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Background: Sand flies are hematophagous arthropods that act as vectors of Leishmania parasites. When hosts are bitten they develop cellular and humoral responses against sand fly saliva. A positive correlation has been observed between the number of bites and antibody levels indicating that anti-saliva antibody response can be used as marker of exposure to sand flies. Little is known about kinetics of antibodies against Phlebotomus perniciosus salivary gland homogenate (SGH) or recombinant salivary proteins (rSP). This work focused on the study of anti-P. perniciosus saliva antibodies in sera of mice and rabbits that were experimentally exposed to the bites of uninfected sand flies., Methodology/principal Findings: Anti-saliva antibodies were evaluated by ELISA and Western blot. In addition, antibody levels against two P. perniciosus rSP, apyrase rSP01B and D7 related protein rSP04 were determined in mice sera. Anti-saliva antibody levels increased along the immunizations and correlated with the number of sand fly bites. Anti-SGH antibody levels were detected in sera of mice five weeks after exposure, and persisted for at least three months. Anti-apyrase rSP01B antibodies followed similar kinetic responses than anti-SGH antibodies while rSP04 showed a delayed response and exhibited a greater variability among sera of immunized mice. In rabbits, anti-saliva antibodies appeared after the second week of exposure and IgG antibodies persisted at high levels, even 7 months post-exposure., Conclusions/significance: Our results contributed to increase the knowledge on the type of immune response P. perniciosus saliva and individual proteins elicited highlighting the use of rSP01B as an epidemiological marker of exposure. Anti-saliva kinetics in sera of experimentally bitten rabbits were studied for the first time. Results with rabbit model provided useful information for a better understanding of the anti-saliva antibody levels found in wild leporids in the human leishmaniasis focus in the Madrid region, Spain.

Background: The presence of oligoclonal IgM bands (OCMB) in cerebrospinal fluid (CSF) is an unfavourable prognostic marker in multiple sclerosis. There is no commercial test to investigate OCMB status. However, a sensitive and specific isoelectrofocusing (IEF) and western blot method was described. We aimed to study the inter-centre reproducibility of this technique, a necessary condition for a reliable test to be incorporated into clinical practice., Methods: The presence of OCMB was analysed by IEF and western blot with prior reduction of pentameric IgM. We assayed the reproducibility of this test in a blinded multicentre study performed in 13 university hospitals. Paired-CSF and serum samples from 52 neurological patients were assayed at every centre., Results: Global analysis rendered a concordance of 89.8% with a kappa value of 0.71., Conclusion: These data indicate that OCMB detection by means of IEF and western blot with IgM reduction shows a good interlaboratory reproducibility and thus can be used in daily clinical setting., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Purpose: To understand the differential responses to doxycycline between Caucasian and Hispanic patients observed in a previous clinical trial., Methods: Primary cultures were established using pterygia excised from male Caucasian (n = 3) and Hispanic (n = 6) patients. The response of these cells to doxycycline was tested in a toxicity assay. In addition, a complete transcriptome was obtained from the nine samples, and the results were analyzed using false discovery rate statistics. Results were confirmed by quantitative RT (qRT)-PCR and Western blotting for a limited set of genes., Results: Caucasian pterygium cells underwent apoptosis upon exposure to doxycycline, whereas Hispanic cells survived the treatment. Transcriptomic analysis showed profound differences between cells of both ethnicities, even before treatment, implicating important cellular pathways such as the mitochondrial oxidative phosphorylation chain, the proteasome, and the components of the extracellular matrix. Following exposure to doxycycline, there was a significant increase in proapoptotic proteins, regulators of the cell cycle, and components of the mitochondrial membrane in Caucasian cells but not in their Hispanic counterparts. There was a good correlation between data obtained by ultrasequencing and those generated by qRT-PCR or Western blotting., Conclusions: The lack of response to doxycycline observed in Hispanic pterygium patients in a previous clinical trial can be explained by the genetic protection afforded to the cells in this ethnic background against apoptosis and cell death. New therapeutic options must be devised for these patients., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

New influenza A viruses with pandemic potential periodically emerge due to viral genomic reassortment. In the face of pandemic threats, production of conventional egg-based vaccines is time consuming and of limited capacity. We have developed in this study a novel DNA vaccine in which viral hemagglutinin (HA) is bivalently targeted to MHC class II (MHC II) molecules on APCs. Following DNA vaccination, transfected cells secreted vaccine proteins that bound MHC II on APCs and initiated adaptive immune responses. A single DNA immunization induced within 8 d protective levels of strain-specific Abs and also cross-reactive T cells. During the Mexican flu pandemic, a targeted DNA vaccine (HA from A/California/07/2009) was generated within 3 wk after the HA sequences were published online. These results suggest that MHC II-targeted DNA vaccines could play a role in situations of pandemic threats. The vaccine principle should be extendable to other infectious diseases.

Background: Our group previously found that up to 7% of amateur anglers in Caceres, Spain may be allergic to the larvae of Protophormia terraenovae (order Diptera, family Calliphoridae) used as live bait for fishing., Objective: To identify the pattern of major allergens in P terraenovae and other species of Calliphoridae., Materials and Methods: Extracts of P terraenovae, Calliphora vomitoria, Lucilia sericata and Lumbricus terrestris were characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis and IgE-immunoblotting techniques in individual sera from 24 patients with a positive skin test result and/or specific IgE determination (enzyme-linked immunosorbent assay [ELISA]) to P terraenovae. ELISA and IgE-immunoblotting inhibition studies were also performed to identify potential cross-reactive allergens between these species., Results: IgE-immunoblotting with P terraenovae showed a band of 15.3 kDa recognized by 15 patients, in addition to 2 further allergens of 22.8 kDa and 69 kDa. For C vomitoria, 5 bands of 73, 46, 40, 28, and 14 kDa were observed. For L sericata, 2 major allergens of 73 kDa and 14 kDa were observed. In the case of L terrestris, IgE from 13 patients recognized 1 allergen of around 15.5 kDa. IgE-immunoblotting and ELISA inhibition revealed the presence of cross-reactivity, mainly between L terrestris and P terraenovae., Conclusions: P terraenovae appears to have species-specific allergens and allergens shared with C vomitoria and L sericata. Striking immunological cross-reactivity was observed between P terraenovae and L terrestris. An allergen of 15-16 kDa could be involved in this phenomenon.

