CSF analysis for protein biomarker identification in patients with leptomeningeal metastases from CNS lymphoma.

INTRODUCTION: Leptomeningeal metastases (LM) from lymphoma remain a difficult complication for oncologist due to the high incidence in morbidity and mortality. Early diagnostic and initiation of treatment are essential to prevent neurological deterioration. Areas covered: In this review, several proteomic approaches are described in order to help and provide the basis for the identification of biomarkers useful in early diagnosis, also in discovery novel targets for therapeutic agents. In fact, the identification of biomarkers will have a high potential to detect leptomeningeal lymphoma, as well as to predict its progression and treatment response. Expert commentary: In the case of LM by Central nervous system (CNS) lymphoma, these studies generated the first insights into the utility of proteomic analysis for biomarker identification and will be demonstrated that identifying specific proteins in cerebrospinal fluid (CSF) had much greater sensitivity for detecting LM in comparison to standard cytological protocols.

The Aotus nancymaae erythrocyte proteome and its importance for biomedical research.

The Aotus nancymaae species has been of great importance in researching the biology and pathogenesis of malaria, particularly for studying Plasmodium molecules for including them in effective vaccines against such microorganism. In spite of the forgoing, there has been no report to date describing the biology of parasite target cells in primates or their biomedical importance. This study was thus designed to analyse A. nancymaae erythrocyte protein composition using MS data collected during a previous study aimed at characterising the Plasmodium vivax proteome and published in the pertinent literature. Most peptides identified were similar to those belonging to 1189 Homo sapiens molecules; >95% of them had orthologues in New World primates. GO terms revealed a correlation between categories having the greatest amount of proteins and vital cell function. Integral membrane molecules were also identified which could be possible receptors facilitating interaction with Plasmodium species. The A. nancymaae erythrocyte proteome is described here for the first time, as a starting point for more in-depth/extensive studies. The data reported represents a source of invaluable information for laboratories interested in carrying out basic and applied biomedical investigation studies which involve using this primate. SIGNIFICANCE: An understanding of the proteomics characteristics of A. nancymaae erythrocytes represents a fascinating area for research regarding the study of the pathogenesis of malaria since these are the main target for Plasmodium invasion. However, and even though Aotus is one of the non-human primate models considered most appropriate for biomedical research, knowledge of its proteome, particularly its erythrocytes, remains unknown. According to the above and bearing in mind the lack of information about the A. nancymaae species genome and transcriptome, this study involved a search for primate proteins for comparing their MS/MS spectra with the available information for Homo sapiens. The great similarity found between the primate's molecules and those for humans supported the use of the monkeys or their cells for continuing assays involved in studying malaria. Integral membrane receptors used by Plasmodium for invading cells were also found; this required timely characterisation for evaluating their therapeutic role. The list of erythrocyte protein composition reported here represents a useful source of basic knowledge for advancing biomedical investigation in this field.

Determining the Plasmodium vivax VCG-1 strain blood stage proteome.

Plasmodium vivax is the second most prevalent parasite species causing malaria in humans living in tropical and subtropical areas throughout the world. There have been few P. vivax proteomic studies to date and they have focused on using clinical isolates, given the technical difficulties concerning how to maintain an in vitro culture of this species. This study was thus focused on identifying the P. vivax VCG-1 strain proteome during its blood lifecycle through LC-MS/MS; this led to identifying 734 proteins, thus increasing the overall number reported for P. vivax to date. Some of them have previously been related to reticulocyte invasion, parasite virulence and growth and others are new molecules possibly playing a functional role during metabolic processes, as predicted by Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis. This is the first large-scale proteomic analysis of a P. vivax strain adapted to a non-human primate model showing the parasite protein repertoire during the blood lifecycle. Database searches facilitated the in silico prediction of proteins proposed for evaluation in further experimental assays regarding their potential as pharmacologic targets or as component of a totally efficient vaccine against malaria caused by P. vivax. BIOLOGICAL SIGNIFICANCE: P. vivax malaria continues being a public health problem around world. Although considerable progress has been made in understanding genome- and transcriptome-related P. vivax biology, there are few proteome studies, currently representing only 8.5% of the predicted in silico proteome reported in public databases. A high-throughput proteomic assay was used for discovering new P. vivax intra-reticulocyte asexual stage molecules taken from parasites maintained in vivo in a primate model. The methodology avoided the main problem related to standardising an in vitro culture system to obtain enough samples for protein identification and annotation. This study provides a source of potential information contributing towards a basic understanding of P. vivax biology related to parasite proteins which are of significant importance for the malaria research community.

