Growth differentiation factor 11 (GDF11) - a promising anti-ageing factor - is highly concentrated in platelets.

Recent research suggests that growth differentiation factor 11 (GDF11) could reverse age-related diseases and that its blood concentration decreases with age. This poses plasma from young donors as a therapeutic GDF11 source to treat age-related diseases. In addition, the tissue source of circulating GDF11 remains unknown. We analysed GDF11 levels in paired samples of serum, plasma and platelet lysate (PL) from 23 volunteers. Plasma and PL were collected by plateletpheresis. Here, we show that GDF11 is highly concentrated in platelets and that the circulating levels reported in previous studies could be biased as a result of serum sample manipulation.

Distribution of immunoreactivity for the adrenomedullin binding protein, complement factor H, in the rat brain.

Adrenomedullin is a multifunctional amidated peptide that has been found in most nuclei of the CNS, where it plays a neuromodulatory role. An adrenomedullin binding protein has recently been found in plasma and characterized as complement factor H. This regulator of the complement system inhibits the progression of the complement cascade and modulates the function of adrenomedullin. Our study shows the ample distribution of factor H immunoreactivity in neurons of telencephalon, diencephalon, mesencephalon, pons, medulla, and cerebellum in the rat CNS, using immunohistochemical techniques for both light and electron microscopy. Factor H immunoreactivity was found in the cytoplasm, but nuclear staining was also a common finding. Some blood vessels and glial cells were also immunoreactive for factor H. Colocalization studies by double immunofluorescence followed by confocal microscopy revealed frequent coexistence of factor H and adrenomedullin immunoreactivities, thus providing morphological evidence for the potential interaction of these molecules in the CNS. The presence of factor H immunoreactivity in glial cells was confirmed by colocalization with glial fibrillary acidic protein. In summary, factor H is highly expressed in the CNS where it could play important roles in regulating adrenomedullin actions and contributing to an intracerebral complement system.

Physiology and pathophysiology of nitric oxide in the nervous system, with special mention of the islands of Calleja and the circunventricular organs.

Nitric oxide (NO) has been recognized as a key regulatory factor in many physiological processes, including central nervous system function, development, and phatophysiology. NO is produced by a class of enzymes known as NO synthases (NOS) and in normal adult animals only the neuronal isoform (nNOS) is detectable. During cortical development, nNOS was found at E14 in neuroblasts of the marginal zone and its expression raised to a zenith by P5, decreasing afterwards until reaching a steady level by P10. At that time, nNOS was found mainly in pyramidal neurons. Interestingly, the inducible isoform of the enzyme (iNOS) was also active from P3 to P7, but it disappeared almost completely by P20. The neurodegeneration observed during normal aging and following hypoxic accidents seems to be the result of cumulative free radical damage, and excessive production of NO may be at the basis of the cascade. After ischemic events we observed an elevation in the number of neurons expressing nNOS coincident with an elevation in Ca2+-dependent NOS activity for up to 120 min. After this period, nNOS activity began to decrease but it was substituted by a rapid increase in Ca2+-independent activity coincident with the histological appearance of previously undetectable iNOS-immunoreactive neurons. These increases in NO production were accompanied by specific patterns of protein nitration, a process that seems to result in loss of protein function. In particular, we observed a correlation between exposure to ischemia-reperfusion and nitration of cytochrome c. This process was coincident with the exit of the cytochrome from the mitochondria to the surrounding cytoplasm, an early event in neuronal apoptosis. Interestingly, most of the morphological and molecular changes associated with ischemic damage were prevented by treatment with inhibitors of NO production, indicating a clear path in the search for efficacious drugs in the battle against cerebrovascular accidents.

Coexistence of translocated cytochrome c and nitrated protein in neurons of the rat cerebral cortex after oxygen and glucose deprivation.

Changes in the distribution of immunoreactive cytochrome c and protein nitration were studied in the rat cerebral cortex after oxygen and glucose deprivation by bright field, confocal and electron microscopy. In control cerebral cortex, nitrotyrosine immunoreactivity indicating protein nitration was found mostly in the neuronal nuclear region, with only a small amount distributed in the cytosol, whereas cytochrome c immunoreactivity was found at the inner membrane and in the intermembrane space of the mitochondria. During the recovery phase after oxygen and glucose deprivation, cytochrome c immunoreactivity was released from the intermembrane space of swollen mitochondria into the surrounding cytosol. The cytosol now also displayed nitrotyrosine immunoreactivity, which had diminished in the nuclear region. Both immunoreactivities were dispersed throughout the soma and processes of the cortical neurons. These changes were largely prevented by the administration of cyclosporin A, which inhibits both the mitochondrial permeability transition and the neuronal isoform of nitric oxide synthase while blocking the induction of the inducible isoform. Ischemia/reperfusion injury increases the production of nitric oxide, reactive oxygen species and intracellular factors that damage the mitochondria and liberate apoptotic factors. We suggest that translocation of cytochrome c from the mitochondria to the cytosol, which has been shown to precede the mitochondrial permeability transition, could result from peroxynitrite-mediated nitration. This phenomenon is attenuated by cyclosporin A administration, suggesting a neuroprotective role for this agent.

