Publications by authors named "Alfredo Prieto"

34 Publications

Ancient genomes in South Patagonia reveal population movements associated with technological shifts and geography.

Nat Commun 2020 08 3;11(1):3868. Epub 2020 Aug 3.

Department of Genetics, Harvard Medical School, Boston, MA, 02115, USA.

Archaeological research documents major technological shifts among people who have lived in the southern tip of South America (South Patagonia) during the last thirteen millennia, including the development of marine-based economies and changes in tools and raw materials. It has been proposed that movements of people spreading culture and technology propelled some of these shifts, but these hypotheses have not been tested with ancient DNA. Here we report genome-wide data from 20 ancient individuals, and co-analyze it with previously reported data. We reveal that immigration does not explain the appearance of marine adaptations in South Patagonia. We describe partial genetic continuity since ~6600 BP and two later gene flows correlated with technological changes: one between 4700-2000 BP that affected primarily marine-based groups, and a later one impacting all <2000 BP groups. From ~2200-1200 BP, mixture among neighbors resulted in a cline correlated to geographic ordering along the coast.
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http://dx.doi.org/10.1038/s41467-020-17656-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7400565PMC
August 2020

Ancient mitochondrial DNA reveals convergent evolution of giant short-faced bears (Tremarctinae) in North and South America.

Biol Lett 2016 Apr;12(4)

Australian Centre for Ancient DNA, School of Biological Sciences, University of Adelaide, Adelaide, South Australia 5005, Australia.

The Tremarctinae are a subfamily of bears endemic to the New World, including two of the largest terrestrial mammalian carnivores that have ever lived: the giant, short-faced bears Arctodus simus from North America and Arctotherium angustidens from South America (greater than or equal to 1000 kg). Arctotherium angustidens became extinct during the Early Pleistocene, whereas Arctodus simus went extinct at the very end of the Pleistocene. The only living tremarctine is the spectacled bear (Tremarctos ornatus), a largely herbivorous bear that is today only found in South America. The relationships among the spectacled bears (Tremarctos), South American short-faced bears (Arctotherium) and North American short-faced bears (Arctodus) remain uncertain. In this study, we sequenced a mitochondrial genome from an Arctotherium femur preserved in a Chilean cave. Our molecular phylogenetic analyses revealed that the South American short-faced bears were more closely related to the extant South American spectacled bear than to the North American short-faced bears. This result suggests striking convergent evolution of giant forms in the two groups of short-faced bears (Arctodus and Arctotherium), potentially as an adaptation to dominate competition for megafaunal carcasses.
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http://dx.doi.org/10.1098/rsbl.2016.0062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881349PMC
April 2016

Mitochondrial genomes reveal the extinct Hippidion as an outgroup to all living equids.

Biol Lett 2015 Mar;11(3)

Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Øster Voldgade 5-7, 1350 Copenhagen K, Denmark Laboratoire AMIS, Université Paul Sabatier 3, Université de Toulouse, UMR CNRS 5288, 37 Allées Jules Guesde, 31073 Toulouse cedex 3, France

Hippidions were equids with very distinctive anatomical features. They lived in South America 2.5 million years ago (Ma) until their extinction approximately 10 000 years ago. The evolutionary origin of the three known Hippidion morphospecies is still disputed. Based on palaeontological data, Hippidion could have diverged from the lineage leading to modern equids before 10 Ma. In contrast, a much later divergence date, with Hippidion nesting within modern equids, was indicated by partial ancient mitochondrial DNA sequences. Here, we characterized eight Hippidion complete mitochondrial genomes at 3.4-386.3-fold coverage using target-enrichment capture and next-generation sequencing. Our dataset reveals that the two morphospecies sequenced (H. saldiasi and H. principale) formed a monophyletic clade, basal to extant and extinct Equus lineages. This contrasts with previous genetic analyses and supports Hippidion as a distinct genus, in agreement with palaeontological models. We date the Hippidion split from Equus at 5.6-6.5 Ma, suggesting an early divergence in North America prior to the colonization of South America, after the formation of the Panamanian Isthmus 3.5 Ma and the Great American Biotic Interchange.
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http://dx.doi.org/10.1098/rsbl.2014.1058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4387498PMC
March 2015

The number of circulating monocytes as biomarkers of the clinical response to methotrexate in untreated patients with rheumatoid arthritis.

J Transl Med 2015 Jan 16;13. Epub 2015 Jan 16.

Department of Medicine, University of Alcalá, Carretera Madrid-Barcelona km 33.600, 28871, Alcalá de Henares, Madrid, Spain.

Background: The aim of this work was to analyze the number and distribution of circulating monocytes, and of their CD14(+high)CD16(-), CD14(+high)CD16(+) and CD14(+low)CD16(+) subset cells, in treatment-naive patients with rheumatoid arthritis (RA), and to determine their value in predicting the clinical response to methotrexate (MTX) treatment.

Methods: This prospective work investigated the number of circulating monocytes, and the numbers of CD14(+high)CD16(-), CD14(+high)CD16(+) and CD14(+low)CD16(+) subset cells, in 52 untreated patients with RA before MTX treatment, and at 3 and 6 months into treatment, using flow cytometry.