Validación de un método por cromatografía líquida de alta resolución (HPLC) para la determinación de ivabradina en comprimidos / Validation of a high resolution liquid chromatography method (HPLC) for the determination of ivabradine tablets

Se presentan los resultados obtenidos en la validación de un método analítico por cromatografía líquida de alta resolución, para la determinación de ivabradina en comprimidos, el cual se diseñó para identificar y cuantificar a dicho analito, con la utilización de una columna RP-18 (150 x 4,6 mm) 5 µm y un detector UV-Vis a una λ: 285 nm. Dicho método se empleará para el control de la calidad y estabilidad de ivabradina comprimidos. El método fue validado siguiendo una metodología de trabajo de acuerdo a los lineamientos de la Conferencia Internacional sobre Armonización de Requisitos Técnicos para el Registro de Medicamentos de uso humano, Validación de procedimientos analíticos (ICH Q2), obteniendo los siguientes resultados: linealidad (r2 = 0,999), precisión Desviación estándar relativa (DSR) = 0,67 % para comprimidos del lote piloto y DSR=0,31% para comprimidos del lote comercial) y exactitud recuperación media=100,2%, demostrando de esta manera que el método propuesto es aplicable a la dosificación de ivabradina en comprimidos, tanto en el análisis del producto terminado cómo en los estudios de estabilidad.

Internal initiation of translation efficiency in different hepatitis C genotypes isolated from interferon treated patients.

Initiation of translation of hepatitis C viral RNA occurs internally and it is mediated by a segment of about 330 nucleotides termed Internal Ribosome Entry Site (IRES) located in the 5' end region. While being the most conserved part of the genome, this region also accumulates nucleotide substitutions which are often covariant. In this study we have examined the activity and sequence variation of IRES elements belonging to genotypes 1b, 2a/2c and 3a in patients that responded or not to interferon therapy. The substitutions found in the IRES region analyzed were predicted to maintain the secondary structure of the RNA. Comparison of their efficiency to promote internal initiation of translation in bicistronic constructs supported the conclusion that for both 1b and 3a genotypes, response to interferon therapy and IRES activity are unrelated, although sequence homology was not always found among isolates from patients with different type of response. IRES activity of the studied genotypes varied about 4-fold under the conditions used in our in vivo assays depending on the cell line used for transfection. Such differences were not evidenced in vitro suggesting that the differences observed depend on trans-acting factors present in the transfected cell.

Response to retreatment with interferon-alpha plus ribavirin in chronic hepatitis C patients is independent of the NS5A gene nucleotide sequence.

OBJECTIVE: Interferon-alpha plus ribavirin is an effective treatment for chronic hepatitis C patients. We evaluated whether the response to this combined therapy correlated with the presence of mutations in a region of 372 nucleotides within the NS5A gene. METHODS: Sixty-two patients, 42 nonresponders and 20 relapsers to a previous course of interferon-alpha, received 3 million units thrice weekly of interferon-alpha-2b and 1-1.2 g daily of ribavirin for 12 months. Basal biochemical and virological (HCV RNA and genotype) parameters were determined. Clinical examinations were carried out at 1, 2, 3, 6, and 12 months. In addition, nucleotide sequencing of the NS5A gene was determined for viral samples obtained from 38 of these patients at the baseline of the combined therapy, as well as in 15 of them before initiating the previous course of interferon as monotherapy. RESULTS: On finishing the 12 months, 36 patients (58.1%) had normal aminotransferases and 25 (40.3%) cleared viremia. Nucleotide sequencing indicated the same level of genetic variability within the group of responder and nonresponder patients all along the 124 amino acid residues of the NS5A gene studied. Neither the type of amino acid substitution nor the number of them was significantly different in one group relative to the other. CONCLUSIONS: Therapy with interferon-alpha-2b plus ribavirin was well tolerated, achieving an end-of-treatment response in 25 (40.3%) patients. Response did not correlate with the presence of mutations in the NS5A gene analyzed, including the interferon sensitivity determining region (ISDR) and its flanking sequences.