Background: Anisakissimplex is the main organism responsible for the zoonotic disease anisakiasis which follows the ingestion of live larvae present in raw or undercooked marine fish. Clinical features include severe epigastric pain, frequently accompanied by severe allergic reactions. We investigated the prevalence of immunoglobulin E (IgE) specific for 5 Anisakis allergens in Italian patients sensitized or allergic to the parasite. The results were compared with those obtained previously in a similar Spanish population., Patients and Methods: We conducted a descriptive, cross-sectional validation study. Asymptomatic Anisakis-sensitized subjects (15 Italian and 17 Spanish) and Anisakis allergic-patients (42 Italian and 35 Spanish) were studied by ImmunoCAP, Western-blotting with nAni s 4 and dot-blotting with rAni s 1, rAni s 5, rAni s 9 and rAni s 10., Results: Anisakis IgE CAP classes 1 or 2 were associated with a high probability of asymptomatic sensitization (66.7%) while CAP classes 4 or above, were associated with a very high probability of allergy to Anisakis (95.2%). The most frequently detected allergen among Italian and Spanish allergic patients was Ani s 1. All of the Spanish patients versus 76.2% of the Italian patients recognized at least one of the allergens tested. Patients suffering from gastrointestinal symptoms only were significantly more frequent among the Italians whereas the Spanish presented more frequently with urticaria, angioedema or anaphylaxis., Conclusions: Anisakis hypersensitivity shows different immunological patterns in different European countries. Allergen component diagnosis might help us to better understand this complex entity. Anisakis-specific IgE levels may have moderate prognostic significance., (Copyright © 2013 S. Karger AG, Basel.)

Background: Previous evidence suggests that 25-hydroxyvitamin D(3) [25(OH)D(3)] protects against several cancers. However, little is known regarding urothelial bladder cancer (UBC). We analyzed the association between plasma 25(OH)D(3) and overall risk of UBC, as well as according to stage and FGFR3 molecular subphenotypes., Methods: Plasma concentrations of 25(OH)D(3) in 1125 cases with UBC and 1028 control subjects were determined by a chemiluminescence immunoassay. FGFR3 mutational status and expression in tumor tissue were assessed. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression adjusting for potential confounders. Analyses were further stratified by tumor invasiveness and grade, FGFR3 expression, and smoking status. Cell proliferation was measured in human UBC cell lines cultured with 1α,25-dihydroxyvitamin D(3)., Results: A statistically significantly increased risk of UBC was observed among subjects presenting the lowest concentrations of 25(OH)D(3) (OR(adj) = 1.83; 95% CI = 1.19 to 2.82; P = .006), showing a dose-response effect (P (trend) = .004). The association was stronger for patients with muscle-invasive tumors, especially among low-FGFR3 expressers (OR(adj) = 5.94; 95% CI = 1.72 to 20.45; P = .005). The biological plausibility of these associations is supported by the fact that, in vitro, 1α,25-dihydroxyvitamin D(3) upregulates FGFR3 expression in UBC cell lines with low levels of wild-type FGFR3., Conclusion: These findings support a role of vitamin D in the pathogenesis of UBC and show that 25(OH)D(3) levels are associated with FGFR3 expression in the tumor. Because FGFR3 mutation and overexpression are markers of better outcome, our findings suggest that individuals with low levels of plasma 25(OH)D(3) may be at high risk of more aggressive forms of UBC.

Sand fly saliva is known to play an important role in the establishment of Leishmania spp. infection. As a consequence, identifying antigenic salivary proteins of different leishmaniasis vectors has currently become a major task in the field of anti-Leishmania vaccine development. The purpose of this work was to improve the knowledge of Phlebotomus perniciosus salivary proteins by combining two-dimensional gel electrophoresis (2DE) methodology, mass spectrometry and Western blotting (WB). Salivary protein profiles of three P. perniciosus colonies from different geographic origins in Spain were compared through SDS-PAGE, leading to a similar pattern with no qualitatively noticeable differences. A gradual increase of the protein content was significantly detected with the age of sand flies, reaching the complete salivary protein profiles at day four. The 2DE revealed a reproducible protein profile that matched the classic monodimensional SDS-PAGE pattern (1DE). More spots rather than protein bands (19 versus 11) were visualized by 2DE and 1DE, respectively, suggesting the presence of either protein isoforms or posttranslational modifications. Sera of mice and hamsters immunized through exposure to sand fly bites following different immunization schedules showed elevated anti-saliva IgG levels. These sera allowed the detection of 5 bands and 16 immunogenic spots in 1DE and 2DE, respectively, followed by WB. These antigens were identified by MALDITOF/TOF as SP03, SP03B, SP08, SP01, SP01B, SP04, SP04B, SP02, Phlebotomus ariasi SP16, and Phlebotomus argentipes SP13. This work is assumed to be the first attempt to establish 2DE proteomic maps of P. perniciosus saliva. All spots were identified as salivary proteins, confirming this technology as an interesting tool to improve sand fly salivary knowledge., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Canine leishmaniasis (CanL) is a widespread disease present in 42 countries. It is considered of epidemiological importance because of its role as a reservoir of human leishmaniasis. Knowledge of the real distribution of CanL and its emergence and/or re-emergence is of great importance in order to determine the extension of the disease. This work reports the detection of CanL in a farm dog located in a Pyrenean area of northwest Catalonia (Spain) where the disease was previously unknown. Since the dog had never left the region and sandfly vectors, Phlebotomus ariasi and P. perniciosus, were present in the farm the case is considered as autochthonous and is the first to be published in this region of Spain., (Copyright © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

Background: Vascular adhesion protein-1 (VAP-1) is a cell surface and circulating enzyme that belongs to the semicarbazide-sensitive amine oxidase (SSAO) family, which oxidatively deaminates primary amines and is implicated in leukocyte extravasation. Our aim was to investigate the alteration of soluble VAP-1/SSAO activity in plasma samples after acute intracerebral hemorrhage (ICH) and its presence in human ICH brain tissue., Methods: VAP-1/SSAO activity was determined in plasma of 66 ICH patients and 58 healthy controls. In addition, we assessed the expression of VAP-1/SSAO in postmortem brain tissue from hemorrhagic stroke patients by Western blot and immunohistochemistry., Results: We observed significantly higher levels of plasma VAP-1/SSAO activity in patients with ICH compared to matched elderly controls (p = 0.001). Plasma VAP-1/SSAO activity <2.7 pmol/min·mg and baseline ICH volume <17 ml were independent predictors of neurological improvement after 48 h (OR 6.8, 95% CI 1.14-41.67, p = 0.035, and OR 10.64, 95% CI 1.1-100, p = 0.041, respectively), after adjustment for baseline stroke severity. We also found that membrane-bound VAP-1/SSAO levels were lower in the perihematoma region than in the corresponding contralateral brain areas of patients deceased due to ICH (p = 0.024)., Conclusions: Our data demonstrate that plasma VAP-1/SSAO activity is increased in ICH and predicts neurological outcome, suggesting a possible contribution of the soluble protein in secondary brain damage. Furthermore, anti-VAP-1/SSAO strategies might be a promising approach to prevent neurological worsening following ICH., (Copyright © 2011 S. Karger AG, Basel.)

