Comparison of three ELISAs for the diagnosis of porcine reproductive and respiratory syndrome.
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Biomedical subjects
Publications and source records attributed to M Martín.
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Previous studies have described a "litter effect" associated with mortality in postweaning multisystemic wasting syndrome (PMWS) affected farms. The main objective of this study was to evaluate litter mortality in different PMWS affected farms and to characterize it in relation to three variables of the sow: parity, porcine circovirus type 2 (PCV2) infectious status and PCV2 antibody titres. The study was performed in seven farms that experienced PMWS in nurseries and/or fattening areas. Fifteen sows from each farm were randomly selected from the same farrowing batch. Serum samples were analyzed for antibodies to PCV2 and for genomic detection of PCV2. Four piglets from each sow (60 piglets per farm) were selected and ear-tagged at birth. Out of 420 initial piglets, 104 (25%) died. Sixty three of them (60%) were necropsied, and 40 (63%) diagnosed as PMWS based on case definition criteria. Our results show that sow PCV2 viremia was significantly related to piglet mortality since more piglets per litter died from viremic than from non-viremic sows. Additionally, a significantly greater proportion of animals died from sows that had low antibody titres against PCV2 (39% vs. 18% from sows with medium to high antibody titres). The present study, of exploratory nature, confirms previous results and further characterizes the so called "litter effect" by establishing that the sow PCV2 status had a significant effect on litter mortality in PMWS affected farms.
One hundred and thirteen finishing pig units and 74 sow units in Catalonia, Spain, were examined to determine the prevalence of salmonella infections and the factors that could be associated with them. Pooled faecal samples were taken from the finishing units, and samples of faeces were collected from individual sows. The Salmonella isolates were serotyped, phage typed and examined for their antimicrobial susceptibility to 18 common antimicrobial drugs. In addition, blood samples from pigs on 141 farms were analysed by ELISA. In both the bacteriological and serological surveys, a questionnaire with 84 questions was completed for each farm. Salmonella species were isolated from 20 per cent of the finishing units and 24 per cent of the sow units; 14 serotypes were detected in the finishing pigs and 11 in the sows. More than 30 per cent of the strains were resistant to tetracycline, sulphonamides, ampicillin or streptomycin, and 69 per cent of the strains were resistant to three or more agents up to 10 compounds. Seventy-seven per cent of the farms had at least one seropositive animal, and 26 per cent of these farms had an individual seroprevalence of 50 per cent or more. The factors associated (P<0.05) with the excretion of Salmonella species in the finishing units were the practice of raising livestock other than pigs (odds ratio [OR]=6.18), the herd size (OR=5.87), and a past history of clinical salmonellosis (OR=4.97). For the sows, the factors associated (P<0.05) with the excretion of Salmonella species were having open-flushed drainage of sewage (OR=34.48), a lack of rodent control measures (OR=0.05) and the number of sows in the unit (OR=9.26). Factors associated with seropositivity in the finishing units were a lack of bird-proof nets (OR=0.30) and the use of water from private wells (OR=3.64).
BACKGROUND: The aim of the study was to analyse the toxicity and health related quality of life (HRQoL) of breast cancer patients treated with FAC (5-fluorouracil, doxorubicin, cyclophosphamide) and TAC (docetaxel, doxorubicin, cyclophosphamide) with and without primary prophylactic G-CSF (PPG). PATIENTS AND METHODS: This was a phase III study to compare FAC and TAC as adjuvant treatment of high-risk node-negative breast cancer patients. After the entry of the first 237 patients, the protocol was amended to include PPG in the TAC arm due to the high incidence of febrile neutropenia. A total of 1047 evaluable patients from 49 centres in Spain, two in Poland and four in Germany were included in the trial. Side-effects and the scores of the EORTC QLQ-C30 and QLQ BR-23 questionnaires were compared in the three groups (FAC, TAC pre-amendment and TAC post-amendment). RESULTS: The addition of PPG to TAC significantly reduced the incidence of neutropenic fever, grade 2-4 anaemia, asthenia, anorexia, nail disorders, stomatitis, myalgia and dysgeusia. Patient QoL decreased during chemotherapy, more with TAC than FAC, but returned to baseline values afterwards. The addition of PPG to TAC significantly reduced the percentage of patients with clinically relevant Global Health Status deterioration (10 or more points over baseline value) at the end of chemotherapy (64% versus 46%, P<0.03). CONCLUSIONS: The addition of PPG significantly reduces the incidence of neutropenic fever associated with TAC chemotherapy as well as that of some TAC-induced haematological and extrahaematological side-effects. The HRQoL of patients treated with TAC is worse than that of those treated with FAC but improves with the addition of PPG, particularly in the final part of chemotherapy treatment.