Results: The absolute number of circulating monocytes, and the numbers of CD14(+high)CD16(-), CD14(+high)CD16(+) and CD14(+low)CD16(+) subset cells, were significantly higher in MTX non-responders than in responders and healthy controls before starting and throughout treatment. Responders showed normal numbers of monocytes, and of their subset cells, over the study period. The pre-treatment absolute number of circulating monocytes, and the numbers of CD14(+high)CD16(-) and CD14(+high)CD16(+) subset cells, were found to be predictive of the clinical response to MTX, with a sensitivity and specificity of >70% and >88%, respectively.

Conclusions: Treatment-naive patients with RA showed an anomalous distribution of circulating monocyte subsets, and an anomalous number of cells in each subset. A higher pre-treatment number of circulating monocytes, and higher numbers of CD14(+high)CD16(-) and CD14(+high)CD16(+) subset cells, predict a reduced clinical response to MTX in untreated patients with RA.
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http://dx.doi.org/10.1186/s12967-014-0375-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310181PMC
January 2015

Flow cytometry enumeration of apoptotic cancer cells by apoptotic rate.

Methods Mol Biol 2015 ;1219:11-20

CNB-CSIC R&D Associated Unit, Department of Medicine, University of Alcalá, Carretera Madrid-Barcelona, Km 33.600, 28871, Alcalá de Henares, Madrid, Spain,

Most authors currently quantify the frequency of apoptotic cells in a given phenotypically defined population after calculating the apoptotic index (AI), i.e., the percentage of apoptotic cells displaying a specific linage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. Firstly, apoptotic cells fragment into apoptotic bodies that later disintegrate. Secondly, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. This chapter describes a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of lineage antigen expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures.
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http://dx.doi.org/10.1007/978-1-4939-1661-0_2DOI Listing
June 2015

Role of circulating lymphocytes in patients with sepsis.

Biomed Res Int 2014 28;2014:671087. Epub 2014 Aug 28.

Laboratory of Immune System Diseases and Oncology, National Biotechnology Center (CNB-CSIC) Associated Unit, Department of Medicine and Medical Specialties, University of Alcala, 28871 Madrid, Spain ; Immune System Diseases and Oncology Service, University Hospital "Príncipe de Asturias", University of Alcala, Alcala de Henares, 28805 Madrid, Spain.

Sepsis is a systemic inflammatory response syndrome due to infection. The incidence rate is estimated to be up to 19 million cases worldwide per year and the number of cases is rising. Infection triggers a complex and prolonged host response, in which both the innate and adaptive immune response are involved. The disturbance of immune system cells plays a key role in the induction of abnormal levels of immunoregulatory molecules. Furthermore, the involvement of effector immune system cells also impairs the host response to the infective agents and tissue damage. Recently, postmortem studies of patients who died of sepsis have provided important insights into why septic patients die and showed an extensive depletion of CD4 and CD8 lymphocytes and they found that circulating blood cells showed similar findings. Thus, the knowledge of the characterization of circulating lymphocyte abnormalities is relevant for the understanding of the sepsis pathophysiology. In addition, monitoring the immune response in sepsis, including circulating lymphocyte subsets count, appears to be potential biomarker for predicting the clinical outcome of the patient. This paper analyzes the lymphocyte involvement and dysfunction found in patients with sepsis and new opportunities to prevent sepsis and guide therapeutic intervention have been revealed.
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http://dx.doi.org/10.1155/2014/671087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4163419PMC
June 2015

Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes.

PLoS One 2013 29;8(10):e78575. Epub 2013 Oct 29.

Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.

Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078575PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3812280PMC
August 2014

Distinctive patterns of naïve/memory subset distribution and cytokine expression in CD4 T lymphocytes in ZAP-70 B-chronic lymphocytic patients.

Cytometry B Clin Cytom 2014 Jan 24;86(1):32-43. Epub 2013 Oct 24.

Laboratory of Immune System Diseases and Oncology, Department of Medicine (CNB/CSIC Associated Unit), University of Alcalá, Alcala de Henares, 28871, Madrid, Spain.

Background: ZAP-70 upregulation in B chronic lymphocytic leukemia (B-CLL) cells is a recognized marker of poor prognosis in these patients; the biological basis of this differential clinical outcome nonetheless remains unknown. ZAP-70 overexpression is considered a surrogate marker of a B-CLL cell subset. To test whether the differential biological characteristics of these patients also include the T helper population, we studied naïve, non-terminated memory (NTEM), terminated memory (TEM) and central memory (CM) cells, and cytokine expression by CD4 T lymphocytes from ZAP-70(+) and ZAP-70(-) B-CLL patients.

Methods: Expression of CD3, CD8, CD45RA, CD27, and CD28 antigens and intracytoplasmic cytokine production (IFNγ, IL-2, IL-4, IL-10, and TNFα) were assessed simultaneously by nine-color flow-cytometry in peripheral blood lymphocytes from B-CLL patients. B cell ZAP-70 expression in B-CLL cells was also analyzed by flow cytometry.

Results: Compared to ZAP-70(-) B-CLL patients, ZAP-70(+) B-CLL patients showed 1) significant reduction in the naïve T helper subset and expansion of NTEM and TEM subsets, 2) a decrease in the percentage of activated CD4 T lymphocytes expressing IFNγ, TNFα, and IL-2, and 3) an increase in the percentage of CD4 T lymphocytes expressing IL-4 or IL-10.