The objective of this article is to describe the prevalence of maternal Hepatitis C infection globally and by HIV serostatus. Unlinked anonymous testing on 474,539 samples from newborn babies was carried out, selected through stratified sampling by year and HIV status, in six Spanish Autonomous Regions, from 2003 to 2006. Results were 0.15% for global anti-hepatitis C virus (HCV), 29.69% and 0.10% for HIV(+) and HIV(-) women, respectively. Anti-HCV prevalence rate is low in these regions compared to Hospital cohorts. Although a high prevalence rate in HIV co-infected women is found, it tends to decrease along the period studied.

An early and accurate diagnosis of multiple sclerosis (MS) is very important, since it allows early treatment initiation, which reduces the activity of the disease. Oligoclonal IgG band (OCGB) detection is a good ancillary tool for MS diagnosis. However, it was argued that its usefulness was limited by the high interlaboratory variability. In the last years, different techniques for OCGB detection have appeared. We performed a blinded aleatorized multicenter study in 19 Spanish hospitals to assess the accuracy and reproducibility of OCGB detection in this new scenario. We studied cerebrospinal fluid (CSF) and serum samples from 114 neurological patients. Every hospital contributed to the study with triplicated pairs of CSF and serum samples of six patients and analyzed 18 different samples. Global analysis rendered a sensitivity of 92.1%, a specificity of 95.1% and a Kappa value of 0.81. This shows that current techniques for OCGB detection have good accuracy and a high interlaboratory reproducibility and thus, represent a good tool for MS diagnosis. When we analyzed separately the different techniques used for OCGB detection, the highest concordance was observed in western blot with alkaline phosphatase detection (kappa=0.91). This indicates that high sensitivity techniques improve the reproducibility of this assay., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Hepatocyte nuclear factor 1-α (HNF-1α) is a homeodomain transcription factor expressed in a variety of tissues (including liver and pancreas) that regulates a wide range of genes. Heterozygous mutations in the gene encoding HNF-1α (HNF1A) cause familial young-onset diabetes, also known as maturity-onset diabetes of the young, type 3 (MODY3). The variability of the MODY3 clinical phenotype can be due to environmental and genetic factors as well as to the type and position of mutations. Thus, functional characterization of HNF1A mutations might provide insight into the molecular defects explaining the variability of the MODY3 phenotype. We have functionally characterized six HNF1A mutations identified in diabetic patients: two novel ones, p.Glu235Gly and c-57-64delCACGCGGT;c-55G>C; and four previously described, p.Val133Met, p.Thr196Ala, p.Arg271Trp and p.Pro379Arg. The effects of mutations on transcriptional activity have been measured by reporter assays on a subset of HNF-1α target promoters in Cos7 and Min6 cells. Target DNA binding affinities have been quantified by electrophoretic mobility shift assay using bacterially expressed glutathione-S-transferase (GST)-HNF-1α fusion proteins and nuclear extracts of transfected Cos7 cells. Our functional studies revealed that mutation c-57-64delCACGCGGT;c-55G>C reduces HNF1A promoter activity in Min6 cells and that missense mutations have variable effects. Mutation p.Arg271Trp impairs HNF-1α activity in all conditions tested, whereas mutations p.Val133Met, p.Glu235Gly and p.Pro379Arg exert differential effects depending on the target promoter. In contrast, substitution p.Thr196Ala does not appear to alter HNF-1α function. Our results suggest that HNF1A mutations may have differential effects on the regulation of specific target genes, which could contribute to the variability of the MODY3 clinical phenotype.

Kennel dogs can serve as sentinels and/or reservoirs of diseases of veterinary and zoonotic interest because they have often roamed free and lived outdoors, thus being exposed to pathogens. We tested dogs from the kennel of Inca (Majorca/Mallorca, Balearic Islands, Spain) for evidence of infection with three protozoan parasites: Leishmania infantum, Toxoplasma gondii, and Neospora caninum. Exposure to L. infantum was found in 56.3% of 48 dogs (37.5% by Western blot, 43.8% by PCR). Only 30% of infected dogs had leishmaniasis-like lesions. Seroprevalence to T. gondii was 58.7% of 46 dogs using the modified agglutination test (MAT, titer 1:25). None of the 44 dogs tested had N. caninum antibodies using a commercial competitive ELISA, probably because the surveyed dogs did not roam in the proximity of cattle farms. Results confirm the endemicity of L. infantum and also the widespread presence of T. gondii in the Mediterranean island of Majorca.

Objective: Oxidative stress is a main mechanism involved in vascular pathologies. Increased thioredoxin (TRX) levels have been observed in several oxidative stress-associated cardiovascular diseases. We aim to test the potential role of TRX as a biomarker of oxidative stress in abdominal aortic aneurysm (AAA)., Methods: TRX levels were analysed in both AAA intraluminal thrombus (ILT) tissue and in tissue-conditioned media by immunohistochemistry, Western blot and ELISA. Moreover, serum TRX levels were assessed in AAA Caucasian patients by ELISA., Results: TRX was mainly localized in the luminal part of ILT in AAA. Compared with the abluminal layer, TRX release was increased in the luminal layer of the ILT of AAA (31+/-9 ng/ml vs. 9+/-3 ng/ml, p<0.05). The interest of this approach is that we can identify proteins potentially released into the blood compartment, which could serve as biomarkers of the pathology. In a training population, serum TRX levels were significantly increased in patients with AAA relative to healthy subjects (50+/-6 ng/ml vs. 26+/-3 ng/ml, p<0.05). These results were validated in a second independent group of patients. Moreover, a positive correlation between TRX and AAA size (rho=0.5, p<0.001) was observed. Finally, in AAA samples with follow-up, TRX was positively associated to aneurismal growth rate (rho=0.25, p=0.027)., Conclusions: TRX release is increased in the luminal part of AAA and TRX serum levels are increased in AAA patients compared with healthy subjects. TRX levels correlates with AAA size and expansion, suggesting its potential role as a biomarker of AAA evolution., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

