Adaptation of the bovine spongiform encephalopathy agent to primates and comparison with Creutzfeldt– Jakob disease: Implications for human health

There is substantial scientific evidence to support the notion that bovine spongiform encephalopathy (BSE) has contaminated human beings, causing variant Creutzfeldt–Jakob disease (vCJD). This disease has raised concerns about the possibility of an iatrogenic secondary transmission to humans, because the biological properties of the primate-adapted BSE agent are unknown. We show that (i) BSE can be transmitted from primate to primate by intravenous route in 25 months, and (ii) an iatrogenic transmission of vCJD to humans could be readily recognized pathologically, whether it occurs by the central or peripheral route. Strain typing in mice demonstrates that the BSE agent adapts to macaques in the same way as it does to humans and confirms that the BSE agent is responsible for vCJD not only in the United Kingdom but also in France. The agent responsible for French iatrogenic growth hormone-linked CJD taken as a control is very different from vCJD but is similar to that found in one case of sporadic CJD and one sheep scrapie isolate. These data will be key in identifying the origin of human cases of prion disease, including accidental vCJD transmission, and could provide bases for vCJD risk assessment.

Does a neuroimmune interaction contribute to the genesis of painful peripheral neuropathies?

Painful peripheral neuropathies are precipitated by nerve injury from disease or trauma. All such injuries will be accompanied by an inflammatory reaction, a neuritis, that will mobilize the immune system. The role of the inflammation itself is difficult to determine in the presence of structural damage to the nerve. A method has been devised to produce a focal neuritis in the rat sciatic nerve that involves no more than trivial structural damage to the nerve. This experimental focal neuritis produces neuropathic pain sensations (heat- and mechano-hyperalgesia, and cold- and mechano-allodynia) in the ipsilateral hind paw. The abnormal pain sensations begin in 1–2 days and last for 4–6 days, with a subsequent return to normal. These results suggest that there is a neuroimmune interaction that occurs at the outset of nerve injury (and perhaps episodically over time in slow developing conditions like diabetic neuropathy) that produces neuropathic pain. The short duration of the phenomena suggest that they may prime the system for more slowly developing mechanisms of abnormal pain (e.g., ectopic discharge in axotomized primary afferent neurons) that underlie the chronic phase of painful neuropathy.

Neurocytopathic effects of beta-amyloid-stimulated monocytes: a potential mechanism for central nervous system damage in Alzheimer disease.

Growing evidence indicates that cells of the mononuclear phagocyte lineage, which includes peripheral blood monocytes (PBM) and tissue macrophages, participate in a variety of neurodestructive events and may play a pivotal role in neurodegenerative conditions such as Alzheimer disease. The present study sought to determine whether exposure of PBM to beta-amyloid peptide (A beta), the major protein of the amyloid fibrils that accumulate in the brain in Alzheimer disease, could induce cytopathic activity in these cells upon their subsequent incubation with neural tissue. PBM were incubated with A beta for 3 days, centrifuged and washed to remove traces of cell-free A beta, and then applied to organotypic cultures of rat brain for varying periods of time. By using a cell-viability assay to quantitate neurocytopathic effect, an increase in the ratio of dead to live cells was detected in cultures containing A beta-stimulated PBM versus control PBM (stimulated with either bovine serum albumin or reverse A beta peptide) as early as 3 days after coculture. The ratio of dead to live cells increased further by 10 days of coculture. By 30 days of coculture, the dead to live cell ratio remained elevated, and the intensity of neurocytopathic effect was such that large areas of brain mass dissociated from the cultures. These results indicate that stimulation of PBM with A beta significantly heightens their neurocytopathic activity and highlight the possibility that inflammatory reactions in the brain play a role in the neurodegeneration that accompanies Alzheimer disease.

Pathogenic autoantibodies are routinely generated during the response to foreign antigen: a paradigm for autoimmune disease.

