccine candidate against Staphylococcus aureus. Unprecedented for staphylococcal lipoproteins, the protein was demonstrated to have a discrete, punctate localization on the bacterial surface. FhuD2 vaccination generated protective immunity against diverse clinical S. aureus isolates in murine infection models. Protection appeared to be associated with functional antibodies that were shown to mediate opsonophagocytosis, to be effective in passive transfer experiments, and to potentially block FhuD2-mediated siderophore uptake. Furthermore, the protein was found to be up-regulated in infected tissues and was required for staphylococcal dissemination and abscess formation. Herein we show that the staphylococcal iron-hydroxamate uptake system is important in invasive infection and functions as an efficacious vaccine target. The extraordinary speed of antibiotic resistance acquisition of Staphylococcus aureus, associated with the severity of staphylococcal infections, dictates the urgent need of a vaccine against this deadly pathogen. However, although several vaccine candidates have been proposed and some of them have been tested in clinical trials using both active and passive immunization modalities [1], an effective vaccine is still missing. In an attempt to develop an S. aureus vaccine, we have been applying a reverse vaccinology approach, mainly based on in silico screening and proteomics, to select surface-exposed proteins. As reported in this work, FhuD2 (ferric hydroxamate uptake) is one of the proteins identified through our screening. FhuD2 was first discovered as a ferric hydroxamate siderophore binding protein that mediates iron uptake through a

Safe recombinant vaccines, based on a small number of antigenic proteins, are emerging as the most attractive, cost-effective solution against infectious diseases. In the present work, we confirmed previous data from our laboratory showing that whole viable bacterial cell treatment with proteases followed by the identification of released peptides by mass spectrometry is the method of choice for the rapid and reliable identification of vaccine candidates in Gram-positive bacteria. When applied to the Group B Streptococcus COH1 strain, 43 surface-associated proteins were identified, including all the protective antigens described in the literature as well as a new protective antigen, the cell wall-anchored protein SAN_1485 belonging to the serine-rich repeat protein family. This strategy overcomes the difficulties so far encountered in the identification of novel vaccine candidates and speeds up the entire vaccine discovery process by reducing the number of recombinant proteins to be tested in the animal model.

Systemic sclerosis (SSc) is an autoimmune disease characterized byendothelial dysfunction and fibrosis of the skin and internal organs [1].Cardiac involvement in the course of SSc could be primary or sec-ondary to pulmonary arterial hypertension and kidney pathology. Pri-mary cardiac involvement may manifest as myocardial involvement,arrhythmias,fibrosisoftheconductionsystem,pacemakerandintracar-diac defibrillator implantation, sudden death and autonomic dysfunc-tion [2,3].Amongtheabnormalities in SSc,QTc intervalprolongation hasbeenreportedandassociatedwithanti-RNApolymeraseIIIantibodies,longerdisease duration, and greater disease severity [4].Accordingly,theaimofthestudyistoassessQTcprolongationinSScand evaluate correlations with clinical variables and complications ofthe disease.The protocol is in accordance with the Declaration of Helsinki andwas approved by the Ethics Committee at our Institution. Twenty Cau-casian subjects fulfilling the American Rheumatism Association (ARA)criteria for classification and diagnosis of SSc [5] and twenty healthysubjectswereexaminedbeforeinclusioninthestudy.All examined pa-tientsunderwentclinicalevaluation,ambulatory24-hourECGmonitor-ing, transthoracic echocardiogram.Scleroderma patients with coronary artery disease, congestiveheartfailure, left ventricular dysfunction, significant valvular abnormalitiesand arrhythmias were not included in the study.Patients with pulmonary function abnormalities were not includedeither. Patients with diabetes mellitus, renal failure, hepatic or thyroiddysfunction, and anemia were excluded.Patients were not takingβ-blockers, antiarrhythmic drugs,ACE-inhibitors or angiotensin receptor antagonists. QTc interval,assessed by 24-hour Holter ECG recording, was defined as prolongedwhen N440 ms [6].Nailfold videocapillaroscopy (NVC) was performed by an opticalprobe, equipped with magnification 200× contact lens and connectedto image analysis software (Pinnacle Studio Version 8). The identifiedpatterns were classified as early, active or late [7]. Skin thickening wasassessed by a modified Rodnan total skin score (mRSS) [8]. In all pa-tientswemeasuredtheDiseaseActivityIndex(DAI)andDiseaseSever-ity Scale (DSS) [9].Thedatawereexpressedasmedianandrange.Multivariateanalysiswas applied for the estimation of relationship of QTc with disease vari-ables. The Mann–Whitney U-test or Kruskal–Wallis were used to testdifferences between two individual study groups. Spearman's rankorder correlation coefficient (r) was used to test for an association be-tweennumericalvariables.p-Valuesb0.05wereconsideredsignificant.Commercial software (SPSS version 20.0) was used for statisticalanalysis.Twentysclerodermapatientswereenrolledinthecurrentstudy.Theepidemiological and clinical features of SSc are shown in Table 1.ThemedianvalueofQTcissignificantly(pb0.0001)increasedinSScpatients than healthy controls [447 (414–566) vs 386 (342–447)].The medianvalueof QTc is significantly (p b 0.01) different in threecapillaroscopicgroups:early425(421 –454),active437(416–467),late471 (445–566).ThemedianvalueofQTcissignificantly(pb 0.05)augmentedin SScpatients with digital ulcers than in SSc patients without digital ulcers[459(422–566)vs436(416–454)].Apositivecorrelation (p b0.05)ex-ists between QTc and mRSS (r = 0.53).NocorrelationswerefoundbetweenQTcanddiseasesubset,diseaseduration, Disease Activity Index, and Disease Severity Scale.Amongthe causes of suddencardiacdeath in SScpatients,ventricu-lar late potentials, autonomic dysfunction, pro-arrhythmogenic drugsand increased QT dispersion were found.

