Development and application of novel ELISA-based analytical tools for assessing nitroxidative distress biomarkers in ischemic stroke: implications for improved diagnosis and clinical management.

Ischemic cerebrovascular accident (iCVA) is a public health issue, whose subjacent events involve the development of nitroxidative distress. Identifying biomarkers that assist in the diagnosis of this disease has clinically relevant implications. The aim of this study was to develop an analytic tool for measuring nitroxidative distress biomarkers, intended for application in clinical practice to enhance patient healthcare. Three enzyme linked immunosorbent assays (ELISA) were developed, with different detection objectives. One of them, in a sandwich format, quantifies the amount of fibrinogen in human plasma, an important glycoprotein involved in the blood coagulation process, contributing to thrombus formation and thereby participating in the mechanism of ischemic stroke. Another ELISA, also in a sandwich format, detects the presence of nitrotyrosine residues in fibrinogen from human plasma, a nitroxidative posttranslational modification resulting from the attack of peroxynitrite by-products on tyrosine residues present in proteins. The third one, in inhibition format, determines human plasma nitrotyrosine total content and was used to analyze human plasma samples from control and iCVA patients. Those two groups of plasma samples were analyzed using inhibition ELISA, revealing statistically significant differences in their nitrotyrosine content and molar ratios of nitrotyrosine to fibrinogen, which were higher in the iCVA group. This study provides evidence that nitroxidative distress occurs in ischemic stroke, as indicated by the detection of the biomarker nitrotyrosine. This finding supports other studies that also identified nitrotyrosine in ischemic stroke, through several different methods. This specific ELISA method is applicable for the rapid analysis of clinical samples, making it a potential clinical tool for assessing iCVA patients.

Identification and relative quantification of 3-nitrotyrosine residues in fibrinogen nitrated in vitro and fibrinogen from ischemic stroke patient plasma using LC-MS/MS.

Ischemic stroke is one of the leading causes of death and disability worldwide. This acute vascular event interferes with blood supply to the brain and induces a burst of free radicals such as nitric oxide and superoxide, producing peroxynitrite, a precursor of strong nitrating agents. Fibrinogen is one of the most abundant plasma proteins; it plays a role in the hemostatic system, mediating clot formation, which can be affected by nitrotyrosine formation. We hypothesized that nitration of fibrinogen by ONOOH and ONOOCO2- radical products could be one of the early events of the ischemic stroke, and protein-bound 3-nitrotyrosine could be a potential biomarker for diagnosis and/or prognosis of this condition. A targeted mass spectrometry approach was developed to analyze the nitration of fibrinogen and its association with ischemic stroke. First, a comprehensive mapping of 3-nitrotyrosine locations and their relative quantification was performed by LC-MS/MS, using in vitro nitrated fibrinogen samples. Twenty different 3-nitrotyrosine residues were identified on fibrinogen nitrated in vitro, varying with the peroxynitrite tofibrinogen molar ratio used. Nine tyrosine residues that were consistently modified at different treatment ratios were chosen to perform a targeted LC-MS/MS analysis in clinical samples. Enriched fibrinogen fractions from clinical samples from 24 ischemic stroke and 12 patients with non-inflammatory conditions were analysed with this method. Three of the nine tyrosine residues analysed (ßY452, ßY475 and γY380) showed a significant difference between the ischemic stroke and non-inflammatory disease groups. ROC curve analysis suggested an association of these residues either individually or in combination with ischemic stroke. Different tyrosine nitration patterns were also observed in fibrinogen modified in vitro and in vivo, suggesting differences in the nitration process in these situations. This is the first study showing a putative association between the nitration profile of specific tyrosine residues in human fibrinogen and ischemic stroke.

Leaf saponins of Quillaja brasiliensis enhance long-term specific immune responses and promote dose-sparing effect in BVDV experimental vaccines.

