Pedro González-Porqué

Intrathecal synthesis of oligoclonal IgM against myelin lipids predicts an aggressive disease course in MS

Oligoclonal IgM bands restricted to cerebrospinal fluid are an unfavorable prognostic marker in MS, the most common demyelinating disease of the CNS. We have attempted to identify the B cell subpopulation responsible for oligoclonal IgM secretion and the specificity of these bands. In addition, we explored the relationship between specificity and disease evolution. Intrathecal B cell subpopulations present in 29 MS patients with oligoclonal IgM bands and 52 without them were analyzed. A considerable increase in CD5(+) B lymphocytes was found in patients with oligoclonal IgM bands. These cells mostly secrete IgM antibodies recognizing nonproteic molecules. We also studied whether oligoclonal IgM bands present in cerebrospinal fluid of 53 MS patients were directed against myelin lipids. This was the case in most patients, with phosphatidylcholine being the most frequently recognized lipid. Disease course of 15 patients with oligoclonal IgM against myelin lipids and 33 patients lacking them was followed. Patients with anti-lipid IgM suffered a second relapse earlier, had more relapses, and showed increased disability compared with those without anti-lipid IgM. The presence of intrathecal anti-myelin lipid IgM antibodies is therefore a very accurate predictor of aggressive evolution in MS.

Intrathecal IgM synthesis is a prognostic factor in multiple sclerosis.

Intrathecal IgM synthesis (ITMS) predicts a worse evolution in the first stages of multiple sclerosis (MS). The aim of this study was the follow‐up of a group of relapsing‐remitting MS patients for a longer time to evaluate whether the ITMS implies a poor prognosis. Oligoclonal IgM bands were performed in 29 MS patients followed up from 5 to 16 years. Time to conversion to secondary‐progressive MS (SPMS), time elapsed to reach a disability of 6 in the Expanded Disability Status Scale (EDSS), percentage of patients with a benign MS, and changes in EDSS score were evaluated. During the follow‐up, 70.8% of patients with ITMS converted to SPMS. None of the patients without ITMS did. At the end of the study, 63.6% of patients with ITMS had reached EDSS 6, whereas none of the patients lacking ITMS reached values above EDSS 3. When patients with benign MS were analyzed, 82% lacked ITMS. All patients with a nonbenign MS had ITMS. At the end of the study, the mean EDSS score was 4.64 in patients with ITMS and 1.31 in those without. The presence of oligoclonal IgM bands in cerebrospinal fluid is an unfavorable prognostic marker in MS. Ann Neurol 2003;53:000–000

Clinically isolated syndromes: a new oligoclonal band test accurately predicts conversion to MS.

Background: Patients with a clinically isolated demyelinating syndrome (CIS) are at risk of developing a second attack, thus converting into clinically definite multiple sclerosis (CDMS). Therefore, an accurate prognostic marker for that conversion might allow early treatment. Brain MRI and oligoclonal IgG band (OCGB) detection are the most frequent paraclinical tests used in MS diagnosis. A new OCGB test has shown high sensitivity and specificity in differential diagnosis of MS. Objective: To evaluate the accuracy of the new OCGB method and of current MRI criteria (MRI-C) to predict conversion of CIS to CDMS. Methods: Fifty-two patients with CIS were studied with OCGB detection and brain MRI, and followed up for 6 years. The sensitivity and specificity of both methods to predict conversion to CDMS were analyzed. Results: OCGB detection showed a sensitivity of 91.4% and specificity of 94.1%. MRI-C had a sensitivity of 74.23% and specificity of 88.2%. The presence of either OCGB or MRI-C studied simultaneously showed a sensitivity of 97.1% and specificity of 88.2%. Conclusions: The presence of oligoclonal IgG bands is highly specific and sensitive for early prediction of conversion to multiple sclerosis. MRI criteria have a high specificity but less sensitivity. The simultaneous use of both tests shows high sensitivity and specificity in predicting clinically isolated demyelinating syndrome conversion to clinically definite multiple sclerosis.