Stimulation of the myelin basic protein gene expression by 9-cis-retinoic acid and thyroid hormone: activation in the context of its native promoter.

Thyroid hormone plays an important role in brain development, in part by regulating myelination. Previous studies have shown that the myelin basic protein (MBP) promoter is activated by thyroid hormone (T3) via a T3-response element (T3RE) at position -186. Surprisingly, although MBP levels are initially decreased in hypothyroid neonates, they approach later control levels, in most brain regions, despite persistent hypothyroidism. We have studied the T3-independent transcriptional regulation of this gene, using transient transfection assays. We found that, in the absence of T3, the RXR ligand, 9-cis-retinoic acid (9cRA) was able to stimulate transcription of the MBP promoter in a dose-dependent manner. This activation was unaffected by the mutation or deletion of the T3RE and required DNA sequences located between positions -162/+60. Accordingly, this MBP promoter fragment bound RXR in vitro. The 9cRA-dependent activation of the MBP promoter required the presence of both, the DNA binding and the ligand-dependent transactivation domain (AF-2) in RXR. Furthermore, as T3, 9cRA was able to stimulate MBP expression in the CG-4 cell line after differentiation to oligodendrocytes and increased the number of cells expressing the MBP protein in primary rat optic nerve glial cell cultures.

Thyroid hormone regulates the expression of the MAL proteolipid, a component of glycolipid-enriched membranes, in neonatal rat brain.

Detergent-insoluble glycosphingolipid-enriched membranes (DIGs) have been involved in the sorting and transport of specific proteins during oligodendrocyte maturation. The MAL (MAL, MVP17, VIP17) proteolipid, an integral membrane protein present in DIGs in mature oligodendrocytes, has been proposed as a component of the machinery for DIG-mediated transport in a restricted pattern of cell types including myelinating cells. We have previously shown that thyroid hormone regulates the expression of the myelin protein genes coordinately, and have suggested a major role for thyroid hormone in the control of oligodendrocytes generation. Here we show that the expression of the MAL gene is down-regulated by hypothyroidism and up-regulated by hyperthyroidism in myelinated regions of the brain. In contrast, adult-onset hypothyroidism has no effect on the steady-state levels of MAL mRNA. Taken together, our results show that MAL expression during oligodendrocyte maturation is modulated by thyroid hormone, suggesting that this hormone could play an important role in the myelin biogenesis during neonatal development.

Hypothyroidism coordinately and transiently affects myelin protein gene expression in most rat brain regions during postnatal development.

To assess the role of thyroid hormone on myelin gene expression, we have studied the effect of hypothyroidism on the mRNA steady state levels for the major myelin protein genes: myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG) and 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in different rat brain regions, during the first postnatal month. We found that hypothyroidism reduces the levels of every myelin protein transcript, with striking differences between the different brain regions. Thus, in the more caudal regions, the effect of hypothyroidism was extremely modest, being only evident at the earlier stages of myelination. In contrast, in the striatum and the cerebral cortex the important decrease in the myelin protein transcripts is maintained beyond the first postnatal month. Therefore, thyroid hormone modulates in a synchronous fashion the expression of the myelin genes and the length of its effect depends on the brain region. On the other hand, hyperthyroidism leads to an increase of the major myelin protein transcripts above control values. Finally, lack of thyroid hormone does not change the expression of the oligodendrocyte progenitor-specific gene, the platelet derived growth factor receptor alpha.

Evidence for the existence of at least two timing mechanisms that contribute to oligodendrocyte generation in vitro.