Conclusions: In conclusion, in early stage B-CLL patients, ZAP-70 upregulation is associated with distinct patterns of activation/differentiation stage subset distribution and of cytokine expression in CD4 T lymphocytes.
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http://dx.doi.org/10.1002/cyto.b.21120DOI Listing
January 2014

Distinctive patterns of naïve/memory subset distribution and cytokine expression in CD4 T lymphocytes in ZAP-70 B-chronic lymphocytic patients.

Cytometry B Clin Cytom 2013 Jul 29. Epub 2013 Jul 29.

Laboratory of Immune System Diseases and Oncology, Department of Medicine (CNB/CSIC Associated Unit), University of Alcalá, Alcala de Henares, 28871, Madrid, Spain; Hematology Service, Hospital Universitario de la Paz, Madrid, Spain.

Background: ZAP-70 upregulation in B chronic lymphocytic leukemia (B-CLL) cells is a recognized marker of poor prognosis in these patients; the biological basis of this differential clinical outcome nonetheless remains unknown. ZAP-70 overexpression is considered a surrogate marker of a B-CLL cell subset. To test whether the differential biological characteristics of these patients also include the T helper population, we studied naïve, non-terminated memory (NTEM), terminated memory (TEM) and central memory (CM) cells and cytokine expression by CD4 T lymphocytes from ZAP-70 and ZAP-70 B-CLL patients.

Methods: Expression of CD3, CD8, CD45RA, CD27, and CD28 antigens and intracytoplasmic cytokine production (IFNγ, IL-2, IL-4, IL-10 and TNFα) were assessed simultaneously by nine-color flow-cytometry in peripheral blood lymphocytes from B-CLL patients. B cell ZAP-70 expression in B-CLL cells was also analyzed by flow cytometry.

Results: Compared to ZAP-70 B-CLL patients, ZAP-70 B-CLL patients showed 1) significant reduction in the naïve T helper subset and expansion of NTEM and TEM subsets, 2) a decrease in the percentage of activated CD4 T lymphocytes expressing IFNγ, TNFα and IL-2, and 3) an increase in the percentage of CD4 T lymphocytes expressing IL-4 or IL-10.

Conclusions: In conclusion, in early stage B-CLL patients, ZAP-70 upregulation is associated with distinct patterns of activation/differentiation stage subset distribution and of cytokine expression in CD4 T lymphocytes. © 2013 Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cytob.21120DOI Listing
July 2013

Early alterations of B cells in patients with septic shock.

Crit Care 2013 May 30;17(3):R105. Epub 2013 May 30.

Introduction: It has recently been proposed that B lymphocytes are involved in sepsis pathogenesis. The goal of this study is to investigate potential abnormalities in a subset distribution and activation of circulating B lymphocytes in patients with septic shock.

Methods: This observational prospective study was conducted in a medical-surgical ICU. All patients with septic shock were eligible for inclusion. B-cell phenotypes (CD19+CD69+, CD19+CD23+, CD19+CD5+, CD19+CD80, CD19+CD86+, CD19+CD40 and CD19+CD95+) were assessed by quantitative flow cytometry upon admission to the ICU and 3, 7, 14 and 28 d later.

Results: Fifty-two patients were included. Thirty-six healthy volunteers matched for age and sex were used as controls. The patients had lymphopenia that was maintained during 28 d of follow-up. In patients with septic shock who died, the percentage of CD19+CD23+ was lower during the 7 d of follow-up than it was in survival patients. Moreover, the percentage of CD80+ and CD95+ expression on B cells was higher in patients who died than in survivors. Receiver operating characteristic curve analysis showed that a CD19+CD23+ value of 64.6% at ICU admission enabled discrimination between survivors and nonsurvivors with a sensitivity of 90.9% and a specificity of 80.0% (P=0.0001).

Conclusions: Patients with septic shock who survive and those who don't have different patterns of abnormalities in circulating B lymphocytes. At ICU admission, a low percentage of CD23+ and a high of CD80+ and CD95+ on B cells were associated with increased mortality of patients with septic shock. Moreover, a drop in circulating B cells persisted during 28 d of ICU follow-up.
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http://dx.doi.org/10.1186/cc12750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4056890PMC
May 2013

Circulating sICAM-1 and sE-Selectin as biomarker of infection and prognosis in patients with systemic inflammatory response syndrome.

Eur J Intern Med 2013 Mar 23;24(2):132-8. Epub 2013 Jan 23.

Intensive Care Unit, Hospital Universitario Príncipe de Asturias, Department of Medicine, University of Alcalá, Alcalá de Henares, Madrid, Spain.

Background: Vascular endothelium activation is a key pathogenic step in systemic inflammatory response syndrome (SIRS) that can be triggered by both microbial and sterile proinflammatory stimuli. The relevance of soluble adhesion molecules as clinical biomarkers to discriminate between infectious and non-infectious SIRS, and the individual patient prognosis, has not been established.

Methods: We prospectively measured by sandwich ELISA, serum levels of soluble E-Selectin (sE-Selectin), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble intercellular adhesion molecule-2 (sICAM-2) at ICU admission and at days 3, 7, 14 and 28 in patients with sepsis and at days 3 and 7 in patients with non-infectious SIRS.