Caffeine is the most widely consumed substance in the world which antagonizes adenosine effects. Adenosine acting through A(1) receptors inhibits glutamate release which binds to metabotropic glutamate receptors (mGluRs). Recently, we have shown that maternal caffeine intake during gestation causes down-regulation of A(1) and metabotropic glutamate receptors in the brain of both rat mothers and fetuses. In the present work we provide evidence that caffeine also affects receptors in hearts, causing a decrease in mGluRs from both maternal and fetal hearts. A decrease in G(q/11) and PLC beta(1) proteins level was also observed in both tissues. However, phospholipase C activity was only affected in fetal heart, being significantly decreased. These results suggest an in vivo cross-talk mechanism between adenosine and glutamate receptors in peripheral tissues. Therefore, special attention should be paid to caffeine ingestion during gestation.
BACKGROUND: The "Alamo" project is a retrospective analysis of 14,854 patients diagnosed of breast cancer between 1990 and 1997 in 50 Spanish hospitals. METHODS: Alamo I (AI) consisted of 4,532 patients diagnosed with breast cancer between 1990 and 1993. Data were collected in 2000. Alamo II (AII) consisted of 10,322 patients diagnosed between 1994 and 1997. Data were collected in 2003. RESULTS: At presentation, there were (AI vs. AII) 17.6% vs. 24.3% at stage I; 55.5% vs. 53.1% at stage II; 18.7% vs. 15% at stage III; 7.2% vs. 5.9 at stage IV. Median age was 57 (AI) vs. 58 years (AII) and 65.9% vs. 67.2% (AI vs. AII) were post-menopausal. Firstline treatment for disease stages I, II and III was surgery in 91% of patients in both studies. Breast conserving surgery rate increased from 20.2% (AI) to 32.7% (AII). Adjuvant systemic treatments were administered to 87.6% (AI) and 92.8% (AII) of patients. Recurrence rate diminished from 36.6% (AI) to 22.5% (AII) and the 9-year survival rate increased from 63.2% (95% CI: 61.5-64.9) to 70.1% (95% CI: 68.5-71.8). CONCLUSION: Breast cancer outcomes in Spain have improved from 1990-1993 to 1994-1997, likely because of breast cancer screening program implementation and new therapies.
ORF5 sequences of porcine reproductive and respiratory syndrome virus (PRRSV) were analysed to determine genetic diversity, codon usage, positive and negative selection sites and potential changes in the predicted glycoprotein 5 (GP5). A hypothetical GP5 containing all selected sites was constructed to determine its characteristics. These sequences corresponded to isolates obtained 10 years apart (1991-1995, 18 strains) and a second set (n = 46) from 2000 to 2005. Similarity to Lelystad virus (LV) decreased from 95.5% in 1991-1995 to 89.5% in 2000-2005. Three highly variable regions were found in ORF5. Codon usage was different in both sets for leucine, glutamine, serine and proline. Thus, 2000-2005 sequences used codons more similar to those present in highly expressed pig genes compared to the 1991-1995 set. Twenty four sites of positive selection and 20 sites of negative selection were found in GP5, most of them in transmembrane regions. Additional glycosylation in N37 of GP5 was common in 2000-2005 but some sequences lack a glycosylation site in N46. The hypothetical GP5 was only 88.1% similar to LV and was less hydrophobic. Taking together these results suggest that PRRSV is still adapting to pig cells.
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The cloning and expression of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, was studied in Escherichia coli. PCR-amplified products of the preenterocin P gene (entP) or entP plus the putative EntP immunity gene (entiP), were cloned in plasmid pETBlue-1 under the control of the inducible T7lac promoter. Although target genes in derivative plasmids pJG01 (entP) and pJG02 (entP plus entiP) did not generate products with antimicrobial activity after an in vitro combined transcription/translation reaction, they were expressed as biologically active products following transformation and induction in the E. coli Tuner(DE3)pLacI host. The use of specific antibodies and an ELISA permitted the detection and quantification of EntP in the supernatant (SN), cellular soluble protein fraction (CSF), and inclusion bodies (IB) of E. coli Tuner(DE3)pLacI cells transformed with either pJG01 or pJG02. Functional EntP from the supernatants of E. coli Tuner(DE3)pLacI (pJG01) cultures grown in a complex medium was recovered, at a high efficiency, by immunoaffinity chromatography in a single step. A purification method based on hydrophobic adsorption and reverse-phase chromatographies also permitted the recovery of active EntP from the supernatants of the same cultures grown in a minimally defined medium. The E. coli Tuner(DE3)pLacI (pJG01) cells would merit consideration as an alternative experimental model for the heterologous production and functional expression of EntP, as well as for the fast and efficient recovery of this bacteriocin from the supernatant of this recombinant producer.
