Pedro Rodrigues

University of Porto, Oporto, Porto, Portugal

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Publications (46)123.86 Total impact

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    ABSTRACT: This paper reviews the family Gymnophallidae, recognizing as valid seven genera; four within the subfamily Gymnophallinae: Gymnophallus Odhner, 1900 (syn. Meiogymnophallus Ching, 1965), Paragymnophallus Ching, 1973, Pseudogymnophallus Hoberg, 1981, and Bartolius Cremonte, 2001, and three in the Parvatrematinae: Parvatrema Cable, 1953, Lacunovermis Ching, 1965, and Gymnophalloides Fujita, 1925. Specimens representing one species of each available genus were chosen from those well-described and non controversial species, for which strong morphological information was available, and used for molecular studies (ITS1-5.8S-ITS2-28S rDNA strands were sequenced). The presence or absence of a pars prostatica differentiates between the 2 subfamilies, Gymnophallinae and Parvatrematinae. The characters used to differentiate genera are: location of the ovary (pre-, post- or inter-testicular), size and location of the genital pore (inconspicuous and located at the anterior margin of the ventral sucker, or conspicuous and located at some distance from the anterior margin of ventral sucker), presence of caecal pockets, and presence or absence of ventral pit (a muscular structure which can be either well-developed, similar in size and musculature to the ventral sucker, or be poorly developed). The characters previously used to distinguish among genera that actually should be considered to separate species include: shape of tegument spines (broad, sharp or serrated), presence of lateral projections on the oral sucker (also called papillae or lips), shape of the seminal vesicle (unipartite or bipartite), shape of the prostatic duct (elongate or oval), presence of papillae on the genital pore, shape of the genital atrium (tubular, wide, oval), shape of the vitellaria (follicular in a variable degree, paired or single), shape of the excretory vesicle (V or Y), extension of uterus (restricted to forebody, at hindbody or extending in both). Additionally, some of these characters may vary with the age of worm. The morphological and molecular information obtained in this study provided strong support for recognizing seven valid genera in the family Gymnophallidae. Copyright © 2014. Published by Elsevier Ireland Ltd.
    Parasitology International 12/2014; 64(2). DOI:10.1016/j.parint.2014.12.003 · 2.11 Impact Factor
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    ABSTRACT: Abstract Morphological and molecular evidence suggest that specimens formerly described as Lacunovermis sp. from Nacella (Patinigera) spp. (Patellogastropoda: Patellidae) belong to a new species of Gymnophalloides Fujita, 1925. Based on the new information, they are identified as G. nacellae n. sp. The new species differs from Gymnophalloides tokiensis, G. seoi, and G. heardi mainly through the presence of a group of papillae located on the ventral surface between oral and ventral suckers. A detailed morphological study revealed the lack of pars prostatica, a character previously reported in G. seoi, which is why it was formerly placed in the subfamily Gymnophallinae. Molecular information proved that G. nacellae is close to G. seoi, being nestled together with Parvatrema representatives. This molecular information, along with the absence of pars prostatica, allows these 2 genera to be placed in Parvatrematinae. An amended diagnosis of Gymnophalloides is provided. Histological sections of mantle epithelium of the limpet show metacercariae attached by their oral and ventral suckers in a similar manner to G. seoi in its host, the oyster Crassostrea gigas. Tissue reaction includes cells of outer mantle epithelium being stretched by sucker attachment, hemocyte infiltration of connective tissue between mantle epitheliums, and abnormal calcareous deposition on the inner surface of the shell.
    Journal of Parasitology 07/2012; 99(1). DOI:10.1645/GE-2909.1 · 1.26 Impact Factor
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    Fish Pathology 01/2012; 47(2):80-82. · 0.88 Impact Factor
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    ABSTRACT: Maritrema portucalensis sp. nov. (Digenea, Microphallidae) from Aveiro estuary, northern Portugal, is described on the basis of excysted metacercariae obtained from the gill lamellae of the shore crab Carcinus maenas (L.). M. portucalensis sp. nov. most closely resembles Maritrema subdolum Jägerskiöld, 1909, a species that also frequently uses C. maenas as second intermediate host. The new species differs from M. subdolum in having a narrower body at the level of testes and a smaller ovary and testes. Also, the digestive caeca presented various folds along their length, thickening in the terminal region. Moreover, the body surface was fully covered by tegumental spines provided with several teeth. To complement our morphological characterization and to identify M. portucalensis metacercariae, the ITS1 rDNA region of the cysts isolated from C. maenas was sequenced and compared with the corresponding available sequences of digenean trematodes belonging to the family Microphallidae. Alignments revealed 28 base-pair differences between the query-ITS1 and that of M. subdolum, and more importantly a 100% similarity with the sequence of Microphallidae sp. no. 15 cercaria from the snail Hydrobia ulvae. The last observation allowed us to establish an unequivocal association between cercaria known as Microphallidae sp. no. 15, and the metacercaria of M. portucalensis sp. nov. characterized in this work, contributing decisively to the clarification of its life cycle.