Results: At ICU admission, sE-Selectin, sVCAM-1 and sICAM-1 in patients with infectious SIRS were significantly higher than those found in patients with non-infectious SIRS. ROC analysis revealed that the AUC for infection identification was best for sICAM-1 (0.900±0.041; 95% CI 0.819-0.981; p<0.0001). Moreover, multivariate analysis showed that 4 variables were significantly and independently associated with mortality at 28 days: male gender (OR 15.90; 95% CI, 2.54-99.32), MODS score (OR 5.60; 95% CI, 1.67-18.74), circulating sE-Selectin levels (OR 4.81; 95% CI, 1.34-17.19) and sVCAM-1 concentrations (OR 4.80; 95% CI, 1.34-17.14).

Conclusions: Patients with SIRS secondary to infectious or non-infectious etiology show distinctive patterns of disturbance in serum soluble adhesion molecules. Serum ICAM-1 is a reliable biomarker for classifying patients with infectious SIRS from those with non-infectious SIRS. In addition, soluble E-Selectin is a prognostic biomarker with higher levels in patients with SIRS and fatal outcome.
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http://dx.doi.org/10.1016/j.ejim.2012.10.009DOI Listing
March 2013

Monocyte populations as markers of response to adalimumab plus MTX in rheumatoid arthritis.

Arthritis Res Ther 2012 Jul 27;14(4):R175. Epub 2012 Jul 27.

Introduction: The treatment of rheumatoid arthritis (RA) patients with anti-tumor necrosis factor alpha (TNFα) biological drugs has dramatically improved the prognosis of these patients. However, a third of the treated patients do not respond to this therapy. Thus, the search for biomarkers of clinical response to these agents is currently highly active. Our aim is to analyze the number and distribution of circulating monocytes, and of their CD14⁺highCD16⁻, CD14⁺highCD16⁺ and CD14⁺lowCD16+ subsets in methotrexate (MTX) non-responder patients with RA, and to determine their value in predicting the clinical response to adalimumab plus MTX treatment.

Methods: This prospective work investigated the number of circulating monocytes, and of their CD14⁺highCD16⁻, CD14⁺highCD16⁺ and CD14⁺lowCD16⁺ subsets, in 35 MTX non-responder patients with RA before and after three and six months of anti-TNFα treatment using multiparametric flow cytometry. The number of circulating monocytes in an age- and sex-matched healthy population was monitored as a control.

Results: Non-responder patients with RA show an increased number of monocytes and of their CD14⁺highCD16⁻, CD14⁺highCD16⁺ and CD14⁺lowCD16⁺ subsets after three months of adalimumab plus MTX treatment that remained significantly increased at six months. In contrast, significant normalization of the numbers of circulating monocytes was found in responders at three months of adalimumab plus MTX treatment that lasts up to six months. CX3CR1 expression is increased in monocytes in non-responders. At three months of anti-TNFα treatment the number of circulating monocytes and their subsets was associated with at least 80% sensitivity, 84% specificity and an 86% positive predictive value (PPV) in terms of discriminating between eventual early responders and non-responders.

Conclusions: The absolute number of circulating monocytes and of their CD14⁺highCD16⁻, CD14⁺highCD16⁺ and CD14⁺lowCD16⁺ subsets at three months of adalimumab plus MTX treatment, have a predictive value (with high specificity and sensitivity) in terms of the clinical response after six months of anti-TNFα treatment in patients with RA.
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http://dx.doi.org/10.1186/ar3928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3580569PMC
July 2012

Sepsis-induced acute respiratory distress syndrome with fatal outcome is associated to increased serum transforming growth factor beta-1 levels.

Eur J Intern Med 2012 Jun 6;23(4):358-62. Epub 2011 Nov 6.

Intensive Care Unit, Hospital Universitario Príncipe de Asturias, Department of Medicine, University of Alcalá, Alcalá de Henares, Madrid, Spain.

Background: TGF-β1 is a promoter of pulmonary fibrosis in many chronic inflammatory diseases. TGF-β1 circulating levels in patients with sepsis-induced Acute Respiratory Distress Syndrome (ARDS) have not been established.

Methods: In this prospective pilot cohort study, serum bioactive TGF-β1 concentration, determined by sandwich ELISA, was analyzed in 52 patients who fulfilled criteria for septic shock at admission and on days 3 and 7.

Results: Of the 52 patients enrolled in the study, 46.1% fulfilled the criteria for ARDS on admission. At ICU admission, there were not statistical differences in TGF-β1 concentrations between septic shock patients with or without ARDS. After 7 days of follow-up in ICU, circulating TGF-β1 levels were significantly higher in patients with sepsis and ARDS than in those without ARDS [55.47 (35.04-79.48 pg/ml) versus 31.65 (22.89-45.63 pg/ml), respectively] (p = 0.002). Furthermore, in septic shock associated ARDS patients, TGF-β1 levels were significantly higher in nonsurvivors than in survivors [85.23 (78.19-96.30 pg/ml) versus 36.41 (30.21-55.47 pg/ml), respectively] (p = 0.006) on day 7 of ICU follow-up.

Conclusions: In patients with septic shock, persistent ARDS is accompanied with increased circulating TGF-β1 levels. Furthermore, ARDS patients with fatal outcome show higher TGF-β1 concentrations than survivors. These results suggest the relevance of TGF-β1 levels found in the pathogenesis of persistent sepsis-induced ARDS.
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http://dx.doi.org/10.1016/j.ejim.2011.10.001DOI Listing
June 2012

The predictive role of early activation of natural killer cells in septic shock.