The aim of this study was to analyze the type of immune response (Th1, Th2) and protein composition of bronchoalveolar lavage (BAL) of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Flow cytometry analysis of intracellular cytokines revealed different patterns: in IPF and SSc Th2 profiles were predominant, whereas in sarcoidosis Th1 prevailed. The proteomic analysis of BAL fluid (BALF) showed that there were quantitative differences between the three diseases. These were more evident between sarcoidosis and IPF, confirming our previous observations, whereas SSc had an intermediate profile between the two, however with some peculiarities. Comparison of BALF protein maps, constructed with the same quantity of total proteins, enabled us to identify the main profiles of the three diseases: an increase in plasma protein prevalent in sarcoidosis and also present in SSc, though for fewer proteins with respect to IPF and a greater abundance of low molecular weight proteins, mainly locally produced, in IPF. These findings are in line with the different pathogenesis of these diseases: IPF is considered a prevalently fibrotic disorder limited to the lung, with intense local production of functionally different proteins, whereas sarcoidosis and SSc are systemic immunoinflammatory diseases.

Human milk is a source of bioactive substances regulating the development and activity of the newborn immune system. Human milk has been found to contain a number of cytokines, including interleukins, growth factors, and colony stimulating factors. In the present study, we assessed 10 specimens of human milk for the presence of macrophage migration inhibitory factor (MIF), a cytokine recently described in several human reproductive organs and tissues. Using biochemical as well as immunologic techniques, we showed that MIF is abundantly present in human milk, mostly distributed in the lipid layer and in the aqueous phase. Fractionation of the lipid layer showed that MIF is highly concentrated inside milk fat globules. In view of its proinflammatory features, we speculate that milk MIF may protect the newborn against infection and play a role in preserving the functionality of the lactating mammary gland. Furthermore, the localization of MIF in lipid globules suggests a possible strategy for the protection of milk cytokines from the gastric barrier.

New generation vaccines are in demand to include only the key antigens sufficient to confer protective immunity among the plethora of pathogen molecules. In the last decade, large-scale genomics-based technologies have emerged. Among them, the Reverse Vaccinology approach was successfully applied to the development of an innovative vaccine against Neisseria meningitidis serogroup B, now available on the market with the commercial name BEXSERO® (Novartis Vaccines). The limiting step of such approaches is the number of antigens to be tested in in vivo models. Several laboratories have been trying to refine the original approach in order to get to the identification of the relevant antigens straight from the genome. Here we report a new bioinformatics tool that moves a first step in this direction. The tool has been developed by identifying structural/functional features recurring in known bacterial protective antigens, the so called "Protectome space," and using such "protective signatures" for protective antigen discovery. In particular, we applied this new approach to Staphylococcus aureus and Group B Streptococcus and we show that not only already known protective antigens were re-discovered, but also two new protective antigens were identified.

The administration of statins in patients with liver disease is not an absolute contraindication. Hepatotoxicity is a rare and often dose-related event and in the literature there are only a few described cases of fatal rhabdomyolysis in patients with chronic liver disease after statin administration. During treatment with 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, the factors responsible for myopathy may either be related to the patient, or due to interactions with other medications that are metabolic substrates of the same isozymes and therefore able to increase blood statin concentration. The most important side effects consist of increased transaminase levels, abdominal pain or muscle weakness, increased serum levels of creatine kinase and rhabdomyolysis. In this article we report a case of fatal rhabdomyolysis with acute renal failure after gastric endoscopy, where midazolam was used as a sedation agent in a patient with chronic liver disease treated with a high dose of atorvastatin. Therefore, we suggest paying particular attention to the potential risks of associating atorvastatin and midazolam in patients with chronic liver disease who need to undergo gastric endoscopy.

