Aptamers binding to Bradykinin

Bradykinin is a peptide of the kinin group, involved in a number of receptor-mediated physiological actions, including inflammation and vasodilation, as well as neuromodulation, neuroprotection and promotion of neurogenesis. Bradykinin is the main ligand of the B2 receptor- the main kinin receptor- which is involved in the cardiac and renal protective effects of kinins in diseases. Antibodies have been considered for a long time as promising therapeutic agents in various fields, especially cancer-related ones. Aptamers, on the other hand, have proven to be an excellent alterative, since they have similar properties to those of monoclonal antibodies, such a high-specificity of recognition and high-affinity binding. Plus, they are developed using in vitro selection procedures and can be reproduced by enzymatic reactions. SELEX is a powerful tool for the development of both DNA and RNA aptamers. The main goal of this project was to design a method to select aptamers against bradykinin using capillary electrophoresis alongside the SELEX technique. The selection was done by comparing the aptamers’ (ssDNA-target complex) electrophoretic mobility with that of the ssDNA and the target, which allowed us to define an appropriate collection window that took into consideration the analytes’ detection time, thus enabling the collection of the desired oligonucleotides. After two selection rounds, the collected pool was sequenced, the affinity was measured and the aptamers’ secondary structure was predicted. We concluded that with only two selection cycles, the original DNA library’s bulk affinity grew around 0.4%. The structural characterization of the aptamers, performed with the aid of the Mfold software, revealed that there are many repetitive motifs amongst them, indicating that the selection process was successful. We have obtained 16 sequences of candidate aptamers as bradykinin ligands of similar sequences and secondary structures whose biological activity should be analyzed after synthesis; mainly in regard to their role as bradykinin inhibitors.

MMP-9/RECK imbalance: a mechanism associated with high-grade cervical lesions and genital infection by Human Papillomavirus (HPV) and Chlamydia trachomatis

"Manuscript"

Seasonal variation of the effect of high-carbohydrate and high-protein diets on the intermediate metabolism of Parastacus brasiliensis (Crustacea, Decapoda, Parastacidae) maintained in the laboratory

The goal of this study was to evaluate the effect of a high-carbohydrate diet (HC) and a high-protein diet (HP) on the metabolism of the crayfish Parastacus brasiliensis (Von Martens, 1869), collected in different seasons and maintained in the laboratory for 15 days. Crayfish were collected monthly from January 2002 to January 2004 at São Francisco de Paula, Southern Brazil, in Guarapirá stream. In the laboratory, the animals were kept submerged in aquariums under controlled conditions. They were fed ad libitum, for 15 days with either a HC or HP diet. At the end of this period, haemolymph samples were collected, as were hepatopancreas, gills, and abdominal muscle that were removed for determination of glycogen, free glucose, lipids, and triglycerides. The haemolymph samples were used for determination of glucose, proteins, lipids, and triglycerides. Statistical analysis (ANOVA) revealed significant seasonal differences in biochemical composition in crayfish maintained on HC or HP diets. Independent of the diets offered to the animals and the controlled conditions for 15 days, the indications of seasonality were unchanged. The observed changes seemed to be related to the reproductive period. Moreover, the HC diet increased all energy reserves in adult parastacids, which may aid in reproduction.

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

Updated response assessment criteria for high-grade gliomas: response assessment in neuro-oncology working group.

Currently, the most widely used criteria for assessing response to therapy in high-grade gliomas are based on two-dimensional tumor measurements on computed tomography (CT) or magnetic resonance imaging (MRI), in conjunction with clinical assessment and corticosteroid dose (the Macdonald Criteria). It is increasingly apparent that there are significant limitations to these criteria, which only address the contrast-enhancing component of the tumor. For example, chemoradiotherapy for newly diagnosed glioblastomas results in transient increase in tumor enhancement (pseudoprogression) in 20% to 30% of patients, which is difficult to differentiate from true tumor progression. Antiangiogenic agents produce high radiographic response rates, as defined by a rapid decrease in contrast enhancement on CT/MRI that occurs within days of initiation of treatment and that is partly a result of reduced vascular permeability to contrast agents rather than a true antitumor effect. In addition, a subset of patients treated with antiangiogenic agents develop tumor recurrence characterized by an increase in the nonenhancing component depicted on T2-weighted/fluid-attenuated inversion recovery sequences. The recognition that contrast enhancement is nonspecific and may not always be a true surrogate of tumor response and the need to account for the nonenhancing component of the tumor mandate that new criteria be developed and validated to permit accurate assessment of the efficacy of novel therapies. The Response Assessment in Neuro-Oncology Working Group is an international effort to develop new standardized response criteria for clinical trials in brain tumors. In this proposal, we present the recommendations for updated response criteria for high-grade gliomas.