We studied the prevalence of Helicobacter pylori in patients with leprosy and the effects of co-infection on the immune response to Helicobacter antigens in the polar groups of leprosy (lepromatous and tuberculoid). We showed that there is no difference in the prevalence of H. pylori in patients with leprosy as compared to a non-leprosy population. We also demonstrated that the immune response to low molecular weight H. pylori antigens (35, 26 and 19 kDa) differs in patients with lepromatous as compared to those with tuberculoid leprosy. In lepromatous leprosy, we show that there is a higher prevalence of the 35 and 26 kDa antigens, but a lower prevalence of the 19 kDa antigen. These immunological results are consistent with previous histopathological studies illustrating a more severe gastrointestinal inflammation in lepromatous patients; importantly, a response to the 35 kDa antigen is recognized as a marker for the development of ulcerative disease.

Background: The occurrence of a second delayed paraneoplastic neurological syndrome (PNS), different from the first, in the same patient, is not well known. However, recognition of this possibility is important in the management of the patient with PNS., Objective: To describe eight patients who presented with two different PNS with a time delay of months to years., Design, Setting and Patients: Retrospective analysis of eight patients, included in the European PNS database, with two different PNS that occurred months to years apart., Results: The median delay between the two different PNS was 15 months. The antibody repertoire did not change between the two episodes. Anti-Hu was detected in five patients, and anti-CV2 and anti-Ri in one each. One patient did not present onconeural antibodies. Lung cancer was diagnosed in six patients. In five patients, the second PNS revealed a cancer relapse (n=4) or a second cancer (n=1) and in three it occurred without evidence of tumour recurrence. In these three patients, all with anti-Hu antibodies, the second episode consisted of a lower motor neuron disease. Median survival was not reached after a median follow-up of 5 years., Conclusions: A second PNS can reveal a cancer relapse but can also arise in its absence. The long survival of patients with a second PNS suggests that the immune response might be more effective in controlling the cancer.

Context: Thyroglobulin (TG) gene mutations cause congenital hypothyroidism (CH) with goiter. A founder effect has been proposed for some frequent mutations. Mutated proteins have a defect in intracellular transport causing intracellular retention with ultrastructural changes that resemble an endoplasmic reticulum storage disease., Objective: To reveal new aspects of thyroglobulin pathophysiology through clinical, cellular, molecular, and genetic studies in a family presenting with CH due to TG mutations from Galicia, an iodine-deficient area of Spain., Design: The included clinical evaluation of family members, DNA sequencing for TG gene mutation and haplotyping analysis, ultrastructural analysis of thyroid tissue specimens from affected subjects, analysis of effects of mutations found on TG gene transcription, and in vitro studies of cellular production and secretion of mutated proteins., Setting: Locations included primary care and university hospitals., Results: Family members with CH, mental retardation, and goiter were compound heterozygous for c.886C-->T (p.R277X) and g.IVS35+1delG. For c.886C-->T, a founder effect cannot be excluded, and its transcription was hardly detectable. g.IVS35+1delG caused an in-frame deletion in exon 35 and produced a protein that, although synthesized, could not be secreted. Ultrastructural analyses showed morphological changes consistent with an endoplasmic reticulum storage disease., Conclusion: The shorter thyroglobulin resulting from the novel g.IVS35+1delG was retained within the endoplasmic reticulum of thyrocytes, and together with p.R227X caused severe hypothyroidism with goiter. p.R277X, the most commonly described TG mutation, is caused by a TG exon-7 highly mutation-prone region, and the possibility that some cases were introduced to South America from Galicia cannot be excluded.

A 27-year-old woman of Moldavian origin presented at the age of 15 with progressive proximal limb weakness and painful cramps in her calf muscles. Clinical examination revealed prominent muscle weakness in proximal muscles of the lower extremities and distal anterior compartment of legs, and mild weakness in shoulder girdle muscles. In addition, she had marked calf hypertrophy, muscle atrophy involving the anterior and posterior compartments of the thighs, and the distal anterior compartment of legs, as well as mild scapular winging and hyperlordosis. A muscle biopsy taken from the biceps brachii showed mild dystrophic changes, absent vacuoles, and abundant lobulated fibers. Immunofluorescence and Western blot assays demonstrated complete telethonin deficiency. Molecular analysis revealed a homozygous Trp25X mutation in the telethonin (TCAP) gene resulting in termination of transcription at an early point. Four families from Brazil with telethonin deficiency have previously been reported and classified as LGMD2G, but the actual frequency of this disease is unknown. With this current identification of a case outside the Brazilian population, telethonin mutation-associated LGMD should be considered worldwide.

Background: In Castilla-La Mancha, Spain, the area with the highest density of vineyards in the world, 2 cases of Vitis vinifera pollen allergy have been previously reported., Objective: To determine the clinical relevance and biochemical characteristics of vine pollen in the Spanish province of Ciudad Real., Methods: We designed a prospective study of patients treated in the allergy units from Puertollano and Ciudad Real for respiratory symptoms of 4 months' duration in the year 2000. Skin prick tests with a standard aeroallergen battery and V vinifera pollen extract were performed on all patients. We also performed conjunctival and bronchial provocation tests and serum specific IgE and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting on the patients who tested positive for V vinifera pollen., Results: We included 200 patients, 98 sensitized to any pollen and 9 to V vinifera pollen. We found 8 of 9 positive conjunctival provocation test results and a positive bronchial provocation result to vine pollen in a vine grower. Serum specific IgE against V vinifera pollen was detected in 9 of 9 patients. Immunoblotting with V vinifera pollen extract showed IgE-binding bands at 45 and 67 kDa., Conclusions: Vine pollen could be the cause of pollinosis in exposed patients living in areas with a high density of vineyards.