Crit Care 2012 Dec 12;16(2):413. Epub 2012 Dec 12.

Recently, several studies about the role of natural killer (NK) cells in sepsis have been highlighted. In an earlier study, we characterized the abnormalities of circulating lymphocytes in 52 patients with septic shock during the first 28 days in the intensive care unit. Our results confirm and expand some previous reports. We found that patients who did not survive exhibited less NK cell (CD3-CD56⁺) depletion than survivors and that these NK cells expressed CD69⁺ and CD57⁺. These data demonstrate that NK cells are key participants in septic shock because patients who survived have more depletion and expressed less early activation and differentiation.
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http://dx.doi.org/10.1186/cc11204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681341PMC
December 2012

Loss of surface antigens is a conserved feature of apoptotic lymphocytes from several mammalian species.

Cell Immunol 2011 24;271(1):163-72. Epub 2011 Jun 24.

Department of Medicine, University of Alcalá, Madrid, Spain.

Human lymphocytes lose the expression of lineage antigens (LAgs) along apoptosis. Our aim was to extent our previous studies of LAg loss to rodent species, quantifying LAg expression on apoptotic murine lymphocytes using flow cytometry to measure alterations in cell permeability, phosphatidylserine exposure and caspase activation of CD3, CD5, CD4, CD8, CD19 and CD28 LAgs in highly purified lymphocyte populations. We found loss of expression by apoptotic cells of all LAgs studied in the three species analyzed except for CD3 antigen in mouse. We also found an early, rapid and dramatic reduction in the expression of CD28 by early apoptotic cells. We found several homologies across the three species in the kinetic of loss of several LAgs such as CD5, CD4 and CD28. These data suggest that the loss of expression of LAgs by apoptotic lymphocytes is a common and conserved feature of lymphocytes undergoing apoptosis in several mammalian species.
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http://dx.doi.org/10.1016/j.cellimm.2011.06.018DOI Listing
December 2011

Mortality in patients with septic shock correlates with anti-inflammatory but not proinflammatory immunomodulatory molecules.

J Intensive Care Med 2011 Mar-Apr;26(2):125-32

Intensive Care Unit, Hospital Universitario Principe de Asturias, Alcala de Henares, Madrid, Spain.

Background: Mortality in patients with septic shock remains unacceptably high and the attempts to antagonize certain proinflammatory cytokines based on the results of animal model studies have failed to improve survival rates. The objective of this article is to examine the pro-/anti-inflammatory cytokine balance in patients with septic shock and its connection with mortality.

Methods: Serum levels of proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin 1β [IL-1β], interferonγ [IFN-γ], and IL-6) and soluble cytokine antagonists (soluble TNF receptor I [sTNF-RI], sTNF-RII, and IL-1Ra) were determined on admission to the intensive care unit (ICU) and 3, 7, 14, and 28 days later in 52 patients with septic shock and in 36 healthy controls. Specific sandwich enzyme-linked immunosorbent assay (ELISA) was used for all determinations.

Results: Serum levels of most of the pro- and anti-inflammatory molecules examined (TNF-α, IL-6, sTNF-RI, sTNF-RII, and IL-1 receptor agonist [IL-1Ra]) were significantly elevated on admission and during the 28-day observation period in patients when compared to controls. Notably, the anti-inflammatory mediators sTNF-RI, sTNF-RII, and IL-1Ra were better predictors of mortality. Receiver-operating characteristic (ROC) analysis revealed that sTNF-RI or sTNF-RII concentrations over 2767 or 4619 pg/mL, respectively, determined a high risk of death (sensitivity: 100%-100%, specificity: 57.1%-71.4%, area under the curve [AUC] 0.759-0.841, respectively), whereas IL-1Ra concentrations below 7033 pg/mL determined a high probability of survival (sensitivity: 60%, specificity: 100%, AUC 0.724). In addition, IFN-γ levels were significantly higher in survivors than in controls during the initial 2 weeks of observation.

Conclusions: Our data show that serum cytokine disturbance patterns have prognostic significance in patients with septic shock admitted to the ICU. The pattern, defined by an early response to continuously elevated anti-inflammatory cytokine serum levels, is associated with an enhanced risk of a fatal outcome for patients.
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http://dx.doi.org/10.1177/0885066610384465DOI Listing
August 2011

Human brucellosis is characterized by an intense Th1 profile associated with a defective monocyte function.

Infect Immun 2010 Jul 19;78(7):3272-9. Epub 2010 Apr 19.

Laboratorio de Enfermedades del Sistema Inmune y Oncología, Unidad Asociada al CNB-CSIC, Universidad de Alcalá, Madrid, Spain.