Serum samples from 361 pigs (194 fattening pigs and 167 sows) were examined by means of two commercial ELISAs (Svanovir; Svanova Biotech and Salmotype; Labor Diagnostik) used for the serological diagnosis of salmonellosis in pigs; 211 of the samples came from farms of known bacteriological status and the other 150 were collected randomly from 60 farms of unknown status. The ELISAs were done according to the manufacturers' directions and the samples were categorised accordingly. The results were compared by using a linear regression analysis and by the calculation of Kappa values. To try to improve the agreement between the tests, the raw optical densities (ODS) were transformed to sample/positive (S/P) ratios by using the positive control as a reference, and cut-off values for these S/P ratios were calculated by means of a receiver operating characteristic (ROC) analysis. All but two of the known infected farms were recognised as such by both tests. However, the correlation of the raw ODS for individual pigs was poor (r=0.546) and had a Kappa value for the results categorised according to the manufacturers' recommendations of 0.191. On some farms the correlation was high (r=0.97) but on others it was low (r=0.05) with no apparent reason for the difference. The S/P ratios did not improve the agreement (Kappa=0.25).
Chronic glutamate treatment during gestational period caused a significant decrease in total metabotropic glutamate receptors (mGluR) number. Similar results were observed on the steady-state level of mGlu(1) receptor detected by immunoblotting assays, suggesting that this is the main receptor subtype modulated by agonist exposure. Furthermore, no variations on mRNA coding mGlu(1) receptor were found, suggesting post-transcriptional modulation as a possible mechanism of the lost of receptor detected at the membrane surface. On the other hand, western-blotting to determine level of G(q/11) protein and phospholipase C beta(1) revealed a significant decrease of both proteins in mothers brain. This decrease was associated with significant variation in glutamate and DHPG-stimulated phospholipase C activity. No significant differences on mGluR transduction pathway components were observed in fetuses brain. These results suggest that glutamate intake during pregnancy causes a down-regulation of different proteins involved in glutamate response mediated by mGluR only in mothers brain without significantly affecting fetuses brain.
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The objective of this study is to compare the predictive accuracy of a neural network (NN) model versus the standard Cox proportional hazard model. Data about the 3811 patients included in this study were collected within the 'El Alamo' Project, the largest dataset on breast cancer (BC) in Spain. The best prognostic model generated by the NN contains as covariates age, tumour size, lymph node status, tumour grade and type of treatment. These same variables were considered as having prognostic significance within the Cox model analysis. Nevertheless, the predictions made by the NN were statistically significant more accurate than those from the Cox model (p < 0.0001). Seven different time intervals were also analyzed to find that the NN predictions were much more accurate than those from the Cox model in particular in the early intervals between 1-10 and 11-20 months, and in the later one considered from 61 months to maximum follow-up time (MFT). Interestingly, these intervals contain regions of high relapse risk that have been observed in different studies and that are also present in the analyzed dataset.
The aim of this work was to study whether caffeine or theophylline chronically consumed during pregnancy affect inhibitory adenylyl cyclase pathway mediated by adenosine, in rat brain of both mothers and full-term fetuses. Immunoblotting analysis revealed a significant decrease in alphaGi(1,2) subunit level (27-29% in mothers, 15-18% in fetuses), associated with a significant increase in the mRNA level coding alphaGi(1) in both maternal and fetal rat brain (12-22%) after methylxanthine intake. No significant differences in alphaGi(3) level were detected in any case. On the other hand, forskolin- and forskolin plus guanosine-5'-O(3-thiotriphosphate) tetralithium salt-stimulated adenylyl cyclase activity was significantly decreased (30-36%) in maternal brain. Moreover, adenylyl cyclase inhibition elicited by N(6)-cyclohexyladenosine, specific adenosine A(1) receptor agonist, was also significantly decreased in caffeine- (40.5%) and theophylline- (55.0%) treated mothers, suggesting a desensitization of adenosine A(1) receptor/adenylyl cyclase pathway in maternal brain. However, no significant differences were detected in fetal brain between control and treated animals. Therefore, caffeine or theophylline chronic intake during pregnancy differently modulates inhibitory adenylyl cyclase pathway mediated by adenosine in maternal and fetal brain causing a loss of the system responsiveness only in maternal brain but down-regulating Gi(1) protein in both mother and fetus brain.