T regulator lymphocytes (Tregs) play a key role in the maintenance of immune tolerance and in the development of autoimmune diseases. Expression of Foxp3 is specific for Tregs, and can be used for the identification of these cells. This study investigated the variations of Tregs Foxp3+ in the kidney biopsies inflammatory infiltrate of different lupus nephritis classes compared to that of ANCA glomerulonephritis, acute tubulointerstitial nephritis and nephroangiosclerosis. Sections of paraffin-embedded tissue have been stained by immunohistochemistry with anti-CD3 and anti-FoxP3 antibodies. We find that the ratio of FoxP3+/CD3+ cells is significantly lower in patients with lupus nephritis class IV and in patients with vasculitides than in the course of nephroangiosclerosis, tubulointerstitial nephritis and lupus nephritis class V. The data presented herein demonstrate a decrease of FoxP3+ Treg cells in the inflammatory infiltrate of lupus nephritis, particularly during the most active phases of lupus nephritis, as observed in the course of a IV class nephritis.

Despite the global medical needs associated with Staphylococcus aureus infections, no licensed vaccines are currently available. We identified and characterized a protein annotated as an epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. In addition, we determined the structure of the recombinant protein (rEpiP) by X-ray crystallography. The crystal structure revealed that rEpiP was cleaved somewhere between residues 95 and 100, and we found that the cleavage occurs through an autocatalytic intramolecular mechanism. The protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine whether the protein acts as a serine protease, we mutated the hypothesized catalytic serine 393 residue to alanine, generating rEpiP-S393A. The crystal structure of this mutant protein showed that the polypeptide chain was not cleaved and was not interacting stably with the active site. Indeed, rEpiP-S393A was shown to be impaired in its protease activity. Mice vaccinated with rEpiP were protected from S. aureus infection (34% survival, P=0.0054). Moreover, the protective efficacy generated by rEpiP and rEpiP-S393A was comparable, implying that the noncleaving mutant could be used for vaccination purposes.—Kuhn, M. L., Prachi, P., Minasov, G., Shuvalova, L., Ruan, J., Dubrovska, I., Winsor, J., Giraldi, M., Biagini, M., Liberatori, S., Savino, S., Bagnoli, F., Anderson, W. F., Grandi, G. Structure and protective efficacy of the Staphylococcus aureus autocleaving protease EpiP.

Systemic sclerosis (SSc) is a systemic disease of connective tissue,characterized by endothelial dysfunction and fibrosis of the skin andvisceral organs [1]. Autonomic dysfunction (AD) is a feature of SSc,starting early in the disease and possibly preceding the developmentof fibrosis [2].Several manifestations of renal involvement occur in SSc and wehave recently demonstrated that renal resistive index (RI) correlateswith glomerular filtration rate (GFR), digital microvascular damage [3]and in monitoring scleroderma renal crisis [4].Accordingly, the aim of the study is to assess intrarenal arterialstiffness by Doppler ultrasound and examine the correlation with ADusing heart rate variability (HRV) analysis.The protocol is in accordance with the Declaration of Helsinki andwas approved by the Ethics Committee at our Institution. NineteenCaucasian subjects fulfilling the American Rheumatism Association(ARA) criteria for classification and diagnosis of SSc [5] and nineteenhealthysubjectswereexaminedbeforeinclusioninthestudy.Allexam-ined patients underwent clinical evaluation, 24-h Holter monitoring,electrocardiography (ECG) and transthoracic echocardiogram.Scleroderma patients with coronary artery disease, congestiveheartfailure, left ventricular dysfunction, significant valvular abnormalitiesand arrhythmias were not included in the study.Patients with pulmonary function abnormalities were not includedeither. Patients with diabetes mellitus, renal failure, hepatic or thyroiddysfunction and anaemia were excluded.Patients were not taking β-blockers, antiarrhythmic drugs, ACE-inhibitors or angiotensin receptor antagonists. GFR was calculatedwith CKD-EPI equation [6].Autonomic nervous activity was evaluated by heart rate variability(HRV) analysis during 24-hour ECG recording. Autonomic nervousactivity was analyzed following the recommendations of the TaskForce of the European Society of Cardiology and the North AmericanSociety of Pacing and Electrophysiology [7]. Total power in the fre-quency range (0–0.40 Hz) was divided into low frequency (LF:0.04–0.15 Hz, modulated mainly by sympathetic system) and highfrequency (HF: 0.15–0.40 Hz, modulated by parasympathetic system).The power of LF and HF components was considered in normalizedunits (nu). LF/HF rate is sympathovagal balance. Artificial data andarrhythmic were excluded. Data analyses were performed with soft-ware Del Mar Avionics Accuplus 363, Irvine California, USA.Renal Doppler ultrasound was performed using a Toshiba AplioUltrasound System SSA-790 (Tokyo, Japan) equipped with convex 3.5-MHz probe. RI was calculated as (peak systolic frequency shift −minimum diastolic frequency shift) / peak systolic frequency shift.All the ultrasound examinations were performed by same blindedphysician in order to reduce variability in the assessment managementof the study.Thedatawereexpressedasmedianandrange.Multivariateanalysiswas applied for the estimation of relationship of HRV variables withrenal Doppler indices or disease variables. The Mann–Whitney U-testor Kruskal–Wallis was used to test differences between two individualstudy groups. Spearman's rank order correlation coefficient (r) wasused to test for an association between numerical variables. p-Valuesb0,05 were considered significant. Commercially software (SPSSversion 20.0) was used for statistical analysis.Nineteen scleroderma patients were enrolled in the current study.Median age of 45 years (range 23–58) and a median SSc duration of6,5 years (range 2–25) were investigated in the Clinical ImmunologyUnit-Scleroderma Center. Six patients had limited cutaneous SSc and