Use of the 2,3-diacyl-trehalose and the purified protein derivative in the serodiagnosis of tuberculosis in AIDS

The effect of the human immunodeficiency virus (HIV) infection on IgG production against purified protein derivative (PPD) and 2,3-diacil-trehalose (SL-IV) was investigated by an enzyme-linked immunosorbent assay (ELISA) test. Comparison between the antigens showed that immunocompetent patients produce preferentially antibodies to SL-IV than to PPD (73.3% versus 63.3%). Combination of these results showed an increase of the sensitivity to 80%, which decreased over the spectrum of immunodepression caused by HIV. In the tuberculous HIV seropositive group the sensitivities of SL-IV and PPD were 36.4% versus 40% and 0% versus 22.2% in the tuberculosis/acquired immunodeficiency syndrome (TB/AIDS) group. Combination of these results gave respectively 54.5% and 20%, showing that serological tests have limited value for diagnosis of tuberculosis in HIV infected patients. High antibody levels were observed in HIV seropositive asymptomatic group, but only two individuals were positive for both antigens. In the follow up, one of them developed tuberculous lymphadenitis, indicating that further work is needed to access the value of serological tests in predicting tuberculosis in HIV infected individuals.

A long-term intake of a protein hydrolysate seems to increase the risk of encephalopathy in mice infected with Schistosoma mansoni

Previous investigations showed that Schistosoma mansoni infection aggravates protein malabsorption in undernourished mice and this can be reverted by administration of casein hydrolysate. The present study was undertaken to evaluate the effects of ingestion of casein hydrolysate for long periods. Albino Swiss mice were divided into eight groups. Diets contained 5% (undernourished ) or 20% (controls) casein levels. For each group there were sub-groups ingesting whole or hydrolysed casein for 12 weeks. Infection with S. mansoni developed in half of the animals under each diet. All undernourished mice developed malabsorption. Low albuminemia was detected in infected animals independently of the protein level in the diet. However, albuminemia was lower in infected controls than in undernourished non-infected mice, suggesting a deficient liver protein synthesis. Infected mice fed on a 20% protein hydrolysed diet exhibited low weight gain and high mortality rates. On the other hand, non-infected mice ingesting the same diet had the highest body weights. We are investigating the hypothesis that infected mice, even when fed normal diets, are unable to metabolise large amounts of amino acids due to the liver lesions related to schistosomiasis and as a result die of hepatic coma. In some of them, the excessive accumulation of ammonia in the blood enhances the outcome of an encephalopathy.

Protein intake and exercise for optimal muscle function with aging: recommendations from the ESPEN Expert Group.

The aging process is associated with gradual and progressive loss of muscle mass along with lowered strength and physical endurance. This condition, sarcopenia, has been widely observed with aging in sedentary adults. Regular aerobic and resistance exercise programs have been shown to counteract most aspects of sarcopenia. In addition, good nutrition, especially adequate protein and energy intake, can help limit and treat age-related declines in muscle mass, strength, and functional abilities. Protein nutrition in combination with exercise is considered optimal for maintaining muscle function. With the goal of providing recommendations for health care professionals to help older adults sustain muscle strength and function into older age, the European Society for Clinical Nutrition and Metabolism (ESPEN) hosted a Workshop on Protein Requirements in the Elderly, held in Dubrovnik on November 24 and 25, 2013. Based on the evidence presented and discussed, the following recommendations are made (a) for healthy older people, the diet should provide at least 1.0-1.2 g protein/kg body weight/day, (b) for older people who are malnourished or at risk of malnutrition because they have acute or chronic illness, the diet should provide 1.2-1.5 g protein/kg body weight/day, with even higher intake for individuals with severe illness or injury, and (c) daily physical activity or exercise (resistance training, aerobic exercise) should be undertaken by all older people, for as long as possible.