Adrenomedullin expression is up-regulated by ischemia-reperfusion in the cerebral cortex of the adult rat.

Changes in the pattern of adrenomedullin expression in the rat cerebral cortex after ischemia-reperfusion were studied by light and electron microscopic immunohistochemistry using a specific antibody against human adrenomedullin (22-52). Animals were subjected to 30 min of oxygen and glucose deprivation in a perfusion model simulating global cerebral ischemia, and the cerebral cortex was studied after 0, 2, 4, 6, 8, 10 or 12 h of reperfusion. Adrenomedullin immunoreactivity was elevated in certain neuronal structures after 6-12 h of reperfusion as compared with controls. Under these conditions, numerous large pyramidal neurons and some small neurons were intensely stained in all cortical layers. The number of immunoreactive pre- and post-synaptic structures increased with the reperfusion time. Neurons immunoreactive for adrenomedullin presented a normal morphology whereas non-immunoreactive neurons were clearly damaged, suggesting a potential cell-specific protective role for adrenomedullin. The number and intensity of immunoreactive endothelial cells were also progressively elevated as the reperfusion time increased. In addition, the perivascular processes of glial cells and/or pericytes followed a similar pattern, suggesting that adrenomedullin may act as a vasodilator in the cerebrocortical circulation. In summary, adrenomedullin expression is elevated after the ischemic insult and seems to be part of CNS response mechanism to hypoxic injury.

Adrenomedullin in the central nervous system.

Adrenomedullin (AM) is a novel vasodilator peptide first purified from human pheochromocytoma by tracing its capacity to stimulate cAMP production in platelets. AM immunoreactivity is widely distributed in the central nervous system (CNS) and in the rat has been demonstrated by immunohistochemical techniques to be present in many neurons throughout the brain and spinal cord, as well as in some vascular endothelial cells and perivascular glial cells. Electron microscopy shows that the immunoreactivity is located mainly in the neuronal cytoplasm, but also occurs in the cell nucleus in some cells of the caudate putamen and olfactory tubercle. Biochemical analyses suggest that higher molecular forms, presumably precursor forms, may predominate over fully processed AM in some brain areas. The expression of AM immunoreactivity is increased in cortical neurons, endothelial cells, and perivascular processes after a simulation of ischemia by oxygen and glucose deprivation. Immunohistochemical, electrophysiological, and pharmacological studies suggest that AM in the CNS can act as a neurotransmitter, neuromodulator, or neurohormone, or as a cytoprotective factor in ischemic/hypoxic conditions, in addition to its vasodilator role.

Neuronal and inducible nitric oxide synthase expression and protein nitration in rat cerebellum after oxygen and glucose deprivation.

A perfusion model of global cerebral ischemia was used for the immunohistochemical study of changes in the glutamate-nitric oxide (NO) system in the rat cerebellum and cerebellar nuclei during a 0-14 h reperfusion period after 30 min of oxygen and glucose deprivation, with and without administration of 1.5 mM N(omega)-nitro-L-arginine methyl ester (L-NAME). While immunostaining for N-methyl-D-aspartate receptor subunit 1 (NMDAR1) showed no marked changes during the reperfusion period, neuronal NO synthase (nNOS) immunostaining increased in stellate and basket cells, granule cells and neurons of the cerebellar nuclei. However, global cerebellar nNOS concentrations determined by Western blotting remained largely unchanged in comparison with actin expression. Inducible NOS (iNOS) immunostaining appeared in Purkinje cells and neurons of the cerebellar nuclei after 2-4 h of reperfusion and intensified during the 6-14 h period. This was reflected by an increase in global cerebellar iNOS expression determined by Western blotting. Immunostaining for protein nitrotyrosine was seen in Purkinje cells, stellate and basket cells, neurons of the cerebellar nuclei and glial cells in controls, and showed a progressive translocation in Purkinje cells and neurons of the cerebellar nuclei from an initial perinuclear or nuclear location towards the periphery. At the end of the reperfusion period the Purkinje cell apical dendrites were notably retracted and tortuous. Prior and concurrent L-NAME administration eliminated nitrotyrosine immunostaining in controls and blocked or reduced most of the postischemic changes observed. The results suggest that while nNOS expression may be modified in certain cells, iNOS is induced after a 2-4 h period, and that changes in protein nitration may be associated with changes in cell morphology.