A sensitive and reproducible method for the detection of oligoclonal IgM bands

We have developed a sensitive and specific isoelectrofocusing (IEF) method for the detection of oligoclonal (OGC) IgM in the cerebrospinal fluid (CSF) and sera. In this procedure, ampholytes, in the range pH 5-8, were used to improve the resolution, and serum was diluted in saline to avoid IgM precipitation. Samples were treated with 50 mM dithiothreitol (DTT) at pH 9.5 to reduce IgM and improve the migration of the protein. Using an anti-IgM labelled with alkaline phosphatase, the limit of detection was found to be 0.1 ng in 5 microl of sample.

Influence of oligoclonal IgM specificity in multiple sclerosis disease course

Oligoclonal IgM bands (OCMB) against myelin lipids predict an aggressive multiple sclerosis (MS) course. However, the clinical significance of OCMB without lipid specificity, present in other MS patients, remains unknown. We describe here a characterization of these antibodies and study their role in MS progression. Fifty-four MS patients showing CSF-restricted OCMB were included in this study at disease onset and followed-up during 61.1 ± 2.7 months. The specificity of OCMB and the CSF B-cell profile were investigated. A second CSF IgM study was performed in a group of eight patients. Thirty-eight patients showed OCMB against myelin lipids (M+L+) and other sixteen had OCMB lacking this specificity (M+L-). The CD5+ B cell subpopulation, responsible for most persistent IgM responses, was considerably higher in M+L+ than in M+L- patients (3.3 ± 0.6% versus 0.8 ± 0.2, P = 0.009). In addition, M+L+ bands persisted during disease course, while M+L- disappeared during follow-up. M+L+ patients suffered more relapses (4.2 ± 0.6 versus 1.6 ± 0.3, P = 0.002) and reached higher disability (EDSS score of 2.2 ± 0.2 versus 1.2 ± 0.2, P = 0.02) than M+L- group. These data corroborate that anti-lipid OCMB associate with an aggressive MS course and show that OCMB that do not recognize myelin lipids represent a transient immune response related to a more benign disease course. Multiple Sclerosis 2008; 14: 183—187. http://msj.sagepub.com

Description of an enzyme-linked immunosorbent assay for the detection of protein tyrosine kinase.

A solid immunoassay for the detection of protein tyrosine kinases has been developed. It is based on the binding of the synthetic polypeptide poly(Glu.Na,Tyr) 4:1 to microELISA wells, where the phosphorylation reaction takes place in the presence of ATP and enzyme. The phosphorylated tyrosine residues produced in the reaction are finally detected, in the same well, by means of an ELISA using monoclonal antiphosphotyrosine antibody, peroxidase-labeled goat anti-mouse IgG antibody, and substrate. The amount of protein tyrosine kinase activity present in the sample is proportional to the color at 492 nm developed in each well.

CSF oligoclonal band patterns reveal disease heterogeneity in multiple sclerosis

Oligoclonal IgG bands (OCGB) are characteristic of multiple sclerosis (MS). Most patients show OCGB exclusively in cerebrospinal fluid (CSF). Others have serum bands with additional ones in CSF. Moreover, IgM bands against myelin lipids (LS-OCMB) associate with aggressive relapsing-remitting MS (RRMS). We studied oligoclonal bands in 424 MS patients. Most primary progressive (PPMS) patients showed serum OCGB with additional bands in CSF. Conversely, most RRMS and secondary progressive (SPMS) patients showed OCGB exclusively in CSF (p<0.0001). Moreover, no PPMS patient presented LS-OCMB, while 31% of RRMS and 60% of SPMS groups showed these antibodies (p<0.0001). This suggests heterogeneous autoimmune mechanisms in MS.