Complement Factor I (CFI) is a regulator of the classical and alternative pathways. CFI has enzymatic activity and is able to cleave C3b and C4b. Homozygous Factor I deficiency is associated with infectious and/or autoimmune diseases. Here we describe the biochemical and genetic characterization in two Spanish families with complete Factor I deficiency. In Family 1, the propositus suffered from several episodes of meningitis for more than a year. Biochemical complement studies showed undetectable Factor I levels in the propositus and in her sister, while their parents and a brother had partial Factor I deficiency and were healthy. In Family 2, three out of five children were homozygous for Factor I deficiency, two of whom suffered from meningitis and the third one from several infections. The parents and the other two siblings were healthy and heterozygous for Factor I deficiency. Molecular studies showed that the two families had different mutations at exon 5 of the Factor I gene, which codifies for module LDLr1. One mutation corresponds to a 772G>A change at the donor splice site that was originally found in a family from Northern England. The second is a new missense mutation 739T>G, that generates a Cys to Gly change.

We evaluated the suitability of high-throughput proteomic methods to monitor terrestrial ecosystems. Free-living Mus spretus from three sites along the "Domingo Rubio" (DR) stream were compared with mice from Doñana Biological Reserve ("Santa Olalla" lagoon (SOL) negative control), using specimens from an industrial settlement (phosphogypsum stacks (PS)) and rice fields ("Matochal" rice fields (ARZ)) as positive controls. Our 2-DE analysis showed 36 spots with significantly altered expression. Sixteen were identified by MALDI-TOF-PMF and peptide matching with Mus musculus databases. Identified proteins play different roles: cytoskeletal dynamics, proteolysis, biotransformation, oxidative-stress adaptation, and metabolism. Animals from different polluted environments showed contrasting differences in their proteomes, with specific increases and decreases in selected groups of proteins that seem to be co-ordinately regulated. Proteomic data were consistent with metal biomonitoring and conventional biomarker responses, indicating that DR (and PS/ARZ) animals sustained a heavier pollutant burden than SOL specimens and suffered a chronic oxidative stress. Whereas some protein expression differences may protect mice from pollutant toxicity, others should make them more susceptible. Transcript expression signatures agree with the documented lack of correlation between mRNA and protein levels. Nonetheless, a positive significant correlation was found between the gpx1 mRNA molecules and the intensity of one of the two identified GPX1 isospots.

Currently, there are around 150,000 HIV-infected patients in Spain. This number, together with the fact that this disease is now a chronic condition since the introduction of antiretroviral therapy, has generated an increasing demand on the clinical microbiology laboratories in our hospitals. This increase has occurred not only in the diagnosis and treatment of opportunistic diseases, but also in tests related to the diagnosis and therapeutic management of HIV infection. To meet this demand, the Sociedad de Enfermedades Infecciosas y Microbiología Clinica (Spanish Society of Infectious Diseases and Clinical Microbiology) has updated its standard Procedure for the microbiological diagnosis of HIV infection. The main advances related to serological diagnosis, plasma viral load, and detection of resistance to antiretroviral drugs are reviewed in this version of the Procedure.

The hantaviruses are involved in a number of clinical syndromes of different severity and prognosis. Hantaviruses are widely distributed around the world, but the spectrum of illnesses they cause outside recognized endemic areas is unclear. A retrospective analysis was performed to detect anti-hantavirus antibodies in the serum of patients with hypertransaminemia of unknown etiology and in that of healthy members of the general population of Madrid (Spain). Antibodies were detected by indirect immunofluorescence and enzyme immunoassay; positive results were confirmed by Western blotting. Of the 182 patients with hypertransaminemia, 11 (6%) were positive for anti-hantavirus IgG antibodies; Western blotting using recombinant Puumala virus N antigen showed one of these patients to have hantavirus-specific IgM antibodies. Among the 146 healthy subjects from the general population, 3 (2%) were positive for anti-hantavirus IgG antibodies. These results show that anti-hantavirus antibodies are more commonly detected in patients with hypertransaminemia than in healthy people.

Visceral leishmaniasis (VL) caused by Leishmania infantum is a common disease in human immunodeficiency virus (HIV)-infected people in the Mediterranean basin. However, most such cases are asymptomatic, and little information about the prevalence of these infections in HIV-infected individuals is available. The aim of this study was to assess the prevalence of subclinical infection and the relationship between several Leishmania infection markers by noninvasive methods in asymptomatic HIV-infected patients from Southern Spain. Ninety-two HIV-infected patients, who were consecutively attended at the participant hospitals in 2004, were invited to participate in this study. These patients were asymptomatic and without any history of cutaneous or visceral leishmaniasis. Leishmania kinetoplast DNA (kDNA) was amplified from peripheral blood samples from 28 (30.4%) of these HIV-infected subjects. Sera from three (3.5%) patients tested positive for Leishmania by an enzyme-linked immunoassay. Two patients (2.4%) showed a specific 16-kDa band by Western blotting. In contrast, none of the patients showed a positive agglutination of urine. The leishmanin skin test was positive for four (4.3%) patients. None of the patients with a PCR-positive result showed a positive reaction by enzyme-linked immunoassay or by specific bands in Western blotting or had a positive leishmanin skin test. In conclusion, L. infantum kDNA was detected in a large proportion of asymptomatic HIV-infected patients, although a demonstrable cellular or humoral immune response to this parasite was not shown. Conversely, Leishmania antigen in urine was not detected in these patients.

Recent studies have revealed elevated expression of transforming growth factor beta1 (TGF-beta1) in gastric mucosa of patients with gastric cancer (GC) and those undergoing ulcer repair. As production of TGF-beta1 is genetically regulated, we aimed to assess whether functional polymorphisms of the TGFB1 gene are involved in susceptibility to and clinical characteristics of Helicobacter pylori-related diseases. DNA from 142 unrelated Spanish patients with GC, 200 with peptic ulcer and 342 healthy controls was typed for the MspA1I T+869C, and the Sau96I G+915C polymorphisms of the TGFB1 gene using polymerase chain reaction and RFLP analysis. H. pylori infection and CagA/VacA antibody status were determined by Western blot in patients and controls. H. pylori infection (odds ratio (OR): 11.44; 95% confidence interval (CI): 4.45-29.42; P<0.001) and non-steroidal anti-inflammatory drugs (OR: 5.07; 95% CI: 2.53-10.16; P<0.001) were identified as independent risks factors for duodenal ulcer (DU), whereas the TGFB1+869(*)C/C genotype was associated with reduced risk of developing the disease (OR: 0.32; 95% CI=0.15-0.68; P=0.003). Our results show that the TGFB1 T+869C gene polymorphism is involved in the susceptibility to DU and provide further evidence that host genetic factors play a key role in the pathogenesis of H. pylori-related diseases.