Hypermethylation of P16ink4a and P15ink4b genes as a marker of disease in the follow-up of non-Hodgkin's lymphomas.

The hypermethylation of p16ink4a and p15ink4b genes have been described as an inactivating mechanism alternative to deletions and mutations that accounts for a relatively high proportion of cancers, including non-Hodgkin's lymphomas (NHLs). To investigate whether detection of abnormal methylation could have clinical applications in the management and follow-up of lymphomas, we have analysed the behaviour and evolution of p16ink4a and p15ink4b methylation in 13 NHL cases undergoing chemotherapy. All cases were also analysed for the presence of monoclonal rearrangements of immunoglobulin or T-cell receptor genes. Six patients showed methylation in at least one of these genes at diagnosis, whereas in two other cases methylation appeared during the treatment. The other five cases were always unmethylated. Methylation was detected when any histological or molecular evidence of disease was present, suggesting a good correlation between methylation and disease. In some cases, we were able to detect methylation in patients at complete remission and without evidence of monoclonal cell population, indicating a high sensitivity of the PCR to detect methylation. These results suggest that p16ink4a and p15ink4b methylation could be good markers of disease and could be helpful in identifying lymphoma patients at risk of relapse.

Identification by comparative genomic hybridization of genetic changes involved in tumoral progression of a T-cell non-Hodgkin lymphoma.

Comparative genomic hybridization (CGH) was used to detect chromosomal imbalances in tumor DNA from two relapsed samples obtained in stages II and IV of a T-cell non-Hodgkin lymphoma in order to identify genetic mechanisms involved in tumor progression of this neoplasm. With conventional cytogenetic techniques (CCT), a complex hyperdiploid karyotype was obtained in stage IV. Using CGH analysis, a normal profile was observed in stage II, whereas gains of 6p11.2, 7q11.2, 7q21-->q32, 7q34, 10p13, Xp11.4, and loss of 4q33-->qter chromosomal regions were detected in stage IV.

[Application of modified comparative genomic hybridization in the genetic analysis of 5 lymphoid neoplasms]. / Aplicación de la hibridación genómica comparativa modificada en 5 neoplasias linfoides.

To analyze the utility of performing modified comparative genomic hybridization (mCGH) as a complementary technique to conventional cytogenetic techniques in the genetic analysis of lymphoid neoplasms. Modified comparative genomic hybridization and subsequent FISH techniques were performed in 5 lymphoid neoplasms cases diagnosed in Fundación Jiménez Díaz. The latter was done in order to confirm the results obtained with mCGH. Gains of chromosomal regions not detected with conventional cytogenetic techniques were detected by mCGH. A good correlation in the results obtained between conventional cytogenetic and mCGH techniques was observed. Nevertheless, mCGH enables the detection and subsequent identification of gains of genetic sequences undetectable with cytogenetic techniques with possible diagnostic and prognostic value.

Neuronal and inducible nitric oxide synthase and nitrotyrosine immunoreactivities in the cerebral cortex of the aging rat.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) and nitrotyrosine immunoreactivities were localized and semiquantitatively assessed in the cerebral cortex of aged rats by means of light microscopic immunocytochemistry and Western blotting, using a new series of specific polyclonal antibodies. In the aged rats the strongly nNOS-immunoreactive multipolar neurons found in layers II-VI of the cortex of young rats were seen in similar numbers, but showed varicose, vacuolated, and fragmented processes, with an irregular outline and loss of spines. A large number of more weakly nNOS-positive neurons, characterized by a ring of immunoreactive cytoplasm, and not seen in young rats, were observed in layers II-VI of aged rat cortex. While no iNOS-immunopositive neurons were found in the cortex of young rats, a large number of such neurons appeared throughout the aged rat cortex. Nitrotyrosine-positive cells outnumbered total NOS-positive neurons in the cortex of young rats, but this relation was inverted in the aged rats, although these showed a slight increase in the number and staining intensity of nitrotyrosine-positive cells. Western blots of brain extracts showed a several-fold increase in both nNOS- and iNOS-immunoreactive bands in the aged rat, but a less marked increase in nitrotyrosine-containing proteins. The results suggest that while nNOS and iNOS expression is substantially increased in the aged rat cortex, this is not necessarily accompanied by a proportionate increase in nitric oxide synthesis. The mechanisms underlying the increased expression of nNOS and iNOS, and the functional implications of this increase, require elucidation.

Increased C-MYC oncogene copy number detected with combined modified comparative genomic hybridization and FISH analysis in a Richter syndrome case with complex karyotype.