Amniotic fluid insulin-like growth factor binding protein 3 concentration as early indicator of fetal growth restriction.

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is an important regulator of fetal growth and its bioavailability depends on insulin-like growth factor binding proteins (IGFBPs). Genes coding for IGF-I and IGFBP3 are polymorphic. We hypothesized that either amniotic fluid protein concentration at the beginning of the second trimester or genotype of one of these two genes could be predictive of abnormal fetal growth. STUDY DESIGN: Amniotic fluid samples (14-18 weeks of pregnancy) from 123 patients with appropriate for gestational age (AGA) fetuses, 39 patients with small for gestational age (SGA) fetuses and 34 patients with large for gestational age (LGA) were analyzed. Protein concentrations were evaluated by ELISA and gene polymorphisms by PCR. RESULTS: Amniotic fluid IGFBP3 concentrations were significantly higher in SGA compared to AGA group (P=0.030), and this was even more significant when adjusted to gestational age at the time of amniocentesis and other covariates (ANCOVA analysis: P=0.009). Genotypic distribution of IGF-I variable number of tandem repeats (VNTR) polymorphism was significantly different in SGA compared to AGA group (P=0.029). 19CA/20CA genotype frequency was threefold decreased in SGA compared to AGA group and the risk of SGA occurrence of this genotype was decreased accordingly: OR=0.289, 95%CI=0.1-0.9, P=0.032. Genotype distribution of IGFBP3(A-202C) polymorphism was similar in all three groups. CONCLUSIONS: High IGFBP3 concentrations in amniotic fluid at the beginning of the second trimester are associated with increased risks of SGA while 19CA/20CA genotype at IGF-I VNTR polymorphism is associated with reduced risks of SGA. Neither IGFBP3 concentrations, nor IGF-I/IGFBP3 polymorphisms are associated with modified risks of LGA.

Classification of human astrocytic gliomas on the basis of gene expression: a correlated group of genes with angiogenic activity emerges as a strong predictor of subtypes.

The development of targeted treatment strategies adapted to individual patients requires identification of the different tumor classes according to their biology and prognosis. We focus here on the molecular aspects underlying these differences, in terms of sets of genes that control pathogenesis of the different subtypes of astrocytic glioma. By performing cDNA-array analysis of 53 patient biopsies, comprising low-grade astrocytoma, secondary glioblastoma (respective recurrent high-grade tumors), and newly diagnosed primary glioblastoma, we demonstrate that human gliomas can be differentiated according to their gene expression. We found that low-grade astrocytoma have the most specific and similar expression profiles, whereas primary glioblastoma exhibit much larger variation between tumors. Secondary glioblastoma display features of both other groups. We identified several sets of genes with relatively highly correlated expression within groups that: (a). can be associated with specific biological functions; and (b). effectively differentiate tumor class. One prominent gene cluster discriminating primary versus nonprimary glioblastoma comprises mostly genes involved in angiogenesis, including VEGF fms-related tyrosine kinase 1 but also IGFBP2, that has not yet been directly linked to angiogenesis. In situ hybridization demonstrating coexpression of IGFBP2 and VEGF in pseudopalisading cells surrounding tumor necrosis provided further evidence for a possible involvement of IGFBP2 in angiogenesis. The separating groups of genes were found by the unsupervised coupled two-way clustering method, and their classification power was validated by a supervised construction of a nearly perfect glioma classifier.