Accuracy of CSF and MRI criteria for dissemination in space in the diagnosis of multiple sclerosis

Demonstration of lesion dissemination in space (DIS) and time (DIT) is necessary for the diagnosis of multiple sclerosis (MS) in clinically isolated syndromes (CIS). The McDonald criteria accepted two methods to demonstrate DIS. The fulfillment of at least three of four MRI Barkhof criteria (MRI-BC) or, alternatively, the finding of at least two MRI lesions on T2-weighted images (T2 lesions) plus the presence of oligoclonal IgG bands (OCGB) in cerebrospinal fluid (CSF). We aimed to evaluate the accuracy of both methods for DIS demonstration to predict conversion of CIS to MS using a new OCGB test. We studied fifty-eight CIS patients with OCGB detection and brain MRI, and followed them up during 6 years. Twenty-eight patients fulfilled MRI-BC. Twenty-five of them converted to MS during follow-up (sensitivity 73.53%, specificity 87.50%, accuracy 79.31%). Thirty-four patients had at least two T2 lesions plus oligoclonal bands. Thirty-three converted to MS during follow-up (sensitivity 94.29%, specificity 95.65%, accuracy 94.82%). The presence of oligoclonal IgG bands plus two T2 lesions accurately predicts CIS conversion to MS. MRI-BC criteria have a high specificity but less sensitivity and accuracy. These results reinforce the role of CSF study in MS diagnosis.

Antiviral activity of lauryl gallate against animal viruses.