The airway surface is covered by a fluid, the airway surface liquid, interposed between the mucous layer and the epithelium. The airway surface liquid contains proteins, secreted by different cell types, that may have pro-/anti-inflammatory or bactericidal functions or have a role in the mucociliary clearance. We have used a proteomics approach to identify the proteins secreted by an isolated in vitro model of human airway epithelium, at resting and under proinflammatory conditions, as a strategy to define the factors involved in epithelial barrier function. To this aim, we have analyzed the airway surface liquid from human bronchial epithelial cells grown as polarized monolayers in the presence and absence of inflammatory stimuli such as IL-4, IL-1beta, TNF-alpha, and IFN-gamma. Two-dimensional electrophoresis followed by mass spectrometry analysis has allowed the identification of approximately 175 secreted protein spots, among which are immune-related proteins, structural proteins, an actin severer, some protease inhibitors, and a metalloproteinase. Comparisons between treated and untreated conditions have shown that the expression of several proteins was significantly modified by the different cytokines. Our results indicate that the surface epithelium is an active player in the epithelial barrier function and that inflammatory conditions may modulate protein secretion.

Macrophage migration inhibitory factor (MIF) was originally identified for its capacity to inhibit the random migration of macrophages in vitro. To date, the role of MIF as a pro-inflammatory cytokine, pituitary hormone, and counter-regulator of glucocorticoid action on the immune response is commonly recognized. Although recent studies suggest an involvement of MIF in reproduction, no data exist on the expression of this cytokine in early human pregnancy. In this study, we evaluated the presence of MIF protein and mRNA in specimens of chorionic villi from first-trimester human placenta. Tissues were obtained at 6-10 wk of gestation and analyzed by Western blotting, reverse transcription-polymerase chain reaction, and immunohistochemistry. Our results demonstrate that human villous tissue is a novel site of MIF synthesis. In addition, immunohistochemical analysis identified MIF protein in the cytotrophoblasts of both the inner layer of villi and in the trophoblastic cell islands. We speculate that in view of its proinflammatory features, MIF might play a critical role in human implantation and in early embryonic development.

RATIONALE: The airway surface liquid, the thin layer of liquid covering the airways, is essential for mucociliary clearance and as a barrier against microbial and other noxious agents. Proteins secreted into the airway surface liquid by epithelial and nonepithelial cells may be important in innate immunity and to improve the fluidity of mucous secretions. OBJECTIVES: We aimed to identify proteins specifically secreted into the airway surface liquid by human bronchial epithelial cells, in resting conditions and after treatment with interleukin 4 (IL-4), a cytokine released in asthma. METHODS AND MAIN RESULTS: By using a proteomics approach, we found that one of the most abundant proteins was gelsolin, which breaks down actin filaments. Gelsolin mRNA and protein secretion were increased threefold in the airway surface liquid of epithelia treated with IL-4. These results were confirmed at the functional level by measuring actin depolymerization using a fluorescence assay. Gelsolin protein was also upregulated in the airways of subjects with asthma. CONCLUSIONS: Our findings indicate that gelsolin is released by epithelial cells into the airways and that its secretion is increased by IL-4 in vitro. In addition, we found that the concentration of both IL-4 and gelsolin were raised in the bronchoalveolar lavage of patients with asthma. These results suggest that gelsolin might improve the fluidity of airway surface liquid in asthma by breaking down filamentous actin that may be released in large amounts by dying cells during inflammation.