The presence of a lethal extracellular 39-kDa protease, a virulence determinant of a Listonella pelagia strain which produces vibriosis in turbot, was determined in the extracellular products (ECP) of 33 Vibrionaceae strains. Both immunological and enzymatic techniques distinguished this specific protease from other Vibrionaceae proteins. It was detected in 15% (5/33) of the ECPs assayed belonging to strains of the Vibrio splendidus-V. lentus related group isolated in Galician aquaculture systems (NW Spain). As these strains were associated with diseased octopus and cultured turbot, were able to colonize the internal organs of fish and produced a lethal ECP for fish, they are a potential risk for the health of reared aquatic organisms.

Background & Aim: The role that H. pylori infection plays in the development of and Barrett's esophagus (BE) is uncertain. We tested the hypothesis that infection with cagA+ Helicobacter pylori strains protects against the development of BE., Methods: We studied 104 consecutive patients, residents in an area with a high prevalence of H. pylori infection, with BE and 213 sex- and age-matched controls. H. pylori infection and CagA antibody status were determined by western blot serology., Results: H. pylori prevalence was higher in patients with BE than in controls (87.5% vs. 74.6%; OR. 2.3; 95% CI: 1.23-4.59). Increasing age was associated with a higher prevalence of H. pylori (p < 0.05). The prevalence of CagA+ H. pylori serology was similar in patients with BE and controls (64.4% vs. 54.5%; NS). Type I H. pylori infection (CagA+ and VacA+) was similar in patients with BE and controls (44.2% vs. 41.3%; NS). Logistic regression analysis identified alcohol (O.R. 7.09; 95% CI 2.23-22.51), and H. pylori infection (OR: 2.41; 95%CI: 1.20-4.84) but not CagA+ serology as independent factors., Conclusion: Neither H. pylori infection nor H. pylori infection by CagA+ strains reduce the risk of BE in a population with high prevalence of H. pylori infection.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by immunodeficiency, thrombocytopenia and eczema. A broad spectrum of mutations in the WASP gene has been identified as causing the disease. In the present paper, we report on a patient affected by WAS with a novel complex mutation, characterized by a small 9 bp deletion followed by an inversion of 151 bp and a gross deletion of 4.3 kb within the Xp11.23 region. The small deletion and the inverted fragment are found in intron 11. The large deletion initiates downstream of exon 11 of the WASP gene, including exon 12, and a genomic region upstream of the promoter of the contiguous SUV39H1 gene. Expression studies of the mRNA of the patient's sample showed the presence of two aberrant transcripts that code for a protein of 519 amino acids. We demonstrate that these two transcripts differ in the 3' UTR region, and result from the use of two alternative polyadenylation signals. The severe phenotype of the patient correlates with the presence of an aberrant protein.

Objective: To study the prevalence and factors associated with HIV and HCV infection among inmates of a Spanish prison., Method: A cross-sectional study was carried out in July 2001. We determined HCV (ELISA and RIBA-3) and HIV (ELISA and Western-blot) serology in the prison population. Study variables included age, sex, nationality and previous intravenous drug use (IDU). In IDU inmates we analyzed the age when intravenous drug use was initiated, years of consumption, age at first admission in prison and syringe sharing with other inmates. The subpopulations of Arab and Romani (gypsy) inmates were studied differentially., Results: A total of 800 inmates (mean age 34.2 6 6.2 years) were evaluated; 74.3% were Spanish and 33.6% IDU. HCV serology was obtained in 730 inmates and HIV serology in 773 with the following seroprevalence results: HCV 38.2%, HIV 19.1% and HCV-HIV co-infection 18.8%. The variables associated with HCV or HIV infection in the univariate analysis were Spanish nationality, previous IDU and coinfection by the other virus. In the multivariate analysis, only coinfection and, particularly, previous IDU (HCV infection: adjusted ORp 104.8 [95% CI: 49.4-222.2]) (HIV infection adjusted ORp 45.1 [95% CI: 14.0-144.9]) maintained an association with the two infections., Conclusions: The prevalence of HIV and HCV infection and coinfection is high in Spanish prisons. Infection by either of these viruses and previous IDU were independently associated with both infections. The percentage of non-Spanish inmates with these infections is low.

A Spanish family is reported with dystrophinopathy of myalgia and cramps syndrome type. There were five affected males and three females, and also six asymptomatic carriers. Muscle biopsy showed a dystrophic pattern, but immunohistochemistry carried out with three anti-dystrophin antibodies was normal. Dystrophin analysis by western blot revealed a dystrophin of reduced quantity and molecular weight. DNA analysis showed a deletion of the dystrophin gene involving exons 45-52. The natural history of this disorder and the large intrafamilial clinical variability are discussed.

Mucopolysaccharidosis IIIA (MPS IIIA; Sanfilippo syndrome) is an autosomal recessive lysosomal disorder caused by the deficiency of sulfamidase (EC 3.10.1.1), required for the degradation of the mucopolysaccharide heparan sulfate. The molecular defects of 26 unrelated Spanish MPS IIIA patients were recently reported by our group. Here we describe the heterologous expression, using a baculovirus system, of the cDNAs corresponding to eight out of the 14 mutant alleles present in this patient group and the characterization of the corresponding mutant enzymes. In particular, we expressed the following alleles: p.S66W, p.R74H, p.Q85R, p.R206P, p.L386R, p.R433W, p.R433Q, and c.1079delC (previously named as c.1091delC), and the two variants of the polymorphism p.R456H. The expression of the mutant alleles and the characterization of the corresponding enzymes revealed that their activity was severely compromised. Only mutations p.S66W and p.R206P retained low levels of residual activity. However, Western blot analysis showed in all cases the presence of the expected two forms of the sulfamidase, the precursor and the mature proteins, indicating a normal processing of the mutant enzyme.

A seroprevalence study was performed to determine the percentage of people exposed to Seoul virus among biologists and police officers (n = 226), whose work might put them into contact with viral reservoirs. Detection of specific IgG antibodies was performed by indirect immunofluorescence and western-blot. There was one positive result among the biologists (4.54%). This study confirms Seoul virus exposure in one of the employment groups studied.