Saponin-based adjuvants are promising adjuvants that enhance both humoral and T-cell-mediated immunity. One of the most used natural products as vaccine adjuvants are Quillaja saponaria bark saponins and its fraction named Quil A®. Despite that, its use has been restricted for human use due to safety issues. As an alternative, our group has been studying the congener species Quillaja brasiliensis saponins and its performance as vaccine adjuvants, which have shown to trigger humoral and cellular immune responses comparable to Quil A® but with milder side effects. Here, we studied a semi purified aqueous extract (AE) and a previously little characterized saponin-enriched fraction (QB-80) from Q. brasiliensis as vaccine adjuvants and an inactivated virus (bovine viral diarrhea virus, BVDV) antigen co-formulated in experimental vaccines in mice model. For the first time, we show the spectra pattern of the Q. brasiliensis saponins by MALDI-TOF, a novel and cost-effective method that could be used to characterize different batches during saponins production. Both AE and QB-80 exhibited noteworthy chemical similarities to Quil A®. In addition, the haemolytic activity and toxicity were assessed, showing that both AE and QB-80 were less toxic than Quil A®. When subcutaneously inoculated in mice, both fractions promoted long-term strong antibody responses encompassing specific IgG1 and IgG2a, enhanced the avidity of IgG antibodies, induced a robust DTH reaction and significantly increased IFN-É£ production in T CD4+ and T CD8+ cells. Furthermore, we have proven herein that AE has the potential to promote dose-sparing, substantially reducing the dose of antigen required for the BVDV vaccines and still eliciting a mixed Th1/Th2 strong immune response. Based on these results, and considering that AE is a raw extract, easier and cheaper to produce than commercially available saponins, this product can be considered as candidate to be escalated from experimental to industrial uses.

The recombinant gut-associated M17 leucine aminopeptidase in combination with different adjuvants confers a high level of protection against Fasciola hepatica infection in sheep.

Fasciola hepatica M17 leucine aminopeptidase (FhLAP) is thought to play a role in catabolizing peptides generated by the concerted activity of gut-associated endopeptidases on host polypeptides, thus releasing amino acids to be used in protein anabolism. In this study, a recombinant functional form of this homo hexameric metallopeptidase produced in Escherichia coli was used in combination with adjuvants of different types in a vaccination trial in Corriedale sheep against experimental challenge with F. hepatica metacercariae. The experimental assay consisted of 6 groups of 10 animals; 5 of the groups (1-5) were subcutaneously inoculated at weeks 0 and 4 with 100 µg of rFhLAP mixed with Freund's complete plus incomplete adjuvant (group 1), Alum (group 2), Adyuvac 50 (group 3), DEAE-D (group 4) and Ribi (group 5); the adjuvant control group (group 6) received Freund's adjuvant. Two weeks after the booster, the sheep were orally challenged with 200 metacercariae. Immunization with rFhLAP induced significant reduction in fluke burdens in all vaccinated groups: 83.8% in the Freund's group, 86.7% in the Alum group, 74.4% in the Adyuvac 50 group, 49.8% in the Ribi group and 49.5% in the DEAE-D group compared to the adjuvant control group. Morphometric analysis of recovered liver flukes showed no significant size modifications in the different vaccination groups. All vaccine preparations elicited specific IgG, IgG1 and IgG2 responses. This study shows that a liver fluke vaccine based on rFhLAP combined with different adjuvants significantly reduced worm burden in a ruminant species that was high in animals that received the enzyme along with the commercially approved adjuvants Alum and Adyuvac 50.

Detection of a new embryonic antigen (ESA-10) in the blood of patients with cancer: preliminary results in the United States.

ESA-10 is an embryonic antigen expressed by tumor cells. A method to detect the antigen in the blood based on alterations in the erythrocyte sedimentation rate that occur when antiserum to ESA-10 is bound to the antigen in blood was devised and used here to determine the sensitivity and predictive value of the test in patients with biopsy proven non-hematologic malignancies, and in normal control subjects. The test was positive in 22 of 24 cancer patients tested, and negative in 30 of 35 control subjects. Of the five positive control subjects, one female had recently given birth and was lactating. Another control subject was recently diagnosed with prostate cancer, just months after having participated in this study. Therefore, this tumor marker test (Turtest(®)) had a sensitivity of 91.7% and a positive predictive value of 81.5% in patients with biopsy proven cancer, and a specificity and negative predictive value in control subjects of 88.2 and 93.8%, respectively, if the control subject who subsequently developed prostate cancer is removed from the control group. Therefore, this simple test has potential as a clinically useful tumor marker with sensitivity and specificity equal to or greater than other commercially available tumor markers and should be explored further in larger studies.