With the final aim to investigate about the possible ability of municipal sludge to adsorb heavy metals from industrial wastes, a characterization study has been performed for anaerobic sludges. The results are compared with those previously obtained for aerobic sludges. In order to evaluate the possibility to mobilize heavy metals (Cu(II), Zn(II), Cr(III), Mn(II), Ni(II), Pb(II), and Cd(II)) from the biomatrix a sequential extraction method was employed. The method is based on the consideration that i) heavy metals are present in the native biomatrix under complex or association forms with inorganic or organic ligands characterized by different hydrophilicity and that ii) the equilibria are likely affected by ionic strength and pH. As extracting reagents, non polar solvents (like CH 2 Cl 2 ) , polar salt (KNO 3 ), acidic media (CO 2 saturated water pH 4 and HCI pH 2) and coordinating agents in alkaline medium (EDTA in NaOH pH=9) were used as extraction agents. In order to check for the possible readsorption of metals taking place during the sequential extraction, a comparison was made by using the extracting reagents sequentially or in a single extraction. In the different extracts obtained, metal analysis was performed by AAS and ligand characterization by FT-IR and ion chromatography. The method has been applied to sludges collected in the municipal waste water treatment plant Consorzio Po-Sangone, serving the town of Torino. The solubilization percentual amount obtained with the use of the mentioned extractants, has reached sequential extraction values around 80.3%, referred to the total DS. Partial values obtained for the single extractions are comparable. The results indicate that species characterized by hydrophilic properties predominate and that preferential solubilizations take place for complexing competition reactions in alkaline medium. Among the metals present (Cd(II), Cu(II), Ni(II), Mn(II), Zn(II), Cr(III) and Pb(II)) it results that only Cu(II),Mn(II), Cr(III) and Zn(II) could be present in non polar species, while all of them seem to be present in prevalently polar complexes formed with proteins and carbohydrates or under ionic association with chloride, nitrate, sulfate, phosphate and hydrogenocarbonate.

Authors present the case of a 37-year-old man admitted to the emergency room of Universidade Federal de São Paulo-Escola Paulista de Medicina, with hyperosmolar coma, following progressive muscle discomfort and loss of renal function, with further need of dialysis therapy. Initial laboratory evaluation showed marked hyperglycemia, hypernatremia, hyperosmolarity, and high levels of creatinine. In the evolution he presented an elevation of creatino-phosphokinase levels in parallel with increasing levels of urea and creatinine. Urinalyses showed progressive increase in proteinuria and hematuria. A muscle biopsy was performed and confirmed the presence of muscular necrosis. The purpose of this paper is to emphasize hyperosmolarity as a newly described cause of rhabdomyolysis. The authors point out its multifactorial physiopathology and also stress the relatively common occurrence of acute renal failure (ARF) following an episode of rhabdomyolysis, and the poor prognosis that this complication represents.

In the human pathogen Staphylococcus aureus, there exists an enormous diversity of proteins containing DUFs (domains of unknown function). In the present study, we characterized the family of conserved staphylococcal antigens (Csa) classified as DUF576 and taxonomically restricted to Staphylococci. The 18 Csa paralogues in S. aureus Newman are highly similar at the sequence level, yet were found to be expressed in multiple cellular locations. Extracellular Csa1A was shown to be post-translationally processed and released. Molecular interaction studies revealed that Csa1A interacts with other Csa paralogues, suggesting that these proteins are involved in the same cellular process. The structures of Csa1A and Csa1B were determined by X-ray crystallography, unveiling a peculiar structure with limited structural similarity to other known proteins. Our results provide the first detailed biological characterization of this family and confirm the uniqueness of this family also at the structural level. We also provide evidence that Csa family members elicit protective immunity in in vivo animal models of staphylococcal infections, indicating a possible important role for these proteins in S. aureus biology and pathogenesis. These findings identify the Csa family as new potential vaccine candidates, and underline the importance of mining the bacterial unknown proteome to identify new targets for preventive vaccines.

Advanced cirrhosis is frequently associated with renal dysfunction. Hepatorenal syndrome (HRS) is characterized by the occurrence of kidney injury in cirrhotic patients in the absence of other identifiable causes. HRS is classified in 2 different types. Type 1 is characterized by acute renal failure and rapid functional deterioration of other organs, usually related to a precipitating event. Type 2 is characterized by slowly progressive renal failure and refractory ascites. Advanced liver disease induces the progression of hemodynamic alterations such as arterial vasodilation of splanchnic circulation and impairment of cardiac function. The resulting ineffective circulating blood volume promotes the activation of both the renin-angiotensin-aldosterone and sympathetic nervous system, by an increase of antidiuretic hormone activity, in an attempt to restore volemia. Despite fluid retention, ascites and dilutional hyponatremia, renal function is often initially preserved by renal production of vasodilators. However, further insults can lead to an imbalance between systemic vasoconstriction and local renal vasodilation, resulting in progressive renal failure. Over the last decade, clinical strategies to prevent HRS have been improved by a better understanding of the natural history of renal failure in cirrhosis, resulting in a reduction of HRS prevalence in cirrhotic patients. Vasoconstrictor drugs may improve renal function, but the effect on mortality has not yet been established. Vaptans, nonpeptide vasopressin receptor antagonists, may also reduce hyponatraemia and ascites, even if the clinical effects in HRS remain unknown. This review updates the pathophysiology, diagnosis and management of HRS.