The homeobox gene extradenticle (exd) acts as a cofactor of the homeotic genes in the specification of larval patterns during embryogenesis. To study its role in adult patterns, we have generated clones of mutant exd- cells and examined their effect on the different body parts. In some regions, exd- clones exhibit homeotic transformations similar to those produced by known homeotic mutations such as Ultrabithorax (Ubx), labial (lab), spineless-aristapedia (ssa) or Antennapedia (Antp). In other regions, the lack of exd causes novel homeotic transformations producing ectopic eyes and legs. Moreover, exd is also required for functions normally not associated with homeosis, such as the maintenance of the dorsoventral pattern, the specification of subpatterns in adult appendages or the arrangement of bristles in the mesonotum and genitalia. Our findings indicate that exd is critically involved in adult morphogenesis, not only in the homeotic function but also in several other developmental processes.Previously published experiments have shown that the endogenous Dfd gene can be ectopically activated by its own (heat-shock-driven) product in a subset of cells of different segments. This results in the differentiation of maxillary structures like cirri and mouth hooks in places where they normally do not appear, and represents a phenomenon of autocatalysis of homeotic gene function that differs from the normal activation process. We show that this out-of-context activation occurs in cells belonging to the anterior compartments of the three thoracic and the A1 to A8 abdominal segments and that it requires the normal function of the polarity genes wingless (wg) and engrailed (en). The wg product, in addition to that of Dfd, appears to be sufficient to activate the endogenous Dfd gene in many embryonic cells. We have studied the effect of several homeotic genes on Dfd activation and phenotypic expression: Scr, Antp, Ubx and Abd-B repress Dfd both transcriptionally and at the phenotypic level, if their products are in sufficient amounts. The endogenous abd-A gene does not have a noticeable effect, but when it is replaced by an hsp70-abd-A gene, which produces a high and uniform level of expression, the phenotypic expression of Dfd is suppressed. Our results also suggest that the differentiation of cirri is induced by Dfd-expressing cells in non-expressing neighboring cells, and that this interaction occurs across the parasegmental border.During evolution, many animal groups have developed specialised outgrowths of the body wall, limbs or appendages. The type of appendage depends on the identity of the segment where they appear, indicating that the Hox genes contribute to appendage specification. Moreover, work carried out principally in Drosophila has identified the gene products and the mechanisms involved in pattern formation in the appendages. In this essay, we compare the morphogenetic processes in the appendages and the body wall; the function of the Hox genes and the response to the signalling molecules involved in local patterning. We speculate that, although the basic mechanisms are similar, there are significant differences in the manner the body trunk and appendages respond to them.[ES] La pared celular es un elemento morfogenetico esencial que determina la forma final de las celulas y que las protege contra la lisis. En S. pombe esta esta constituida por ? y s-glucano y manoproteinas y tanto la sintesis como remodelacion de su estructura requiere de diferentes enzimas estrictamente reguladas. En S. pombe existe poca informacion de como se lleva a cabo la incorporacion del material de membrana y sobre la regulacion de las enzimas implicadas en la sintesis y remodelacion de la pared celular por los mecanismos de transporte vesicular. Para abordar el estudio de como el trafico vesicular mediado por clatrina afecta a la morfogenesis de S. pombe y en particular cual es su papel en la regulacion de la sintesis de la pared celular se ha analizado el papel tanto de la clatrina, mediante el analisis de diferentes mutantes de la cadena ligera de la clatrina, como el del adaptador AP-2, que interviene en el proceso de endocitosis mediada por clatrina. Se ha demostrado que la delecion de la cadena ligera de la clatrina resulta letal para las celulas de S. pombe y que esta letalidad se rescata al incubar las celulas en un medio suplementado con sorbitol. En este caso aunque las celulas pueden sobrevivir poseen graves defectos morfologicos, en crecimiento, en trafico vesicular, en desarrollo sexual, etc. Se ha podido comprobar que la ausencia de Clc1p afecta drasticamente a la estabilidad de Chc1p hecho que hace que, a diferencia de otros organismos, la supervivencia de S. pombe sea mas dependiente de la presencia clatrina. Ademas se ha demostrado que la letalidad causada por la ausencia de Clc1p se debe principalmente a defectos graves en la sintesis de la pared celular que afectan directamente a la sintesis del glucano. Los resultados obtenidos muestran que una reduccion en la cantidad de clatrina causa un leve impacto en el transporte vesicular en general y en otros procesos y elementos biologicos, pero afecta gravemente a la secrecion de enzimas de sintesis/remodelacion de la pared celular, como las s(1,3)glucan sintasa y endoglucanasas. En cuanto al complejo adaptador AP-2 se