The immune system's ability to distinguish self and nonself is essential for both host defense against foreign agents and protection of self-antigens from autoimmune destruction. Such discrimination is complicated by extensive structural homology shared between foreign and self antigens. One hypothesis to explain the development of an autoimmune response is that some B cells activated by foreign antigen acquire, through somatic mutation, specificity for both the eliciting foreign antigen and self antigen. If such clones arise frequently, there must be a mechanism for their elimination. We have analyzed the extent of autoreactivity arising in a nonautoimmune host during the response to a foreign antigen. To overcome the process of apoptosis in primary B cells that might routinely eliminate autoreactive clones, we generated B-cell hybridomas from spleen cells of immunized mice by using a fusion partner constitutively expressing bcl-2. Multiple lines were obtained that recognize simultaneously the hapten phosphorylcholine and the self antigen double-stranded DNA. This dual specificity was not present early but was detected by day 10 after immunization. Some of these cross-reactive antibodies deposit in kidneys in a pattern similar to what is seen in autoimmune disease. These results demonstrate that autoantibodies arise at a high frequency as part of a response to foreign antigen. It has previously been shown that autoreactivity is regulated by central deletion; these data demonstrate a need for negative selection in peripheral lymphoid organs also, to regulate autoantibodies acquiring their self-specificity by somatic mutation.

Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor alpha.

Cytokines are now recognized to play important roles in the physiology of the central nervous system (CNS) during health and disease. Tumor necrosis factor alpha (TNF-alpha) has been implicated in the pathogenesis of several human CNS disorders including multiple sclerosis, AIDS dementia, and cerebral malaria. We have generated transgenic mice that constitutively express a murine TNF-alpha transgene, under the control of its own promoter, specifically in their CNS and that spontaneously develop a chronic inflammatory demyelinating disease with 100% penetrance from around 3-8 weeks of age. High-level expression of the transgene was seen in neurons distributed throughout the brain. Disease is manifested by ataxia, seizures, and paresis and leads to early death. Histopathological analysis revealed infiltration of the meninges and CNS parenchyma by CD4+ and CD8+ T lymphocytes, widespread reactive astrocytosis and microgliosis, and focal demyelination. The direct action of TNF-alpha in the pathogenesis of this disease was confirmed by peripheral administration of a neutralizing anti-murine TNF-alpha antibody. This treatment completely prevented the development of neurological symptoms, T-cell infiltration into the CNS parenchyma, astrocytosis, and demyelination, and greatly reduced the severity of reactive microgliosis. These results demonstrate that overexpression of TNF-alpha in the CNS can cause abnormalities in nervous system structure and function. The disease induced in TNF-alpha transgenic mice shows clinical and histopathological features characteristic of inflammatory demyelinating CNS disorders in humans, and these mice represent a relevant in vivo model for their further study.

Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.

Both stem cells and mast cells express c-kit and proliferate after exposure to c-kit ligand. Mutations in c-kit may enhance or interfere with the ability of c-kit receptor to initiate the intracellular pathways resulting in cell proliferation. These observations suggested to us that mastocytosis might in some patients result from mutations in c-kit. cDNA synthesized from peripheral blood mononuclear cells of patients with indolent mastocytosis, mastocytosis with an associated hematologic disorder, aggressive mastocytosis, solitary mastocytoma, and chronic myelomonocytic leukemia unassociated with mastocytosis was thus screened for a mutation of c-kit. This analysis revealed that four of four mastocytosis patients with an associated hematologic disorder with predominantly myelodysplastic features had an A-->T substitution at nt 2468 of c-kit mRNA that causes an Asp-816-->Val substitution. One of one patient examined who had mastocytosis with an associated hematologic disorder had the corresponding mutation in genomic DNA. Identical or similar amino acid substitutions in mast cell lines result in ligand-independent autophosphorylation of the c-kit receptor. This mutation was not identified in the patients within the other disease categories or in 67 of 67 controls. The identification of the point mutation Asp816Val in c-kit in patients with mastocytosis with an associated hematologic disorder provides insight not only into the pathogenesis of this form of mastocytosis but also into how hematopoiesis may become dysregulated and may serve to provide a means of confirming the diagnosis, assessing prognosis, and developing intervention strategies.

Vector-mediated delivery of 125I-labeled beta-amyloid peptide A beta 1-40 through the blood-brain barrier and binding to Alzheimer disease amyloid of the A beta 1-40/vector complex.