Background: Only limited data on the seroprevalence of herpes simplex virus type 2 (HSV-2) are available from European countries. Until recently, serologic tests for HSV-2 serotyping have been hampered by cross-reactivity to type-common antigens. The present study aims at providing data on the prevalence of HSV-2 infection in a group of STD clinic attendees using a reliable type-specific immunoassay., Goal: To evaluate the seroprevalence of HSV-2 and the accumulated incidence of clinical genital herpes infection in a sample of Spanish sexually transmitted disease (STD) clinic attendees., Study Design: The study consisted of two parts. First, a cross-sectional study of HSV-2 seroprevalence was conducted in patients with STDs. Second, a prospective cohort study was undertaken to evaluate the accumulated incidence of infection by HSV-2 and of clinical episodes of genital herpes in HSV-2-negative patients included in the first study during a follow-up period of 6 to 18 months., Results: Of the 374 patients (129 men, 245 women) studied, 25% were seropositive for HSV-2 (12% of men, 30% of women). Antibodies to HSV-2 were related to female gender (odds ratio, 2.7; P < 0.001) and to the number of sexual partners (odds ratio, 4.1; P < 0.001). Fifty-two percent of patients (145 of 281 patients) who were initially seronegative returned to the clinic for a second serologic testing, of whom 1% (2 of 145 patients) had seroconverted. None of the patients developed genital herpes during the follow-up period., Conclusion: The relatively high seroprevalence (25%) and the low rate (4%) of HSV-2 previously reported in the general population in Spain suggest that the virus circulation may be restricted to certain risk groups. Therefore, future healthcare measures may target specific groups, such as patients with STDs.

Objective: To estimate the prevalence of HTLV infection among pregnant women in Spain., Methods: A commercial ELISA incorporating HTLV-I and HTLV-II antigens was used for HTLV antibody screening. Repeatedly reactive samples were further examined by western blot. Moreover, confirmation with PCR was performed when cells were available., Results: 20,366 pregnant women in 12 different Spanish cities were tested in a 3 year period (July 1996 to August 1999). 32 samples were repeatedly reactive by ELISA, and 10 of them were confirmed as positive by western blot (eight for HTLV-II and two for HTLV-I). In addition, three of 13 women who had an indeterminate western blot pattern yielded positive results for HTLV-II by PCR. All 11 HTLV-II infected women had been born in Spain, and all but one were former drug users. Seven of them were coinfected with HIV-1. One HTLV-I infected woman was from Peru, where HTLV is endemic and where she most probably was infected during sexual intercourse., Conclusion: The overall prevalence of HTLV infection among pregnant women in Spain is 0.064% (13/20,366), and HTLV-II instead of HTLV-I is the most commonly found variant. A strong relation was found among HTLV-II infection and specific epidemiological features, such as Spanish nationality and injecting drug use. Although HTLV-II can be vertically transmitted, mainly through breast feeding, both the low prevalence of infection and its lack of pathogenicity would not support the introduction of HTLV antenatal screening in Spain.

Propionic acidaemia (PA) is an autosomal recessive disorder caused by mutations in either of the PCCA or PCCB genes which encode the alpha and beta subunits, respectively, of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). In this work we have examined the biochemical findings and clinical outcome of 37 Spanish PA patients in relation to the mutations found in both PCCA and PCCB genes. We have detected 27 early-onset and 101 late-onset cases, showing remarkably similar biochemical features without relation to either the age of onset of the disease or the defective gene they have. Twenty-one of the patients have so far survived and three of them, now adolescents, present normal development. Different biochemical procedures allowed us to identify the defective gene in 9 PCCA deficient and 28 PCCB deficient patients. Nine putative disease-causing mutations accounting for 77.7% of mutant alleles were identified among PCCA deficient patients, each one carrying a unique genotypic combination. Of PCCB mutant alleles 98% were characterised. Four common mutations (ins/del, E168K, 1170insT and A497V) were found in 38/52 mutant chromosomes investigated, whereas the remainder of the alleles harbour 12 other different mutations. By examining the mutations identified both in PCCA and PCCB genes and the clinical evolution of patients, we have found a good correlation between certain mutations which can be considered as null with a severe phenotype, while certain missense mutations tend to be related to the late and mild forms of the disease. Expression studies, particularly of the missense mutations identified are necessary but other genetic and environmental factors probably contribute to the phenotypic variability observed in PA.

An outbreak of trichinellosis caused by wild boar meat occurred in the Iruela (Jaen) in southern Spain in February 1996. Thirty-five people were diagnosed on the basis of epidemiologic data, but only 24 patients agreed to participate in this study. Twenty-three (96%) had symptoms suggestive of trichinellosis. Immunofluorescent and Western blot test results for trichinellosis were positive in 18 persons, and 15 had circulating Trichinella spiralis antigens. These findings suggest that results of tests for circulating antigens in conjunction with clinical presentation are useful for the diagnosis of trichinellosis.

Background: Soybean hulls were identified as the etiologic agent responsible for the asthma outbreaks that occurred in Barcelona and Cartagena, Spain., Objective: To identify the main soybean hull allergens using the sera of 18 asthmatic epidemic patients from Barcelona and to compare the results to those previously reported., Methods: Specific IgE to a soybean hull allergen extract was determined by radioimmunoassay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a 10-20% Tris-tricine gradient gel followed by Western blot., Results: Specific IgE, which was detected in 15 of the 18 (83.3%) sera, indicated two nonoverlapping band patterns on the SDS-PAGE/Western blot: (1) pattern 1, present in 11 sera, consisted of three bands with estimated molecular weights (MWs) of 8, 7.5 and 7 kD, which are the MWs described for the allergens Gly m 2, Gly m 1A and Gly m 1B, respectively, and (2) pattern 2, present in 3 sera, consisted of a band with an estimated MW of 8.2-8.3 kD and four additional bands in a MW range of 25-36 kD. The remaining positive sera indicated very faint bands., Conclusions: This study confirms that soybean hulls contain three main allergens with MWs of 8, 7.5 and 7 kD, and indicates additional higher MW allergens, which selectively bind specific IgE of the sera that do not react with the three low MW components. This dichotomous and nonoverlapping pattern of allergen recognition has not been previously described.