ha comprobado, que a diferencia de lo que se conoce hasta el momento en otros organismos unicelulares, este forma un complejo con la clatrina y se ha demostrado que tiene un papel en la endocitosis general de S. pombe. Asi mismo se ha descubierto que AP-2 puede estar interviniendo en la sintesis de la pared celular ya que su ausencia afecta a la actividad s-glucan sintasa y hace que S. pombe sea hiper-sensible a compuestos que afectan a la integridad de la pared celular.We characterized a novel protein of the Ras family, p19 (H-RasIDX). The c-H-ras proto-oncogene undergoes alternative splicing of the exon termed IDX. We show that the alternative p19 mRNA is stable and as abundant as p21 (p21 H-Ras4A) mRNA in all of the human tissues and cell lines tested. IDX is spliced into stable mRNA in different mammalian species, which present a high degree of nucleotide conservation. Both the endogenous and the transiently expressed p19 protein are detected in COS-1 and HeLa cells and show nuclear diffuse and speckled patterns as well as cytoplasmic localization. In yeast two-hybrid assays, p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multiprotein complexes in different signaling pathways. This observation suggests that p19 and p21 play differential and complementary roles in the cell.Resumen del trabajo presentado al Congreso Nacional de Biotecnologia, celebrado en Murcia del 18 al 21 de junio de 2017.A. G. G. thanks Ramon Areces Foundation for a grant. J. C. thanks NIH-CA24487 for financial support.Ministerio de Educacion y Ciencia and grant S-0505/MAT-0283 from Comunidad Autonoma de Madrid to M.S. and by an Institutional grant from Fundacion Ramon Areces to the Centro de Biologia Molecular “Severo Ochoa”We report a genetic and molecular study of UbxMX6 and Ubx195rx1, two mutations in the Ultrabithorax (Ubx) locus which appear to have a strong effect on the activity of the homologous Ubx gene. These mutations show the characteristic embryonic and adult phenotypes of Ubx null alleles, and also fail to produce any detectable Ubx product. Yet, genetic and phenotypic analyses involving a large number of trans heterozygous combinations of UbxMX6 and Ubx195rx1 with different classes of Ubx mutations, indicate that they hyperactivate the homologous gene. This effect is induced on wildtype or mutant forms of Ubx, provided that the pairing in the bithorax region is normal, i.e. these mutations have a strong positive effect on transvection. We also show that, unlike all the other known cases of transvection in Ubx, this is not zeste-dependent. Southern analyses indicate that UbxMX6 is a 3.4 kb deletion, and Ubx195rx1 is an approximately 11 kb insertion of foreign DNA, both in the promoter region. We speculate that the region altered in the mutations may have a wildtype function to ensure cis-autonomy of the regulation of Ubx transcription.Resumen del trabajo presentado al Congreso Nacional de Biotecnologia, celebrado en Murcia del 18 al 21 de junio de 2017.The pannier (pnr) gene of Drosophila encodes a zinc-finger transcription factor of the GATA family and is involved in several developmental processes during embryonic and imaginal development. We report some novel aspects of the regulation and function of pnr during embryogenesis. Previous work has shown that pnr is activated by decapentaplegic (dpp) in early development, but we find that after stage 10, the roles are reversed and pnr becomes an upstream regulator of dpp. This function of pnr is necessary for the activation of the Dpp pathway in the epidermal cells implicated in dorsal closure and is not mediated by the JNK pathway, which is also necessary for Dpp activity in these cells. In addition, we show that pnr behaves as a selector-like gene in generating morphological diversity in the dorsoventral body axis. It is responsible for maintaining a subdivision of the dorsal half of the embryo into two distinct, dorsomedial and dorsolateral, regions, and also specifies the identity of the dorsomedial region. These results, together with prior work on its function in adults, suggest that pnr is a major factor in the genetic subdivision of the body of Drosophila.10th International Symposium on Reproductive Physiology of Fish (10th ISRPF), Expanding the khowledge base of reproductive success: from genes to the environment, 25-30 May 2014, Olhao, Portugal.-- 1 pageBy using a hsp70-Ubx fusion gene, we have ectopically expressed a Ubx product in the embryonic head primordia and studied the developmental effects on the larval head. We find that after high and persistent levels of Ubx product, the head is replaced by three (C1, C2 and C3) abdominal-like denticle belts. The C2 and C3 belts are the homeotic transformations of parasegments 1 and 2, respectively, while the C1 belt probably derives from the transformation and subsequent fusion of the most anterior procephalic primordia. On the basis of their response to the Ubx product and other arguments, we propose that the larval head is made of two genetically distinct components; one is the procephalon and the anterior region of the mandibular lobe, and the other is part of the parasegmental trunk and includes parasegments 1 and 2. Our results also indicate that most or all the larval head structures derive from precursor cells of ventral origin.The Iroquois (Iro) family of genes are