We have been studying oligodendrocyte generation in vitro to obtain insights into how the timely generation of these cells might be regulated. Our studies suggest the existence of timing mechanisms quite different from those of existing models, wherein it is proposed that timely oligodendrocyte generation is associated with synchronous and symmetric differentiation controlled by cell-intrinsic biological clocks. Our results are most consistent with the hypothesis that the propensity of a clone of dividing oligodendrocyte type-2 astrocyte (O-2A) progenitors initially to generate at least one oligodendrocyte may be regulated by cell-intrinsic mechanisms, but that cell-extrinsic signals regulate the extent of further oligodendrocyte generation. In cultures of embryonic rat cortex grown in the presence of platelet-derived growth factor (PDGF), oligodendrocytes appeared in a timely manner in the absence of clonal differentiation. In contrast with previous suggestions, the presence or absence of thyroid hormone (T3) did not alter the probability of individual clones of O-2A progenitors generating at least one oligodendrocyte in vitro at a time equal to the rat's day of birth. Instead, T3 increased the proportion of oligodendrocytes generated within clones. For postnatally derived progenitor cells, the initial appearance of oligodendrocytes also was followed by further asymmetric generation of these cells, with the ratio of progenitors to oligodendrocytes within clones being regulated by environmental signals. T3 and ciliary neurotrophic factor increased oligodendrocyte generation, while neurotrophin-3 (NT-3) suppressed oligodendrocyte generation. Also in contrast to previous reports, NT-3 was not required for the promotion of extensive division of O-2A progenitor cells by PDGF.

Microtubule-associated protein 1B (MAP1B) is present in glial cells phosphorylated different than in neurones.

A panel of four anti-MAP1B antibodies have been used to study the presence and post-translational modification of MAP1B in primary cultures of glial cells. Two antibodies (150 and 125) recognize phosphorylated epitopes whereas the other two (531 and 842) recognize non-phosphorylated phosphorylatable epitopes on the MAP1B molecule. Immunofluorescence and Western blot analysis with antibodies 531 and 842 revealed the presence of small amounts of MAP1B-like immunoreactivity in type 1 astrocytes and a greater content in more differentiated glial cells found in long-term cultures. By immunofluorescence, these latter cells gave positive immunostaining with antibody 125, which recognizes a phosphorylated epitope phosphorylated by casein kinase II. Antibody 150, which reacts to a phosphorylated epitope on the MAP1B molecule, did not show any detectable immunoreactivity in glial cells cultures, either by immunofluorescence or Western blot. All four antibodies recognized hippocampal neurones in culture, with especially intense immunostaining in cell bodies and axons, and reacted strongly with protein present in hippocampal neurones extracts showing an electrophoretic mobility similar to that of brain MAP1B. In mixed optic nerve glial cell cultures, anti-galactocerebroside (GalC) positive cells gave also positive staining with antibodies 531 and 125. We propose that MAP1B is present in cultures of glial cells in moderate amounts and with a phosphorylation state different than in neurones. Thus, less differentiated glial cells, such as type 1 astrocytes, have a small amount of MAP1B, mainly in a non-phosphorylated form, which is spread diffusely in the cytoplasm and probably does not interact with microtubules. More differentiated glial cells, such as oligodendrocytes, show a greater content in MAP1B which, at least in part, is phosphorylated by a casein kinase II-like activity.

Thyroid hormone regulation of RC3, a brain-specific gene encoding a protein kinase-C substrate.

RC3 is a brain-specific mRNA expressed in discrete neuronal groups of the forebrain that encodes a 78-amino acid protein, also called neurogranin, a calmodulin-binding, protein kinase-C substrate. Expression of RC3 mRNA was studied in normal and hypothyroid animals during the first month of life. Hypothyroid rats were produced by administration of methyl-mercapto-imidazol to the pregnant dams and subsequent surgical thyroidectomy on postnatal day 5 of the neonates. As studied by slot-blotting of total cerebrum poly(A)+ RNA, RC3 mRNA accumulates in normal brain from the fifth to seventh postnatal day, reaching maximal levels around days 10-12. RC3 mRNA accumulation in hypothyroid animals was blunted, and the maximal levels attained were about 30-50% of normal values. The effect of hypothyroidism on steady state mRNA levels was also observed by Northern blotting of RNA from cerebral cortex and striatum. As studied by immunoblotting using a polyclonal antibody, hypothyroidism also led to clear decreases in the amount of the RC3 protein in extracts from cerebral cortex, striatum, and hippocampus. A single administration of 10 micrograms T4 to hypothyroid rats on postnatal day 12 led to a steady increase in striatal RC3 mRNA from levels that were about 40% of normal to about 70% of normal at 16 h and 115% of normal at 48 h. In contrast to the effect on RC3, hypothyroidism did not affect developmental expression of the mRNA encoding GAP-43, another brain protein kinase-C substrate of axonal localization. RC3 is, thus, one of the few known neuronal genes whose expression is influenced by thyroid hormone in the brain. Thyroid hormone is required for an appropriate level of expression, not for the developmentally programmed timing of expression of the RC3 gene.