    Acta Parasitologica 12/2011; 56(4). DOI:10.2478/s11686-011-0068-0 · 0.97 Impact Factor
  • J V Neves, C Caldas, J M Wilson, P N S Rodrigues
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    ABSTRACT: Hepcidin, an antimicrobial peptide described as a key regulator of iron metabolism, is known to respond in mammals to several stimuli, including iron overload, anemia, hypoxia and inflammation, through a number of molecular pathways. In order to understand the molecular pathways involved in the regulation of hepcidin expression in teleost fish, we have isolated for European sea bass (Dicentrarchus labrax) several coding sequences of known molecules involved on these pathways in mammals, namely jak3, stat3, tmprss6, bmp6, bmpr2, hjv, smad4, smad5, tfr1 and tfr2. The transcription levels of the isolated genes were evaluated by real-time PCR on fish subjected to experimental iron modulation (overload/deficiency) or infection with Photobacterium damsela. Results show that genes associated with the major pathway of the inflammatory response (IL6/JAK/STAT pathway) in mammals are also modulated in sea bass, being up-regulated during infection. Similarly, genes of the pathways classically associated with the response to variations in iron status (the HJV/BMP/SMAD and HFE/TfR pathways) are also modulated, mostly through down-regulation in iron deficiency and up-regulation during iron overload. Interestingly, many of these genes are also found to be up-regulated during infection, which may indicate a crosstalk between the known pathways of hepcidin regulation. These observations suggest the evolutionary conservation of the mechanisms of hepcidin regulation in teleost fish.
    Fish &amp Shellfish Immunology 12/2011; 31(6):1154-61. DOI:10.1016/j.fsi.2011.10.006 · 3.03 Impact Factor
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    ABSTRACT: The morphology, ultrastructure, genetics, and morphometrics of a species of Diplostomum von Nordmann 1832 (Digenea: Diplostomidae), isolated from the European flounder (Platichthys flesus (L.)) caught off the northwest coast of Portugal, are characterized. The metacercarial stage was found unencysted in the lens capsule of the eye. Light microscopical observations revealed the existence of some variability in specimen shape and size, with two morphotypes, referred to as "round" and "long", being apparent. Scanning electron microscopy revealed a smooth, unarmed tegument, with the lappet region being the most irregular and porose. Both the oral and ventral suckers were provided with a series of papillae, which presented very distinctive ultrastructural features and were particularly conspicuous in the case of the ventral sucker. The two morphotypes detected were found to have 100% genetic correspondence in the 18S + ITS1 + 5.8S region of the rDNA. Since the genetic data for this metacercaria differed from those of the species of Diplostomum available in GenBank, a description of a new genotype (accession number GQ370809) is provided. The molecular phylogenetic analyses, in conjunction with principal components and cluster analyses based on morphometric data, revealed the existence of consistent differences between the Diplostomum sp. metacercariae from flounder compared with Diplostomum spathaceum, Diplostomum mergi, Diplostomum pseudospathaceum, and Diplostomum paracaudum. The latter of these species was found to be the most similar to the present material. Our results do not support an evolutionary separation of the European and North American species of Diplostomum.
    Parasitology Research 05/2011; 110(1):81-93. DOI:10.1007/s00436-011-2453-x · 2.33 Impact Factor
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    ABSTRACT: Ammonia is a toxic by-product of amino acid catabolism and a common environmental pollutant that has been associated with increased disease susceptibility in fish although the mechanism is not well understood. We addressed the hypothesis that elevated environmental ammonia acts by impairing the acute phase response (APR). Specifically, we determined the impact of sub-lethal acute (24 h) and chronic (14 d) ammonia exposure on acute phase protein gene expression in zebrafish (Danio rerio) in response to a challenge with bacterial lipopolysaccharide (LPS: i.p. 10 μg/g after 24h). A panel of LPS-responsive genes (SAA, HAMP, LECT2, Hp and IL1β) were identified and evaluated by real-time quantitative PCR. Ammonia was found to impair induction of SAA, HAMP and LECT2 by 50-90%. Both short (15 min, 1h and 24h) and long-term (14 days) exposure to high environmental ammonia concentrations significantly elevated whole-body cortisol levels compared with control fish. Our results reveal for the first time that exposure to high environmental levels of ammonia suppresses the innate immune response in fish. We hypothesize that high environmental ammonia-mediated elevation of cortisol levels in zebrafish may be playing a key role in this immunosuppression, while the mechanisms involved remains to be elucidated.