The brain amyloid of Alzheimer disease (AD) may potentially be imaged in patients with AD by using neuroimaging technology and a radiolabeled form of the 40-residue beta-amyloid peptide A beta 1-40 that is enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. Transport of 125I-labeled A beta 1-40 (125I-A beta 1-40) through the BBB was found to be negligible by experiments with both an intravenous injection technique and an internal carotid artery perfusion method in anesthetized rats. In addition, 125I-A beta 1-40 was rapidly metabolized after either intravenous injection or internal carotid artery perfusion. BBB transport was increased and peripheral metabolism was decreased by conjugation of monobiotinylated 125I-A beta 1-40 to a vector-mediated drug delivery system, which consisted of a conjugate of streptavidin (SA) and the OX26 monoclonal antibody to the rat transferrin receptor, which undergoes receptor-mediated transcytosis through the BBB. The brain uptake, expressed as percent of injected dose delivered per gram of brain, of the 125I,bio-A beta 1-40/SA-OX26 conjugate was 0.15 +/- 0.01, a level that is 2-fold greater than the brain uptake of morphine. The binding of the 125I,bio-A beta 1-40/SA-OX26 conjugate to the amyloid of AD brain was demonstrated by both film and emulsion autoradiography performed on frozen sections of AD brain. Binding of the 125I,bio-A beta 1-40/SA-OX26 conjugate to the amyloid of AD brain was completely inhibited by high concentrations of unlabeled A beta 1-40. In conclusion, these studies show that BBB transport and access to amyloid within brain may be achieved by conjugation of A beta 1-40 to a vector-mediated BBB drug delivery system.

Persistent high frequency of human immunodeficiency virus-specific cytotoxic T cells in peripheral blood of infected donors.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTLs) are thought to play a major role in the immune response to HIV infection. The HIV-specific CTL response is much stronger than previously documented in an infectious disease, yet estimates of CTL frequency derived from limiting-dilution analysis (LDA) are relatively low and comparable to other viral infections. Here we show that individual CTL clones specific for peptides from HIV gag and pol gene products are present at high levels in the peripheral blood of three infected patients and that individual CTL clones may represent between 0.2% and 1% of T cells. Previous LDA in one donor had shown a frequency of CTL precursors of 1/8000, suggesting that LDA may underestimate CTL effector frequency. In some donors individual CTL clones persisted in vivo for at least 5 years. In contrast, in one patient there was a switch in CTL usage suggesting that different populations of CTLs can be recruited during infection. These data imply strong stimulation of CTLs, potentially leading some clones to exhaustion.

Análise transcriptômica de miRNAs em pacientes sob dupla terapia de antiagregação

A terapia antiagregante é comumente indicada na prevenção e tratamento de doenças cardiovasculares. A dupla antiagregação com clopidrogrel e ácido acetilsalicílico (AAS) tem sido frequentemente adotada em pacientes com Doença Arterial Coronariana (DAC), mas apresenta ineficácia em uma parcela significativa da população com genótipo de respondedores. Essa falha terapêutica nos leva a questionar se outros mecanismos moleculares podem estar influenciando na resposta a esses fármacos. Recentes estudos sugerem que pequenas sequências de RNA não codificantes denominadas microRNAs (miRNAs) podem estar fortemente relacionadas com resposta ao tratamento fármaco-terapêutico, controlando as proteínas envolvidas na farmacocinética e farmacodinâmica. Entretanto, os principais miRNAs que atuam na dinâmica da resposta medicamentosa ainda não foram bem definidos. O objetivo deste estudo foi avaliar o perfil de miRNAs no sangue total periférico, procurando melhor esclarecer os mecanismos envolvidos na resposta aos antiagregantes plaquetários AAS e clopidogrel. Para isso, selecionou-se pacientes com DAC, os quais apresentavam diferentes respostas à dupla terapia de antiagregação determinadas pelo teste de agregação plaquetária. Baseados nos fenótipos, os perfis de expressão de miRNAs foram comparados entre os valores da taxa de agregação categorizados em tercis (T) de resposta. O grupo T1 foi constituído de pacientes respondedores, o T2 de respondedores intermediários e o T3 de não respondedores. Os perfis de miRNAs foram obtidos após sequenciamento de última geração e os dados obtidos foram analisados pelo pacote Deseq2. Os resultados mostraram 18 miRNAs diferentemente expressos entre os dois tercis extremos. Dentre esses miRNAs, 10 deles apresentaram importantes alvos relacionados com vias de ativação e agregação plaquetária quando analisados pelo software Ingenuity®. Dos 10 miRNAs, 4 deles, os quais apresentaram-se menos expressos no sequenciamento, demonstraram os mesmos perfis de expressão quando analisados pela reação em cadeia pela polimerase quantitativa (qPCR): hsa-miR-423-3p, hsa-miR-744-5p, hsa-miR- 30a-5p e hsa-let-7g-5p. A partir das análises de predição de alvos, pôde-se observar que os quatro miRNAs, quando menos expressos simultaneamente, predizem ativação da agregação plaquetária. Além disso, os miRNAs hsa-miR- 423-5p, hsa-miR-744-5p e hsa-let-7g-5p mostraram correlação com o perfil lipídico dos pacientes que, por sua vez, apresentou influência nos valores de agregação compreendidos no T3 de resposta a ambos os medicamentos. Sendo assim, conclui-se que maiores taxas de agregação plaquetária podem estar indiretamente relacionadas com os padrões de expressão de hsa-miR- 423-3p, hsa-miR-744-5p e hsa-let-7g-5p. Sugere-se que a avaliação do perfil de expressão destes 3 miRNAs no sangue periférico de pacientes com DAC possa predizer resposta terapêutica inadequada ao AAS e ao clopidogrel