Aluminum hydroxide associated to Schistosoma mansoni 22.6 kDa protein abrogates partial protection against experimental infection but not alter interleukin-10 production

The need to develop a vaccine against schistosomiasis led several researches and our group to investigate proteins from Schistosoma mansoni as vaccine candidates. Sm22.6 is a protein from S. mansoni that shows high identity with Sj22.6 and Sh22.6 (79 and 91%, respectively). These proteins are associated with high levels of IgE and protection to reinfection. Previously, we have shown that Sm22.6 induced a partial protection of 34.5% when used together with Freund's adjuvant and produced a Th0 type of immune response with interferon-g and interleukin-4. In this work, mice were immunized with Sm22.6 alone or with aluminum hydroxide adjuvant and high levels of IgG, IgG1, and IgG2a were measured. Unfortunately, no protection was detected. Since IL-10 is a modulating cytokine in schistosomiasis, we also observed a high level of this molecule in splenocytes of vaccinated mice. In conclusion, we did not observe the adjuvant effect of aluminum hydroxide associated with rSm22.6 in protective immunity.

The BRAIN TRIAL: a randomised, placebo controlled trial of a Bradykinin B2 receptor antagonist (Anatibant) in patients with traumatic brain injury

BACKGROUND Cerebral oedema is associated with significant neurological damage in patients with traumatic brain injury. Bradykinin is an inflammatory mediator that may contribute to cerebral oedema by increasing the permeability of the blood-brain barrier. We evaluated the safety and effectiveness of the non-peptide bradykinin B2 receptor antagonist Anatibant in the treatment of patients with traumatic brain injury. During the course of the trial, funding was withdrawn by the sponsor. METHODS Adults with traumatic brain injury and a Glasgow Coma Scale score of 12 or less, who had a CT scan showing an intracranial abnormality consistent with trauma, and were within eight hours of their injury were randomly allocated to low, medium or high dose Anatibant or to placebo. Outcomes were Serious Adverse Events (SAE), mortality 15 days following injury and in-hospital morbidity assessed by the Glasgow Coma Scale (GCS), the Disability Rating Scale (DRS) and a modified version of the Oxford Handicap Scale (HIREOS). RESULTS 228 patients out of a planned sample size of 400 patients were randomised. The risk of experiencing one or more SAEs was 26.4% (43/163) in the combined Anatibant treated group, compared to 19.3% (11/57) in the placebo group (relative risk = 1.37; 95% CI 0.76 to 2.46). All cause mortality in the Anatibant treated group was 19% and in the placebo group 15.8% (relative risk 1.20, 95% CI 0.61 to 2.36). The mean GCS at discharge was 12.48 in the Anatibant treated group and 13.0 in the placebo group. Mean DRS was 11.18 Anatibant versus 9.73 placebo, and mean HIREOS was 3.94 Anatibant versus 3.54 placebo. The differences between the mean levels for GCS, DRS and HIREOS in the Anatibant and placebo groups, when adjusted for baseline GCS, showed a non-significant trend for worse outcomes in all three measures. CONCLUSION This trial did not reach the planned sample size of 400 patients and consequently, the study power to detect an increase in the risk of serious adverse events was reduced. This trial provides no reliable evidence of benefit or harm and a larger trial would be needed to establish safety and effectiveness. TRIAL REGISTRATION This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN23625128.

Recommendations of the GARIN group for managing non-critically ill patients with diabetes or stress hyperglycaemia and artificial nutrition.

BACKGROUND & AIMS By means of this update, the GARIN working group aims to define its position regarding the treatment of patients with diabetes or stress hyperglycaemia and artificial nutrition. In this area there are many aspects of uncertainty, especially in non-critically ill patients. METHODS Bibliographical review, and specific questions in advance were discussed and answered at a meeting in the form of conclusions. RESULTS We propose a definition of stress hyperglycaemia. The indications and access routes for artificial nutrition are no different in patients with diabetes/stress hyperglycaemia than in non-diabetics. The objective must be to keep pre-prandial blood glucose levels between 100 and 140 mg/dl and post-prandial levels between 140 and 180 mg/dl. Hyperglycemia can be prevented through systematic monitoring of capillary glycaemias and adequately calculate energy-protein needs. We recommend using enteral formulas designed for patients with diabetes (high monounsaturated fat) to facilitate metabolic control. The best drug treatment for treating hyperglycaemia/diabetes in hospitalised patients is insulin and we make recommendations for adapt the theoretical insulin action to the nutrition infusion regimen. We also addressed recommendations for future investigation. CONCLUSIONS This recommendations about artificial nutrition in patients with diabetes or stress hyperglycaemia can add value to clinical work.