Modified comparative genomic hybridization (mCGH) was performed in a Richter syndrome case with a complex karyotype to identify and map gains of DNA sequences with possible importance in the pathogenesis and progression of the tumor. The mCGH analysis revealed a more intense signal on part of the long arm of one pair of chromosomes belonging to group C. The G-banding study showed that the increased DNA-sequence copy number originated from the 8q22-->qter chromosomal region. This increase was confirmed by performing a fluorescence in situ hybridization analysis on tumor metaphases by first using a chromosome 8-specific library and subsequently a C-MYC probe, which revealed positive staining on six different regions located on six different chromosomes, each one bearing a single copy of the C-MYC oncogene. These results show the existence of C-MYC oncogene copy-number increases and confirm the usefulness of mCGH in the genetic analysis of malignancies.

[Hospital use of fresh frozen plasma]. / Uso hospitalario del plasma fresco congelado.

PURPOSE: The aim of this work is double. On the one hand, to assess if the measures to strictly control the clinical indications of fresh-frozen plasma (FFP) transfusion may lead to a decrease of its use, and on the other to assess the importance of FFP with regard to other blood components, along with disclosing the number and characteristics of the more patients and those who receive only FFP. MATERIAL AND METHODS: Starting from data of the blood bank and the hospital records, an analysis of the use of FFP in the General Hospital was carried out, and it was correlated with the use of other blood components, mostly red cells (RC), and the hospital indices expressed as DRG. An analysis was also performed of the use of FFP in 1996 with regard to the number of transfused patients, mean consumed units in general and according to patient-groups, association with RC use and identification of high-use patients (defined as requiring over 3 FFP units). RESULTS: A decrease in the use of FFP between 1992 and 1996 was appreciated, from 1,385 to 760 units. This decrease, when correlated with the use of RC, was from 17.8 to 9.2 FFP units/ 100 RC units during this period. The FFP units/100 RC units varied from 6 to 2 in three years; this index has been stable since then. With regard to the use in 1996, 162 patients received FFP, which represents 4% of all the transfusions in the hospital. Of these, 15 patients received only plasma (9% of the patients receiving FFP and 0.3% of all transfusions). Other blood components, mainly RC, were associated to FFP in 96% of the cases. The patients consuming more FFP units were those of heart surgery and intensive care units, with significant differences with respect to others. CONCLUSIONS: This study shows a steady decrease in the use of FFP, which is stable in the last years. The patients receiving only FFP represented a low number with respect to all the patients transfused. The follow-up of these patients might provide valuable data about the benefit of adding additional security processes to standard FFP.

[Polycythemia vera as a multiphasic clonal panmyelopathy: diagnostic profile, chronic pathological progression and effect of therapy on the survival of 74 cases]. / La policitemia vera como panmielopatía clonal multifásica: perfil diagnóstico, evolución patocrónica y efecto de la terapéutica sobre la supervivencia de 74 casos.

PURPOSE: 1. To recognise the clinico-biological profile of a group of patients diagnosed of polycythaemia vera (PV) in our centre in the last 30 years. 2. To identify the evolutive patterns of haematological transformation. 3. To evaluate the effect of therapy on the survival. PATIENTS AND METHODS: The clinical records of 74 patients (median age 62 years, male/female = 0.94, followed-up for 6-357 months, median 64 months) were reviewed. Clinico-biological data at diagnosis, therapy, complications and evolution of the haematological picture were evaluated in each case. The actuarial survival in the series was compared to that of the normal population. RESULTS: The clinico-analytical data and diagnostic features were identical to other series reported. Mild increases of bone marrow reticulin was present in two thirds of the cases, overt myelofibrosis being found in only 10% of the patients. Abnormal karyotype was seen in 9% of the patients (11q-, -Y). Phlebotomy was the only treatment in eight cases, without increased incidence of thrombotic phenomena. The remainders received myelosuppressive therapy (32P, busulphan, pipobroman, hydroxyurea, etc.), thrombotic complications appearing in 8 cases and haemorrhagic complications in 4 others. One of these latter patients developed oesophageal carcinoma. The haematological picture evolved into toxic aplastic anaemia in 2 cases; myelofibrosis with myeloid metaplasia (MF/MM) in 8; myelodysplastic sindromes (MDS) in 5, three of them RAEB; and acute myelogenous leukaemia in 3 cases, two of them as the final stage of previous MF/MM and MDS/ RAEB. The actuarial survival was 71% at ten years and 46% at fifteen years, and the median survival as a whole was 13.5 years. CONCLUSIONS: 1: The treatment, mostly myelosuppressive, given to these patients attained a survival similar to that of the general population. 2: Of the cases with known evolution, 15.6% developed MF/MM, its incidence being higher in patients treated only with phlebotomy (37%). 3: The incidence of malignant evolution, i.e., to RAEB/AML, amongst those patients followed-up was 10.6%.