Patients with nephrotic syndrome are at increased risk for thromboembolic events such as deep venous and arterial thrombosis, renal vein thrombosis and pulmonary embolism. This thrombophilic phenomenon has been attributed to a "hypercoagulable" state in which an imbalance between naturally occurring pro-coagulant/pro-thrombotic factors and anti-coagulant/antithrombotic factors promotes in situ thrombosis in deep veins or arteries. Management of thromboembolic events may be divided in prophylactic and therapeutic strategies. Hypoalbuminemia is the most significant independent predictor factor of thrombotic risk, especially for values

A case of two incidental familial intracranial aneurysms associated with asymptomatic coarctation of the aorta is reported. Priority was assigned to aneurysm surgery, which was accomplished by means of two distinct procedures, both requiring moderate, deliberate hypotension. Invasive monitoring always included radial and femoral as well as pulmonary artery pressures, together with mixed venous oximetry and thermodilution cardiac outputs. Spinal somatosensory-evoked potentials were also continuously registered, to prevent spinal cord ischemia. In fact, even moderate hypotension may critically reduce perfusion in the lower, already hypotensive district, because of coarctation.

SpyCEP is a 170-kDa multidomain serine protease expressed on the surface of the human pathogen Streptococcus pyogenes, which plays an important role in infection by catalyzing cleavage and inactivation of the neutrophil chemoattractant interleukin-8. In this study, we investigated the biochemical features and maturation process of SpyCEP, starting from a recombinant form of the protease expressed and purified from Escherichia coli. We show that active recombinant SpyCEP differs from other bacterial proteases in that it is constituted by 2 noncovalently linked fragments derived from autocatalytic processing, an N-terminal fragment of 210 aa bearing one of the 3 catalytic triad residues, and a 1369-residue C-terminal polypeptide containing the remaining 2 catalytic amino acids. The same type of organization is present in the enzyme obtained from S. pyogenes. Furthermore, N-terminal SpyCEP is not involved in the folding of the mature enzyme. The 2 protease fragments were separately expressed in E. coli as soluble polypeptides that, when combined, reconstituted a fully active enzyme complex. Therefore, SpyCEP appears to possess a completely new structural architecture that has not been described so far for other microbial proteases.

Extraintestinal pathogenic Escherichia coli are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis, or septicemia. In this study, we focused our attention on the identification of the outer membrane proteins of the pathogen in consideration of their important biological role and of their use as potential targets for prophylactic and therapeutic interventions. To this aim, we generated a DeltatolR mutant of the pathogenic IHE3034 strain that spontaneously released a large quantity of outer membrane vesicles in the culture supernatant. The vesicles were analyzed by two-dimensional electrophoresis coupled to mass spectrometry. The analysis led to the identification of 100 proteins, most of which are localized to the outer membrane and periplasmic compartments. Interestingly based on the genome sequences available in the current public database, seven of the identified proteins appear to be specific for pathogenic E. coli and enteric bacteria and therefore are potential targets for vaccine and drug development. Finally we demonstrated that the cytolethal distending toxin, a toxin exclusively produced by pathogenic bacteria, is released in association with the vesicles, supporting the recently proposed role of bacterial vesicles in toxin delivery to host cells. Overall, our data demonstrated that outer membrane vesicles represent an ideal tool to study Gram-negative periplasm and outer membrane compartments and to shed light on new mechanisms of bacterial pathogenesis.