Features of bacterial growth and polysaccharide production of Streptococcus pneumoniae serotype 14.

The effect of several cultivation conditions on the kinetics of bacterial growth and polysaccharide production of Streptococcus pneumoniae serotype 14 was studied. The presence in the supernatant of serotype-specific CPS (capsular polysaccharide) during growth was followed by size-exclusion HPLC and, in parallel, confirmed by using a specific latex reagent. The agitation level did not affect the production behaviour, whereas pH maintenance above 6 strongly enhanced both growth and CPS production throughout the cultivation period in flasks. Production of high-molecular-mass polysaccharide was found to be maximal between 5 and 6 h of cultivation, at the end of the exponential phase. By laser light scattering, 90% of this purified CPS product showed a M(w) (molecular mass) range from 350 to 1500 kDa, with an average M(w) of 921 kDa. Extending the culture to 24 h gave rise to a clear shift of the M(w) distribution of the polysaccharide to values lower than 100 kDa. These findings may have strong implications for the large-scale manufacture of the polysaccharide and the associated conjugate vaccine.

Purification, characterization, and immunolocalization of paramyosin from the adult stage of Fasciola hepatica.

Paramyosin, a vaccine candidate in different helminthiases, was purified from the adult liver fluke Fasciola hepatica using two different procedures. The first started with a crude extraction of paramyosin in high-salt buffer followed by gel filtration chromatography and two precipitation-solubilization cycles; in the second, anion exchange chromatography replaced the gel filtration step. In both cases, the apparent molecular weight of the purified protein determined by sodium dodecyl sulfate gel electrophoresis under reducing and non-reducing conditions was 97 kDa and 200 kDa, respectively. The molecular weights were consistent with the presence of a dimeric protein linked by disulfide bridges. Western blot analysis showed that the dimeric and monomeric forms were both recognized by an antiserum raised against the F. hepatica 97 kDa band (alpha-FhPmy), and by an anti- Schistosoma mansoni paramyosin immune serum. Immunohistochemistry using alpha-FhPmy demonstrated the localization of paramyosin within the subtegumental muscle and in muscle cells surrounding the gut of adult parasites. We also observed labeling of extramuscular structures like testes, surface lamellae of the gut and the tegument of adult flukes.

Humoral immune response against fasciola hepatica in bovines: primary characterization of protection induced by irradiated metacercariae

There is an increasing awareness of fasciolosis as an important human and veterinary disease. In order to study the humoral inmune response of bovines, 5-month old calves were experimentally inmunized using 1000 ç irradiated metacercariae per animal, and challenged six weeks later with 500 normal ones per calf. Profiles of antibody responses of non inmunized animals following challenge, measured by ELISA, using both excretion/secretion ans somatic antigens, showed a sharp increase in antibody titres in the first 3 weeks post-infection which were maintained throughout the infection. Animals which instead were inmunized with inactivated parasites had similar antibody responses, but the antibody titres were higher. No booster effect was observed when these inmunized animals were challenger. Result of western blot analysis are consistent with these observations: antibodies against high molecular weight parasite-specific bands, ranging from 72,5 to 40 KD, appeared in week 2 PI in inmunized animals, and were mantained throughout infection, importanfly, inmunization resulted in a 84,2 percent reduction in parasite burden, indicating that high circulating antibody titres against these antigens probably are involved in a protective response. The results presented here indicate that, irradiated metacercariae of fasciola hepatica can be used as useful tool to generate a normal antigenic stimulus for the development of circulation antibodies, and toidentify antigens invloved in triggering a protective inmune response