In animal models, a defective Th1 response appears to be critical in the pathogenesis of brucellosis, but the Th1 response in human brucellosis patients remains partially undefined. Peripheral blood from 24 brucellosis patients was studied before and 45 days after antibiotherapy. Twenty-four sex- and age-matched healthy donors were analyzed in parallel. Significantly increased levels of interleukin 1beta (IL-1beta), IL-2, IL-4, IL-6, IL-12p40, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), but not of IL-10, in serum and/or significantly increased percentages of samples with detectable levels of these cytokines, measured by enzyme-linked immunosorbent assays (ELISA), were found for untreated brucellosis patients, but these levels were reduced and/or normalized after treatment. Flow cytometry studies showed that the intracytoplasmic expression of IFN-gamma, IL-2, and TNF-alpha, but not that of IL-4, by phorbol myristate-activated CD4(+) CD3(+) and CD8(+) CD3(+) T lymphocytes was significantly increased in untreated brucellosis patients and was also partially normalized after antibiotherapy. The percentage of phagocytic cells, the mean phagocytic activity per cell, and the phagocytic indices for monocytes at baseline were defective and had only partially reverted at follow-up. T lymphocytes from untreated brucellosis patients are activated in vivo and show Th1 cytokine production polarization, with strikingly high serum IFN-gamma levels. In spite of this Th1 environment, we found deficient effector phagocytic activity in peripheral blood monocytes.
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http://dx.doi.org/10.1128/IAI.01385-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2897366PMC
July 2010

IFNbeta therapy progressively normalizes the increased ex vivo T lymphocyte apoptosis observed in active patients with multiple sclerosis.

Clin Immunol 2009 Aug 13;132(2):195-202. Epub 2009 May 13.

Neuroimmunology Unit, Clínica Puerta de Hierro, Universidad Autónoma de Madrid, Spain.

Abnormal apoptosis has been reported in circulating T lymphocytes in patients with multiple sclerosis. The effects of 12 months of IFNbeta treatment in T and B lymphocyte spontaneous ex vivo apoptosis were studied in patients with MS. Peripheral blood mononuclear cells were obtained from 48 patients before and at 1, 6 and 12 months after treatment with IFNbeta. Spontaneous ex vivo apoptosis was quantified by four-color flow cytometry. A significant reduction and normalization of the percentage of apoptotic cells was found in all T lymphocyte subsets. B cell apoptosis values were unaffected by therapy; Relapses of the clinical activity of the disease were associated to transitory upturns of lymphocyte apoptosis. In conclusion, IFNbeta therapy progressively normalizes the increased ex vivo T lymphocyte apoptosis observed in MS. However, it is not clear if this reduction in spontaneous T lymphocyte apoptosis is due to direct effect of IFNbeta or secondary to decreased clinical and sub-clinical activity.
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http://dx.doi.org/10.1016/j.clim.2009.04.006DOI Listing
August 2009

Clinical relevance of the severe abnormalities of the T cell compartment in septic shock patients.

Crit Care 2009 25;13(1):R26. Epub 2009 Feb 25.

Department of Medicine (CNB-CSIC) Associated Unit, Laboratory of Immune System Diseases and Oncology, National Biotechnology Center, University of Alcalá, Alcalá de Henares, Madrid, Spain.

Introduction: Given the pivotal role of T lymphocytes in the immune system, patients with septic shock may show T cell abnormalities. We have characterised the T cell compartment in septic shock and assess its clinical implications.

Methods: T lymphocytes from the peripheral blood of 52 patients with septic shock and 36 healthy control subjects were analysed on admission to the intensive care unit, baseline, and 3, 7, 14 and 28 days later. T cell phenotypes (CD3+CD4+/CD3+CD8+, CD45RA+/CD45RO+, CD62L+/CD28+) were assessed by quantitative flow cytometry.

Results: CD3+, CD3+CD4+ and CD3+CD8+ lymphocyte counts were significantly lower in patients with septic shock than control subjects. In surviving patients, CD3+CD4+ lymphocytes had normalised after 14 days, yet CD3+CD8+ numbers were still low. Non effector CD45RA+CD45RO- subsets of CD3+CD4+ and CD3+CD8+ were persistently low during patient follow up. CD3+CD8+CD28+ and CD3+CD8+CD62L+ were reduced in patients versus controls and survivors versus nonsurvivors in the first three days. A prediction receptor operative curve revealed that for the CD3+CD8+CD28+ subset, a cutoff of 136 cells/ml showed 70% sensitivity and 100% specificity for predicting death and the area under the curve was 0.84 at admission. Corresponding values for CD3+CD8+CD62L+ were 141 cells/ml, 60% sensitivity, 100% specificity and an area under the curve of 0.75.

Conclusions: A severe redistribution of T lymphocyte subsets is found in septic shock patients. A different kinetic pattern of T cell subset involvement is observed in surviving and nonsurviving patients, with lower numbers of circulating CD3+CD8+CD28+ and CD3+CD8+CD62L+ associated with a better disease outcome.
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http://dx.doi.org/10.1186/cc7731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2688144PMC
March 2010

Inhaled IL-2 induces systemic immunomodulation in patients with renal cell carcinoma and lung metastasis.

Cancer Immunol Immunother 2009 Feb 1;58(2):235-45. Epub 2008 Jul 1.

CNB-CSIC R&D Associated Unit, Department of Medicine, University of Alcalá, Madrid, Spain.