Background Identifying the impact of coexisting patellofemoral and tibiofemoral osteoarthritis (OA) seems to be important, since the knees with structural changes in both compartments are commonly observed and are more likely to be painful and with function impairments. Objectives To compare the symptoms and physical function in individuals with patellofemoral and tibiofemoral osteoarthritis coexisting and individuals with tibiofemoral osteoarthritis isolated. Methods It were selected men and women (40-65 years old). They were diagnosed with knee OA according to the ACR ( American College of Rheumatology ) criteria and classified regarding the level of severity disease using Kellgren and Lawrence (1957). They were divided into two groups: patellofemoral and tibiofemoral osteoarthritis (PFTFOA) and tibiofemoral osteoarthritis (TFOA) isolated. All the participants had to present OA grade 2 in the tibiofemoral compartment, uni or bilaterally, and to be included in the PFTFOA group, they had also to present signs of OA in patellofemoral compartment. Self-reported symptoms and physical function were assessed by the subscores of Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Performance-based tests to assess physical functional were used (30s Chair Stand Test, 40m Fast-paced Walk Test and Stair Climb Test). Results Fifty eight subjects (23 male and 35 female) with mild knee OA were included in this study. The PFTFOA was composed by 30 subjects (12 male, 18 female, mean age 55±7, height 1,66±0,08, weight 79,6±13,5 and BMI 28,9±3,8) and the TFOA was composed by 28 subjects (11 male, 17 female, mean age 53±7, height 1,67±0,11, weight 75,6±14,1 and BMI 27±3,3). The groups were homogeneous for age (P=0.35), height (P=0.59), weight (P=0.15) and BMI (P=0.05). There were no differences between groups for self-reported pain (P=0.22), stiffness (P=0.20), physical function (P=0.71) and total WOMAC score (P=0.57). There were no differences between groups for Performance-based tests using 30s Chair Stand Test (P=0.63), 40m Fast-paced Walk Test (P=0.89) and Stair Climb Test (P=0.15). Conclusions The findings from the current study showed no significant differences between the groups PFTFOA and PFOA in regard to pain, stiffness and physical function, as evaluated by self-reported and performance-based tests. References Dobson et al., 2013, OARSI recommended performance-based tests to assess physical function in osteoarthritis of the hip or knee: authors9 reply: Osteoarthritis and Cartilage, v. 21, p. 1625-1626. Duncan et al. 2008, How do pain and function vary with compartmental distribution and severity of radiographic knee osteoarthritis?: Rheumatology (Oxford), v. 47, p. 1704-7. Farrokhi et al. 2013, Severity of Coexisting Patellofemoral Disease is Associated with Increased Impairments and Functional Limitations in Patients with Knee Osteoarthritis: Arthritis Care Res (Hoboken), v. 65, p. 544-51. Stefanik et al. 2013, Using magnetic resonance imaging to determine the compartmental prevalence of knee joint structural damage: Osteoarthritis Cartilage, v. 21, p. 695-9. Acknowledgements The authors would like to acknowledge the supported financially by Higher Education Personnel Training Coordination (Capes) and by Support Foundation to Research of Sao Paulo State (FAPESP). Disclosure of Interest None declared

Among the causes of malnutrition in hemodialysis (HD) patients, inadequate dietary intake (IDI) seems to be one of the most frequent and important. Although it has been hypothesized that IDI might be secondary to uremia, anorexia, underlying illness, psychosocial conditions, loss of dentures, depression, aging, or chronic inflammation, definite data on the etiology of IDI in HD patients are still lacking. The goal of this study was to measure the actual dietary energy and protein intakes in stable HD patients and to evaluate which demographic, clinical, dialytic, and humoral variables were associated with a dietary intake lower than recommended by international guidelines.Thirty-seven patients maintained on regular HD, 3 times per week for 4 hours per session, were included in the study. In addition to epidemiologic data, patients were scrutinized for dry weight, weight change in the last 6 months, height, Body Mass Index, Kt/V, serum leptin, leptin-BMI ratio, presence of anorexia, and dietary energy and protein intake. Anorexia was assessed by means of a questionnaire in which the presence of major symptoms, namely meat aversion, taste and smell alterations, nausea and/or vomiting, and early satiety, was investigated. Dietary intake was recorded for 3 days after questionnaire administration by means of 3-day diet diaries.Overall, the mean (+/- standard deviation) dietary energy and protein intakes were 24.9 +/- 10.1 kcal/kg/day and 0.64 +/- 0.4 g protein/kg/day, respectively. Twenty-six patients (70.2%) had energy and protein intakes lower than recommended, 7 (18.9%) had adequate energy intake but inadequate protein intake, 1 (2.7%) had adequate protein intake and inadequate energy intake, and 3 (8.1%) had both adequate energy and adequate protein intakes. Anorexia was present in 14 of the 26 (53%) patients with low protein and energy intakes, and was absent in the other groups ( P =.003). The age of patients with inadequate energy and protein intakes was significantly higher than the age of patients with adequate energy and protein intakes (62.1 +/- 10.4 versus 37 +/- 20.8, P.001) and the age of patients with only adequate energy intake (40.5 +/- 10.4, P.001). Twenty-seven patients (73%) had an energy intake30 kcal/kg/day, and 10 (27%) had an energy intakeor =30 kcal/kg/day. Compared with patients with energy intakesor =30 kcal/kg/day, patients with energy intakes30 kcal/kg/day were significantly older ( P =.0001) and more frequently were anorexic (P.05). Compared with patients with protein intakesor =1.2 g/kg/day, patients with protein intakes1.2 g/kg/day were significantly older (P.001). Limiting the analysis to the 33 patients with protein intakes1.2 g/kg/day, we found a significant negative correlation between age and energy intake ( r =-0.612; P.001) and between age and protein intake ( r =-0.723; P.001). Correlations between both energy and protein intakes and age, dialytic age, Kt/V, C-reactive protein, parathyroid hormone, and leptin-BMI were not statistically significant.This study shows that dietary energy and protein intakes are inadequate in the majority of HD patients and are negatively related to the presence of anorexia and age. These data may be potentially useful in the identification of nutritional strategies as well as in improving food intake in HD patients.