Prophylactic isolated limb perfusion for localized, high-risk limb melanoma: results of a multicenter randomized phase III trial. European Organization for Research and Treatment of Cancer Malignant Melanoma Cooperative Group Protocol 18832, the World Health Organization Melanoma Program Trial 15, and the North American Perfusion Group Southwest Oncology Group-8593.

PURPOSE: Patients with primary cutaneous melanoma > or = 1.5 mm in thickness are at high risk of having regional micrometastases at the time of initial surgical treatment. A phase III international study was designed to evaluate whether prophylactic isolated limb perfusion (ILP) could prevent regional recurrence and influence survival. PATIENTS AND METHODS: A total of 832 assessable patients from 16 centers entered the study; 412 were randomized to wide excision (WE) only and 420 to WE plus ILP with melphalan and mild hyperthermia. Median age was 50 years, 68% of patients were female, 79% of melanomas were located on a lower limb, and 47% had a thickness > or = 3 mm. RESULTS: Median follow-up duration is 6.4 years. There was a trend for a longer disease-free interval (DFI) after ILP. The difference was significant for patients who did not undergo elective lymph node dissection (ELND). The impact of ILP was clearly on the occurrence-as first site of progression - of in-transit metastases (ITM), which were reduced from 6.6% to 3.3%, and of regional lymph node (RLN) metastases, with a reduction from 16.7% to 12.6%. There was no benefit from ILP in terms of time to distant metastasis or survival. Side effects were higher after ILP, but transient in most patients. There were two amputations for limb toxicity after ILP. CONCLUSION: Prophylactic ILP with melphalan cannot be recommended as an adjunct to standard surgery in high-risk primary limb melanoma.

Four main virotypes among extended-spectrum-β-lactamase-producing isolates of Escherichia coli O25b:H4-B2-ST131: bacterial, epidemiological, and clinical characteristics.

A total of 1,021 extended-spectrum-β-lactamase-producing Escherichia coli (ESBLEC) isolates obtained in 2006 during a Spanish national survey conducted in 44 hospitals were analyzed for the presence of the O25b:H4-B2-ST131 (sequence type 131) clonal group. Overall, 195 (19%) O25b-ST131 isolates were detected, with prevalence rates ranging from 0% to 52% per hospital. Molecular characterization of 130 representative O25b-ST131 isolates showed that 96 (74%) were positive for CTX-M-15, 15 (12%) for CTX-M-14, 9 (7%) for SHV-12, 6 (5%) for CTX-M-9, 5 (4%) for CTX-M-32, and 1 (0.7%) each for CTX-M-3 and the new ESBL enzyme CTX-M-103. The 130 O25b-ST131 isolates exhibited relatively high virulence scores (mean, 14.4 virulence genes). Although the virulence profiles of the O25b-ST131 isolates were fairly homogeneous, they could be classified into four main virotypes based on the presence or absence of four distinctive virulence genes: virotypes A (22%) (afa FM955459 positive, iroN negative, ibeA negative, sat positive or negative), B (31%) (afa FM955459 negative, iroN positive, ibeA negative, sat positive or negative), C (32%) (afa FM955459 negative, iroN negative, ibeA negative, sat positive), and D (13%) (afa FM955459 negative, iroN positive or negative, ibeA positive, sat positive or negative). The four virotypes were also identified in other countries, with virotype C being overrepresented internationally. Correspondingly, an analysis of XbaI macrorestriction profiles revealed four major clusters, which were largely virotype specific. Certain epidemiological and clinical features corresponded with the virotype. Statistically significant virotype-specific associations included, for virotype B, older age and a lower frequency of infection (versus colonization), for virotype C, a higher frequency of infection, and for virotype D, younger age and community-acquired infections. In isolates of the O25b:H4-B2-ST131 clonal group, these findings uniquely define four main virotypes, which are internationally distributed, correspond with pulsed-field gel electrophoresis (PFGE) profiles, and exhibit distinctive clinical-epidemiological associations.