The peripheral blood lymphocytes of eight patients with metastatic renal cell carcinoma, and of eight healthy volunteers were analyzed by four-color flow cytometry to characterize the immunophenotypic alterations manifested, determine the prevalence of lymphocyte apoptosis, and detect evidence of the systemic effect of inhaled IL-2. The T, B and NK lymphocytes of untreated patients were found to have undergone profound changes characterized by an increase in susceptibility to both spontaneous and mitogen-induced ex vivo apoptosis, a modified distribution of the main lymphocyte populations in the peripheral blood, and alterations in activation status. An increase in the proportion of regulatory T cells was also seen in these patients. Treatment with inhaled IL-2, however, normalized the rate of apoptosis in all the lymphocyte subpopulations studied, as well as their distribution and activation status. These findings demonstrate that inhaled IL-2 has systemic immunomodulatory effects.
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http://dx.doi.org/10.1007/s00262-008-0546-xDOI Listing
February 2009

Ancient DNA clarifies the evolutionary history of American Late Pleistocene equids.

J Mol Evol 2008 May 9;66(5):533-8. Epub 2008 Apr 9.

Université de Lyon, Paleogenetics and Molecular Evolution, Institut de Génomique Fonctionnelle de Lyon, CNRS UMR 5242 - INRA - Université Claude Bernard Lyon I, Lyon Cedex 07, France.

Hippidions are past members of the equid lineage which appeared in the South American fossil record around 2.5 Ma but then became extinct during the great late Pleistocene megafaunal extinction. According to fossil records and numerous dental, cranial, and postcranial characters, Hippidion and Equus lineages were expected to cluster in two distinct phylogenetic groups that diverged at least 10 MY, long before the emergence of the first Equus. However, the first DNA sequence information retrieved from Hippidion fossils supported a striking different phylogeny, with hippidions nesting inside a paraphyletic group of Equus. This result indicated either that the currently accepted phylogenetic tree of equids was incorrect regarding the timing of the evolutionary split between Hippidion and Equus or that the taxonomic identification of the hippidion fossils used for DNA analysis needed to be reexamined (and attributed to another extinct South American member of the equid lineage). The most likely candidate for the latter explanation is Equus (Amerhippus) neogeus. Here, we show by retrieving new ancient mtDNA sequences that hippidions and Equus (Amerhippus) neogeus were members of two distinct lineages. Furthermore, using a rigorous phylogenetic approach, we demonstrate that while formerly the largest equid from Southern America, Equus (Amerhippus) was just a member of the species Equus caballus. This new data increases the known phenotypic plasticity of horses and consequently casts doubt on the taxonomic validity of the subgenus Equus (Amerhippus).
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http://dx.doi.org/10.1007/s00239-008-9100-xDOI Listing
May 2008

Flow cytometry enumeration of apoptotic cancer cells by apoptotic rate.

Methods Mol Biol 2008 ;414:23-33

CNB-CSIC R&D Associated Unit, Department of Medicine, University of Alcalá, Alcalá de Henares, Maddrid, Spain.

Most authors currently quantify the frequency of apoptotic cells in a given phenotypically defined population after calculating the apoptotic index (AI), that is, the percentage of apoptotic cells displaying a specific lineage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. First, apoptotic cells fragment into apoptotic bodies that later disintegrate. Second, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. This chapter will describe a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of LAg expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures.
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http://dx.doi.org/10.1007/978-1-59745-339-4_3DOI Listing
February 2008

Increased spontaneous ex vivo apoptosis and subset alterations in peripheral blood T cells from patients with multiple sclerosis.

J Clin Immunol 2006 Mar 2;26(2):101-12. Epub 2006 May 2.

Laboratorio de Inmunología Clínica, Unidad Asociada I+D Consejo Superior Investigaciones Científicas (Centro Nacional de Biotecnología), Departamento de Medicina, Universidad de Alcalá, Madrid, Spain.

In order to characterize the immunophenotype and the lymphocyte apoptosis in multiple sclerosis (MS) patients, the peripheral blood lymphocytes of 46 MS patients and 12 healthy volunteers were studied by flow cytometry. Immunophenotypic alterations included significant increases in T CD4+ lymphocytes and reductions in the percentages of CD3+ and CD8+ T cells. After 24 h of culture spontaneous apoptosis was increased in almost T lymphocyte subsets from MS patients and it was significantly higher in those patients who had suffered more than two relapses in the two previous years. The incidence of spontaneous apoptosis was not dependent on FasL-Fas interactions and correlated with the percentages of cells positive for active caspases but not with percentages of Fas+ cells. The increased susceptibility to apoptosis of peripheral blood T lymphocytes from MS patients is difficult to reconcile with the previously proposed role of a defective lymphocyte apoptosis in the pathophysiology of MS.
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http://dx.doi.org/10.1007/s10875-006-9007-5DOI Listing
March 2006

Treatment with AM3 restores defective T-cell function in COPD patients.

Chest 2006 Mar;129(3):527-35

Departamento de Medicina, Universidad de Alcalá, Carretera Madrid-Barcelona, Km 33,600, E-28871 Alcalá de Henares, Madrid, Spain.

Background: Lymphocyte alterations have been associated with an increased prevalence of acute respiratory infections in COPD patients. AM3 is an oral immunomodulator that normalizes the defective functions of peripheral blood natural killer and phagocytic cells in COPD patients and improves their health-related quality of life.

Objectives: To characterize putative systemic abnormalities of the T-cell compartment in COPD patients, and to investigate whether AM3 can restore such abnormalities.

Design: The study was a randomized, prospective, double-blind, placebo-controlled trial in a cohort of COPD patients. The results were also compared to those of nonsmoker and ex-smoker healthy control subjects.