Glutamate is the main excitatory neurotransmitter in the cerebral cortex. Altered glutamatergic transmission has been suggested as having a central role in many neurodegenerative diseases. Metabotropic glutamate receptors (mGluRs) are coupled to intracellular signal transduction via G proteins, and they mediate slower responses than ionotropic glutamate receptors. Group I mGluRs are positively coupled to phospholipase C beta1 (PLCbeta1). Creutzfeldt-Jakob disease (CJD) is a human transmissible spongiform encephalopathy associated with a dysfunction in the membrane glycoprotein PrP which is converted into an abnormal isoform, with a predominant beta-sheet structure, that is pathogenic and partially resistant to protease digestion. Proteins associated with the signal transduction of group I mGluRs were examined in the frontal cortex (area 8) of 12 cases with sCJD and four age-matched controls, by means of gel electrophoresis and Western blotting of total homogenates. Densitometric analysis of the bands demonstrated decreased expression levels of PLCbeta1 and PLCgamma, a non-related phospholipase which is a substrate of tyrosine kinase, in CJD cases when compared with controls. Novel protein kinase C delta (nPKCdelta) has also been found to be significantly decreased in CJD cases. However, no modifications in mGluR1 cPKCalpha expression levels are found in CJD when compared with controls. No modifications in PLCbeta1 solubility in PBS-, deoxycholate- and sodium dodecylsulphate-soluble fractions have been observed in CJD when compared with controls. Finally, no interactions between PLCbeta1 and PrP, as revealed by immunoprecipitation assays, have been found in CJD and controls. The present results show, for the first time, reduced expression levels of phospholipases, particularly PLCbeta1, which may interfere with group I mGluR signaling in the cerebral cortex in CJD. These abnormalities are not the result of abnormal PLC solubility or interactions with PrP. Selective involvement of group I mGluRs may have functional effects on glutamatergic transmission modulation and processing in CJD.
Both the age of the population and anesthetic and surgical techniques are advancing. Currently, 40% of surgical activity is performed in patients older than 65 years, who present a higher surgical risk than younger patients. The aim of treatment in the elderly is to provide the best possible quality of life, even though this represents a surgical challenge because of associated comorbidity and reduced cardiopulmonary reserve. From the moment at which laparotomy becomes an increased stress in the elderly, laparoscopic surgery can be particularly advantageous in this population. Therefore, minimally invasive surgery may have a greater impact in these individuals than in younger patients in reducing postoperative pain, cardiorespiratory complications, hospital stay, and recovery time before resuming physical activity. The recent advances in anesthesia, together with improved patient selection and perioperative cardiac care, and the general adoption of minimally invasive access have enabled more complex gastrointestinal procedures to be performed in the elderly. The factors that could influence the development of this type of approach in the elderly, as well as the precautions that should be taken, should be further analyzed.
Mitochondrial involvement has not been identified in the programmed cell death (PCD) of leaf senescence which suggests that processes such as those involving reactive oxygen species (ROS) are controlled by chloroplasts. We report that transgenic tobacco (DeltandhF), with the plastid ndhF gene knocked-out, shows low levels of the plastid Ndh complex, homologous to mitochondrial complex I, and more than a 30-day-delay in leaf senescence with respect to wt. The comparison of activities and protein levels and analyses of genetic and phenotypic traits of wtxDeltandhF crosses indicate that regulatory roles of mitochondria in animal PCD are assumed by chloroplasts in leaf senescence. The Ndh complex would increase the reduction level of electron transporters and the generation of ROS. Chloroplastic control of leaf senescence provides a nonclassical model of PCD and reveals an unexpected role of the plastid ndh genes that are present in most higher plants.
Pregnant rats were treated throughout the gestational period with either caffeine or theophylline, and its effect on the metabotropic glutamate receptor (mGluRs) signal transduction pathway was studied in both maternal and fetal brain. In maternal brain, radioligand binding assays showed that chronic treatment with methylxanthines caused a significant decrease in the total number of mGluRs. This decrease was accompanied by an increase in receptor affinity. Immunodetection showed that mGluR1a and phospholipase C beta1 (PLCbeta1) were significantly decreased in response to chronic methylxanthine treatment, whereas alphaG(q/11) was not affected. A loss was also detected of PLC stimulation mediated by (S)-3,5-dihydroxyphenylglycine (DHPG), a selective Group I mGluR agonist, suggesting desensitization of the mGluR/PLC pathway. In fetal brain, a loss in total mGluRs was observed in fetuses from mothers treated with caffeine or theophylline, without variation in receptor affinity. A decrease in mGluR1a, alphaG(q/11) and PLCbeta1 levels was also observed in response to treatment. However, changes detected in this immature tissue were not associated with variations in PLC activity. These results suggest that chronic caffeine or theophylline treatment down-regulates several mGluR/PLC transduction pathway components in both maternal and fetal brain, causing a loss of receptor responsiveness only in maternal brain.