Background: This study was carried out to know the prevalence of human immunodeficiency virus type 1 (HIV-1) and Mycobacterium tuberculosis infection in a penitentiary center, the risk factors and the prevention measures to reduce this infection., Subjects and Methods: 1,173 men from a penitentiary center between 1989-1995 were included. Demographic data, risk factors and penitentiary variables were studied. Antibodies against HIV were found and confirmed using Western blot; tuberculin skin reaction (PPD) and CD4 lymphocyte cant were studied., Results: The mean age was 31.8 (SD 8.8 years). They were 61.5% Spanish people and 48.1% were in prison for second time. The 49.7% used illegal drugs. Overall prevalence of HIV-1 infection was 24.3%. The differences of percentages for HIV-1 infection in intravenous drug users (IVDU) were 67.7% and in non IVDU were 3.8% with a significant statistical difference (chi 2 = 557.5; p < 0.0001). Using a multivariate test (stepwise method) we found that the probability to be HIV-1 positive in a penitentiary center was 21.9 times higher in IVDU, 5.6 times in Spanish prisoners, 2.6 times in subjects with more than one prison stay and 1.7 times if they had tatoos. The global prevalence of tuberculosis infection (PPD positive) was a 43.8%, and was associated to the second time inmates in this penitentiary center (chi 2 = 22.1; p < 0.001). A 54.4% of HIV-1 positive people showed a CD4 T lymphocyte count > or = 500 x 10(6)/l., Conclusions: We found a high prevalence of HIV-1 and tuberculosis infection and drug use in this penitentiary center. HIV-1 was transmitted and related by sharing syringes between IVDU independent of the stay in prison. The diagnosis of HIV-1 infection was made soon. The treatments of drug users in penitentiary centers and programs for lowering the risk are very important.

Three members of a Basque family carrying a novel six R2 octapeptide repeat 144-bp insertion in the prion protein gene (PRNP) showed a slowly progressive dementia associated with cerebellar signs, myoclonic jerks, and seizures. Although postmortem examination revealed only focal and minimal spongiform degeneration in one subject with a 4-year course, significant astrogliosis and neuronal loss were associated with pronounced spongiform degeneration in the patient with a duration of symptoms of 10 years. Prion protein (PrP)-immunoreactive patches with a unique morphology were present in the molecular layer of the cerebellum in both subjects. Western blot analysis demonstrated the presence of protease-resistant prion protein (PrPres) with the same characteristics (size and ratio of the three differently glycosylated isoforms) of that found in typical sporadic Creutzfeldt-Jakob disease (CJD129M/M, PrPres type 1). The amount of PrPres correlated with presence and severity of spongiform degeneration in the cerebral cortex. The findings suggest that a relatively low rate of PrPres deposition is the cause of the lack of spongiform degeneration in subjects carrying a 144-bp insertion in PRNP. The presence of PrP-immunoreactive patches with unique morphology in the molecular layer of the cerebellum is a hallmark of certain prion encephalopathies with insertional mutations and is useful in the diagnosis of this subtype of human prion disease.

In order to determine the seroprevalence of hepatitis E virus, 1,993 sera (453 from healthy pregnant women, 491 from Moroccan subjects, 492 from blood donors, 321 from children, and 236 from intravenous drug users) were studied. IgG was measured by enzyme immunoassay (EIA), and positive results were confirmed by Western blot. The EIA detected antibodies in 3.96% of the subjects (5.6% of the Moroccans and drug users and 1.8% of the children). Fifty-four percent of these results were confirmed by Western blot, 11.4% were found to be negative, and 34.2% indeterminate. The overall prevalence after confirmation by Western blot decreased to 2.15%. When studying the Western blot pattern of the positive samples, 95% showed antibodies to SG-3, 65% to 8-5, and only 9.3% to CKS fusion protein. In the indeterminate Western blots, the results for these proteins were 96.3%, 62.9%, and 37%, respectively. When the epidemiological data were analysed, no statistically significant differences between women and men or between different age groups were found.

To assess the spread of human T-cell leukemia virus (HTLV) type I and II in different population groups at potential risk of infection in Spain, a total of 756 subjects were studied: 453 belonging to groups at risk for retrovirus infection, 255 with diseases potentially linked to HTLV-I/II infection and 48 immigrants from endemic areas. An HTLV-I viral-lysate enzyme immunoassay (EIA) with a recombinant transmembrane envelope protein incorporated was used to screen serum samples. Reactive specimens were confirmed by Western blot strips spiked with recombinant proteins that differentiated HTLV-I from HTLV-II. Infection was then verified by the polymerase chain reaction (PCR). Serum samples from 19 of the 756 subjects analyzed (2.5%) were reactive for HTLV by EIA. One of these was from an intravenous drug user (IVDU) in whom HTLV-II infection was confirmed by Western blot and PCR; a specimen from another IVDU showed Western blot reactivity for both retroviruses, but PCR results were negative. Lastly, Western blot confirmed the presence of HTLV in one of the immigrant subjects. Western blot did not verify HTLV infection in the remaining 16 cases, indicating a high rate of nonspecific anti-HTLV reactivity when a second-generation EIA screening test was applied. These results suggest that HTLV is present in Spain among populations at high risk for HTLV, although at a very low rate and restricted to intravenous drug users and individuals immigrating from endemic areas.

The prevalence of anti-Borrelia burgdorferi antibodies was studied in Granada, Spain, between January 1991 and November 1993 in 354 patients with suspected Lyme disease (group 1); in 50 patients either with syphilis (n = 32) or without syphilis but with a positive Rapid Plasma Reagin test (n = 18) (group 2); and in 150 healthy subjects (group 3). In addition, intrathecal antibody production was evaluated by EIA in CSF samples obtained from 117 patients in group 1. Anti-Borrelia burgdorferi antibodies were detected by EIA in 58 patients (16.4%) in group 1, 29 (8.2%) of whom were positive by Western blot. Intrathecal antibody production was detected in one patient. In group 2, 8 (16%) patients had a positive EIA result, but none of these was confirmed by Western blot. western blot was negative for all subjects in group 3. The results of this study indicate that anti-Borrelia burgdorferi antibodies are not uncommon in our area, although Lyme disease is rare.

Up to October 1994, twenty-four HTLV-I and 113 HTLV-II infected persons have been identified in Spain. A third of HTLV-I carriers were immigrants coming from endemic regions. In contrast, most HTLV-II-infected persons were Spanish native drug users. Four cases of HTLV-I-associated myelopathy (TSP) were found, and three patients suffered lymphoma (ATL). A former drug user carrying HTLV-II infection developed a severe proximal myopathy. Studies performed on more than 100,000 blood donors allowed the identification of only two persons with HTLV-I infection.