Setting: Outpatient departments of four hospitals.

Patients: Seventy COPD patients were randomized to receive either AM3 or a placebo orally for 90 consecutive days. Populations of 36 healthy nonsmokers and 36 healthy ex-smokers were used as control subjects.

Measurements: Peripheral blood mononuclear cell (PBMC) proliferation and production of interleukin (IL)-2, IL-4, IL-12p40, tumor necrosis factor-alpha, and interferon (IFN)-gamma proteins in response to the T-cell polyclonal mitogens were assessed at baseline and at the end of treatment.

Results: The proliferative response was significantly decreased in COPD patients. Decreased production of IFN-gamma was the only defect in the profiles of the cytokine measures, and was selectively observed in COPD patients, but not in nonsmoker and ex-smoker healthy control subjects. Treatment with AM3 significantly restored the PBMC proliferative response to polyclonal mitogens and significantly promoted stimulated IFN-gamma production in these patients. The normalization of these proliferative responses was not related to significant variations in the numbers of peripheral blood monocytes, CD3+, CD4+, CD8+ cells or of any major naïve/memory/activated T-cell subset. The increased IFN-gamma production in the AM3 study arm was associated with an increase in the mean of number of IFN-gamma molecules produced per CD8+ T cells.

Conclusions: PBMCs of COPD patients showed clear functional T-lymphocyte abnormalities that are rescued by AM3 treatment.
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http://dx.doi.org/10.1378/chest.129.3.527DOI Listing
March 2006

Accurate apoptosis measurement requires quantification of loss of expression of surface antigens and cell fragmentation.

Cytometry A 2006 Apr;69(4):240-8

CNB-CSIC R&D Associated Unit, Department of Medicine, University of Alcalá, Alcalá de Henares, Madrid, Spain.

Background: The use of ratiometric cell enumeration methods emerges as a more accurate method of measurement of the occurrence of apoptosis in cell cultures. These new flow cytometry methods were used to quantify the impact of cell fragmentation and loss of lineage antigen (LAg) expression on measurement of apoptosis.

Methods: Highly purified human lymphocyte populations were negatively sorted and cultured for 24 h. Apoptotic cells were identified using annexin V, 7-amino-actinomycin D and their LAgs were stained with antibodies. A new indicator, the apoptotic rate, was used to determine apoptosis occurrence and its validity compared with the widely accepted percentage of apoptotic cells (apoptotic index, AI).

Results: Loss of LAg expression and cell fragmentation were observed under all conditions assayed and for all cell populations studied.

Conclusions: Current methods for quantifying of apoptosis involving AI systematically underestimate apoptosis occurrence in all populations and conditions, especially among cells undergoing spontaneous apoptosis.
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http://dx.doi.org/10.1002/cyto.a.20251DOI Listing
April 2006

Evolution, systematics, and phylogeography of pleistocene horses in the new world: a molecular perspective.

PLoS Biol 2005 Aug 28;3(8):e241. Epub 2005 Jun 28.

Ancient Biomolecules Centre, Department of Zoology, University of Oxford, United Kingdom.

The rich fossil record of horses has made them a classic example of evolutionary processes. However, while the overall picture of equid evolution is well known, the details are surprisingly poorly understood, especially for the later Pliocene and Pleistocene, c. 3 million to 0.01 million years (Ma) ago, and nowhere more so than in the Americas. There is no consensus on the number of equid species or even the number of lineages that existed in these continents. Likewise, the origin of the endemic South American genus Hippidion is unresolved, as is the phylogenetic position of the "stilt-legged" horses of North America. Using ancient DNA sequences, we show that, in contrast to current models based on morphology and a recent genetic study, Hippidion was phylogenetically close to the caballine (true) horses, with origins considerably more recent than the currently accepted date of c. 10 Ma. Furthermore, we show that stilt-legged horses, commonly regarded as Old World migrants related to the hemionid asses of Asia, were in fact an endemic North American lineage. Finally, our data suggest that there were fewer horse species in late Pleistocene North America than have been named on morphological grounds. Both caballine and stilt-legged lineages may each have comprised a single, wide-ranging species.
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http://dx.doi.org/10.1371/journal.pbio.0030241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1159165PMC
August 2005

Involvement of Bax, Bcl-2 and caspase 3 in hydroxyurea- or etoposide-induced apoptosis of mouse interleukin-3-dependent lymphoma cells.

Anticancer Res 2005 Mar-Apr;25(2A):999-1007

Departamento de Bioquímica y Biología Molecular, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.

Background: The apoptosis action induced by hydroxyurea or etoposide in interleukin 3-dependent lymphoma cells (DA-1) was studied.

Materials And Methods: The conditions to study apoptosis of these cells were 17 hours of cell treatment with concentrations of 1.25 mM hydroxyurea or 100 microM etoposide using flow cytometry, fluorometry and immunoblots techniques.

Results: Time-dependent reductions of cell viability after these treatments were observed. Caspase 3 activity was highly activated in both cases. Chemical treatments or interleukin 3 withdrawal rendered level changes in Bax and Bcl-2 expression.

Conclusion: These results support the implication of these factors in DA-1 cell apoptosis induced by these chemical treatments or interleukin 3 depletion.
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June 2005