Neonatal hypothyroidism affects the timely expression of myelin-associated glycoprotein in the rat brain.

Congenital hypothyroidism strongly affects myelination. To assess the role of thyroid hormone on myelin gene expression, we have studied the effect of hypothyroidism on the steady state levels of myelin-associated glycoprotein (MAG) and its mRNA in rat brain during the first postnatal month. As studied by immunoblot analysis of several brain regions, MAG increased from days 10-15 onwards, reaching constant levels by days 20-25. Hypothyroid samples showed a delay in the accumulation of MAG that was more severe in rostral regions, such as cortex and hippocampus. The effect of hypothyroidism on the accumulation of the protein correlated with mRNA levels. MAG mRNA started to accumulate in the cerebrum of normal animals by postnatal day 7, reaching maximal levels by day 20. Hypothyroid rats showed a delay of several days in the onset of mRNA expression, increasing thereafter at the same rate as in normal animals, and eventually reaching similar values. When individual brain regions were analyzed, we found strong regional differences in the effect of hypothyroidism. The cerebral cortex was most affected, with messenger levels lower than in normal animals at all ages. In more caudal regions differences between control and hypothyroid rats were evident only at the earlier stages of myelination, with spontaneous recovery at later ages. By run on analysis, we found no differences in transcriptional activities of the MAG gene in normal, hypothyroid, or T4-treated rats. Therefore, the effects of hypothyroidism on MAG mRNA and protein levels were most likely caused by decreased mRNA stability. We propose that thyroid hormone contributes to enhanced myelin gene expression by affecting the stability of newly transcribed mRNA in the early phases of myelination.

Adult rat brain is sensitive to thyroid hormone. Regulation of RC3/neurogranin mRNA.

The mammalian brain is considered to be poorly responsive to thyroid hormone after the so called "critical periods" of brain development, which occur in the rat before postnatal days 15-20. In a previous work (Muñoz, A., A. Rodriguez-Peña, A. Perez-Castillo, B. Ferreiro, J.G. Sutcliffe, and J. Bernal. 1991. Mol. Endocrinol. 5:273-280) we have identified one neuronal gene, RC3, whose expression is influenced by early neonatal hypothyroidism and thyroid hormone treatment. In the present work we show that adult-onset hypothyroidism leads to a reversible decrease of RC3 mRNA. Rats thyroidectomized on postnatal day 40 and killed three months later showed a decreased RC3 mRNA concentration in the cerebral cortex and striatum. The same effect was observed in animals made hypothyroid on postnatal day 32 and killed on postnatal day 52. RC3 expression was normal when hypothyroid animals were treated with T4 five days before being killed. In contrast, the mRNA encoding myelin proteolipid protein showed no changes in either experimental situation. RC3 mRNA levels were not affected by food restriction demonstrating that the effect of hypothyroidism was not related to the lack of weight gain. The control of RC3 mRNA is so far the only molecular event known to be regulated by thyroid hormone once the critical periods of brain development are over and could represent a molecular correlate for the age-independent, reversible alterations induced by hypothyroidism in the adult brain.

Influence of thyroid hormone on brain gene expression.

Brain development in mammals is dependent on thyroid hormone. In the rat, the T3 receptor is present as early as the 14th day of fetal life and increases during the periods of neuroblast proliferation and oligodendrocyte differentiation. The mRNAs encoding the receptor isoforms can also be demonstrated by in situ hybridization, with different distribution of the alpha or beta forms. Expression of several genes was studied in hypothyroid animals during the neonatal period. The lack of thyroid hormone affects the expression of most oligodendrocyte genes in a time- and region-dependent fashion. The major role of thyroid hormone on myelin genes is on their timing of expression. Among the neuronal genes studied, thyroid hormone influences the expression of RC3, a brain specific gene encoding a protein kinase C substrate. Thyroid hormone is not require for the timing of expression of this gene, but is needed for its full expression both during the neonatal period and in adult animals.