We previously reported that photodynamic therapy (PDT) using Purpurin-18 (Pu-18) induces apoptosis in HL60 cells. Using flow cytometry, two-dimensional electrophoresis coupled with immunodetection of carbonylated proteins and mass spectrometry, we now show that PDT-induced apoptosis is associated with increased reactive oxygen species generation, glutathione depletion, changes in mitochondrial transmembrane potential, simultaneous downregulation of mitofilin and carbonylation of specific proteins: glucose-regulated protein-78, heat-shock protein 60, heat-shock protein cognate 71, phosphate disulphide isomerase, calreticulin, beta-actin, tubulin-alpha-1-chain and enolase-alpha. Interestingly, all carbonylated proteins except calreticulin and enolase-alpha showed a pI shift in the proteome maps. Our results suggest that PDT with Pu-18 perturbs the normal redox balance and shifts HL60 cells into a state of oxidative stress, which systematically induces the carbonylation of specific chaperones. As these proteins normally produce a prosurvival signal during oxidative stress, we hypothesize that their carbonylation represents a signalling mechanism for apoptosis induced by PDT.

Background and Aims: Hyperleptinemia is a common feature in hemodialysis (HD) patients. However, the role of increased serum leptin levels in the pathogenesis of HD-related anorexia is still controversial. The purpose of the present prospective study was to ascertain whether hyperleptinemia is causally implicated in the pathogenesis of HD-related anorexia. Methods: We measured the serum leptin levels and the serum leptin/body mass index (BMI) ratio in 24 healthy subjects and in 49 end-stage renal disease patients on maintenance HD. HD patients were subdivided into anorexic (14/49, 28.5%) and non-anorexic (35/49, 71.5%) according to a questionnaire discriminating for the presence of anorexia-related symptoms. Results: Calorie (kcal/kg/day) and protein (g/ kg/day) intakes were significantly lower in anorexic than in non-anorexic patients (20.1 ± 1.1 vs. 27.9 ± 1.3, p = 0.004, and 0.82 ± 0.05 vs. 1.19 ± 0.05, p = 0.001, respectively). Accordingly, serum albumin, total lymphocyte count, mid-arm muscle circumference, and the protein equivalence of nitrogen appearance (PNA) were significantly lower in anorexic patients. The serum leptin concentration (ng/ml) was significantly higher in HD patients than in controls, in males (15.33 ± 3.4 vs. 3.7 ± 0.3, p = 0.003) and in females (42.3 ± 7.2 vs. 10.5 ± 1.3, p = 0.03). Similarly, serum leptin/BMI ratio was significantly higher in HD patients than in controls, in males (0.56 ± 0.1 vs. 0.16 ± 0.02, p = 0.0028) and in females (1.8 ± 0.2 vs. 0.4 ± 0.04, p < 0.0001). However, serum leptin levels were similar in anorexic and in non-anorexic patients, in males (15.3 ± 5.6 vs. 16.9 ± 4.2, p = 0.85) and in females (46.6 ± 12.9 vs. 47.4 ± 9.4, p = 0.96). No differences were observed between the 2 groups in the serum leptin/BMI ratio, in males (0.59 ± 0.2 vs. 0.58 ± 0.14, p = 0.92) and in females (1.5 ± 0.4 vs. 1.8 ± 0.3, p = 0.94). Similarly, no statistically significant differences in terms of serum leptin levels and leptin/BMI ratio were observed between patients with dietary energy intake of Conclusion: The present results indicate that mechanisms other than increases in serum leptin levels might be involved in the pathogenesis of HD-related anorexia.

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed.

The THIN-B metallo-β-lactamase, a subclass B3 enzyme produced by the environmental species Janthinobacterium lividum , was overproduced in Escherichia coli by means of a T7-based expression system. The enzyme was purified (>95%) by two ion-exchange chromatography steps and subjected to biochemical analysis. The native THIN-B enzyme is a monomeric protein of 31 kDa. It exhibits the highest catalytic efficiencies with carbapenem substrates and cephalosporins, except for cephaloridine, which acts as a poor inactivator. Individual rate constants for inactivation by chelators were measured, suggesting that inactivation occurred by a mechanism involving formation of a ternary complex.