Immune biomarkers in acute graft-versus-host disease

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Hipertensão arterial pulmonar : patologia e abordagens terapêuticas

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Hipertensão arterial mascarada no idoso

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Hemochromatosis and alcoholic liver disease

The close association of excessive alcohol consumption and clinical expression of hemochromatosis has been of widespread interest for many years. In most populations of northern European extraction, more than 90% of patients with overt hemochromatosis are homozygous for the C282Y mutation in the HFE gene. Nevertheless, the strong association of heavy alcohol intake with the clinical expression of hemochromatosis remains. We (individually or in association with colleagues from our laboratories) have performed three relevant studies in which this association was explored. In the first, performed in 1975 before the cloning of the HFE gene, the frequency of clinical symptoms and signs was compared in patients with classical hemochromatosis who consumed 100 g or more of alcohol per day versus in nondrinkers or moderate drinkers who consumed less than 100 g of alcohol per day. The results showed no difference between the two groups except for features of complications of alcoholism in the first group, especially jaundice, peripheral neuritis, and hepatic failure. Twenty-five percent of those with heavy alcohol consumption showed histologic features of alcoholic liver disease (including cirrhosis) together with heavy iron overload. It was concluded that these patients had the genetic disease complicated by alcoholic liver disease. In the second study (2002), 206 subjects with classical HFE-associated hemochromatosis in whom liver biopsy had been performed were evaluated to quantify the contribution of excess alcohol consumption to the development of cirrhosis in hemochromatosis. Cirrhosis was approximately nine times more likely to develop in subjects with hemochromatosis who consumed more than 60 g of alcohol per day than in those who drank less than this amount. In the third study (2002), 371 C282Y-homozygous relatives of patients with HFE-associated hemochromatosis were assessed. Eleven subjects had cirrhosis on liver biopsy and four of these drank 60 g or more of alcohol per day. The reason why heavy alcohol consumption accentuates the clinical expression of hemochromatosis is unclear. Increased dietary iron or increased iron absorption is unlikely. The most likely explanation would seem to be the added co-factor effect of iron and alcohol, both of which cause oxidative stress, hepatic stellate cell activation, and hepatic fibrogenesis. In addition, the cumulative effects of other forms of liver injury may result when iron and alcohol are present concurrently. Clearly, the addition of dietary iron in subjects homozygous for hemochromatosis would be unwise. (C) 2003 Elsevier Inc. All rights reserved.

Chronic graft-versus-host disease after granulocyte colony-stimulating factor-mobilized allogeneic stem cell transplantation: The role of donor T-cell dose and differentiation

The use of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood as a source of stem cells has resulted in a high incidence of severe chronic graft-versus-host disease (cGVHD), which compromises the outcome of clinical allogeneic stem cell transplantation. We have studied the effect of G-CSF on both immune complex and fibrotic cGVHD directed to major (DBA/2 --> B6D2F1) or minor (B10.D2 --> BALB/c) histocompatibility antigens. In both models, donor pretreatment with G-CSF reduced cGVHD mortality in association with type 2 differentiation. However, after escalation of the donor T-cell dose, scleroderma occurred in 90% of the recipients of grafts from G-CSF-treated donors. In contrast, only 11% of the recipients of control grafts developed scleroderma, and the severity of hepatic cGVHD was also reduced. Mixing studies confirmed that in the presence of high donor T-cell doses, the severity of scleroderma was determined by the non-T-cell fraction of grafts from G-CSF-treated donors. These data confirm that the induction of cGVHD after donor treatment with G-CSF is dependent on the transfer of large numbers of donor T cells in conjunction with a putatively expanded myeloid lineage, providing a further rationale for the limitation of cell dose in allogeneic stem cell transplantation. (C) 2004 American Society for Blood and Marrow Transplantation.