found in nematodes, insects and vertebrates. They usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that possess a characteristic homeodomain. The Iro genes function early in development to specify the identity of diverse territories of the body, such as the dorsal head and dorsal mesothorax of Drosophila and the neural plate of Xenopus. In some aspects they act in the same way as classical selector genes, but they display specific properties that place them into a category of their own. Later in development in both Drosophila and vertebrates, the Iro genes function again to subdivide those territories into smaller domains.The pannier (pnr) gene encodes a GATA transcription factor and acts in several developmental processes in Drosophila, including embryonic dorsal closure, specification of cardiac cells and bristle determination. We show that pnr is expressed in the mediodorsal parts of thoracic and abdominal segments of embryos, larvae and adult flies. Its activity confers cells with specific adhesion properties that make them immiscible with non-expressing cells. Thus there are two genetic domains in the dorsal region of each segment: a medial (MED) region where pnr is expressed and a lateral (LAT) region where it is not. The homeobox gene iroquois (iro) is expressed in the LAT region. These regions are not formed by separate polyclones of cells, but are defined topographically. We show that ectopic pnr in the wing induces MED thoracic development, indicating that pnr specifies the identity of the MED regions. Correspondingly, when pnr is removed from clones of cells in the MED domain, they sort out and apparently adopt the LAT fate. We propose that (1) the subdivision into MED and LAT regions is a general feature of the Drosophila body plan and (2) pnr is the principal gene responsible for this subdivision. We argue that pnr acts like a classical selector gene but differs in that its expression is not propagated through cell divisions.We have developed a specific polyclonal antibody that recognizes the protein products of the abdominal-A (abd-A) gene, a member of the bithorax complex of Drosophila. The normal expression domain extends from parasegments 7 to 13, in good correspondence with previous genetic and molecular results. However, while the anterior border of expression is precisely demarcated by a parasegmental boundary, the posterior border does not coincide with a lineage boundary. Within the normal domain, the expression of abd-A shows intrametameric modulation; the amount of product is higher in posterior compartments and in the most anterior cells of the anterior compartments and then gradually decreases. We have examined the effect on abd-A expression of a number of mutations, some mapping within and others outside the abd-A transcription unit. Those mapping to the transcription unit eliminate or severely reduce the amount of abd-A antigen, while those mapping outside produce an abnormal distribution of abd-A protein. Finally, we show that the abd-A gene is down-regulated in part of the Abdominal-B (Abd-B) domain, precisely in those regions where the Abd-B gene is expressed at high levels.Resumen del trabajo presentado al Yeast Genetics Meeting, celebrado en Stanford, California (USA) del 22 al 26 de agosto de 2018.The effect of the anti-tumoral drug lauryl gallate on the infectivity of the African swine fever virus among other DNA (Herpes simplex and Vaccinia) and RNA (Influenza, Porcine transmissible gastroenteritis and Sindbis) viruses, involved in animal and human diseases, is analyzed. Viral production was strongly inhibited in different cell lines at non-toxic concentrations of the drug (1-10 μM), reducing the titres from 3 to more than 5 log. units depending on the multiplicity of infection. In our model system (African swine fever virus in Vero cells), the addition of the drug 1 h before virus adsorption, completely abolished virus productivity in a one-step growth virus cycle. Interestingly, no inhibitory effect was observed when lauryl gallate was added after 5 to 8 hpi. Both cellular and viral DNA synthesis and late viral transcription were inhibited by the drug, but, however, the early viral protein synthesis and the virus-mediated increasing of p53 remained unaffected. Activation of the apoptotic effector caspase-3 was not detected after lauryl gallate treatment of Vero cells, and, furthermore, the presence of the drug abrogated the activation of this protease induced by the virus infection. The overall results likely indicate that a cellular factor/function might be the target of the antiviral action of alkyl gallates.Tesis Doctoral presentada por Eduardo Rodenas Martinez en el Centro Andaluz de Biologia del Desarrollo, centro mixto CSIC-UPO.Resumen del trabajo presentado al Yeast Genetics Meeting, celebrado en Stanford, California (USA) del 22 al 26 de agosto de 2018.

Intrathecal synthesis of soluble class I antigens in multiple sclerosis

We have studied the intrathecal synthesis of soluble class I antigens (sHLA), reflected by the index IH = (CSF sHLA/serum sHLA)/(CSF albumin/serum albumin), in multiple sclerosis (MS). IH was increased in patients in relapse, but normal in patients in remission; these findings show that there is a high lymphocyte activation within the central nervous system in patients with clinically active MS.