    Developmental and comparative immunology 05/2011; 36(2):279-88. DOI:10.1016/j.dci.2011.04.008 · 3.71 Impact Factor
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    ABSTRACT: The SLC11A1/Nramp1 and SLC11A2/Nramp2 genes belong to the SLC11/Nramp family of transmembrane divalent metal transporters, with SLC11A1 being associated with resistance to pathogens and SLC11A2 involved in intestinal iron uptake and transferrin-bound iron transport. Both members of the SLC11 gene family have been clearly identified in tetrapods; however SLC11A1 has never been documented in teleost fish and is believed to have been lost in this lineage during early vertebrate evolution. In the present work we characterized the SLC11 genes in teleosts and evaluated if the roles attributed to mammalian SLC11 genes are assured by other fish specific SLC11 gene members. Two different SLC11 genes were isolated in the European sea bass (Dicentrarchus. labrax), and named slc11a2-α and slc11a2-β, since both were found to be evolutionary closer to tetrapods SLC11A2, through phylogenetic analysis and comparative genomics. Induction of slc11a2-α and slc11a2-β in sea bass, upon iron modulation or exposure to Photobacterium damselae spp. piscicida, was evaluated in in vivo or in vitro experimental models. Overall, slc11a2-α was found to respond only to iron deficiency in the intestine, whereas slc11a2-β was found to respond to iron overload and bacterial infection in several tissues and also in the leukocytes. Our data suggests that despite the absence of slc11a1, its functions have been undertaken by one of the slc11a2 duplicated paralogs in teleost fish in a case of synfunctionalization, being involved in both iron metabolism and response to bacterial infection. This study provides, to our knowledge, the first example of this type of sub-functionalization in iron metabolism genes, illustrating how conserving the various functions of the SLC11 gene family is of crucial evolutionary importance.
    BMC Evolutionary Biology 04/2011; 11:106. DOI:10.1186/1471-2148-11-106 · 3.41 Impact Factor
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    ABSTRACT: The present study describes the anatomy and surface topography of the metacercaria of Microphallusprimas (Jägerskiöld, 1909) infecting the shore crab Carcinus maenas (L.) in Aveiro estuary, northern Portugal. The metacercaria species identification resulted from the combined use of morphological and molecular data, particularly the 28S rDNA gene. The metacercariae encysted preferentially in the host's hepatopancreas and also in the gonads. Isolated cysts were present in two distinct forms, spherical and oval, and were shown to be the identical species by the internal transcribed spacer 1 (ITS1) sequence. Chemically excysted metacercariae were studied by light (LM) and scanning electron microscopy (SEM). Their specific characteristics observed include the particular aspect of the vesiculo-prostatic pouch surrounded by a very thin membrane, the presence of a prominent muscular papilla, and an obvious metraterm. The dorsal and ventral tegumental surfaces of the metacercaria were densely packed with similar squamous spines, which decreased in number and size towards the hindbody. The edges of the posterior and ventral face of the body were coated with numerous microvilli, whose function remains unknown. In order to identify the species of metacercariae, we compared a 28S partial rDNA sequence of the two forms of cysts with the same 28S partial region of M. primas available in GenBank. With this comparison, we determined that the sequences had a 100% similarity and therefore belonged to the same species, i.e., M. primas.
    Folia parasitologica 03/2011; 58(1):48-54. DOI:10.14411/fp.2011.005 · 1.21 Impact Factor
  • VIII International Symposium of Fish Parasites., Viña del Mar, Chile; 01/2011
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    ABSTRACT: The digenean trematode Diphtherostomum brusinae (Stossich, 1888) Stossich, 1903 presents a complex life cycle that may involve more than one intermediate host. The present study represents the first description of the metacercariae from D. brusinae infecting the labial palps of a new intermediate host, Mytilus galloprovincialis, in the Aveiro estuary, Portugal. The morphology of this parasitic stage was studied by light (LM) and scanning electron microscopy (SEM) and some differences were reported (body and sucker sizes, and spine distribution and shape). In this work, the 18S partial region of the ribosomal DNA was sequenced from D. brusinae metacercariae isolated from M. galloprovincialis collected in different localities of the Aveiro estuary. In addition, sequences from the same region of the 18S rDNA were obtained from D. brusinae cercariae and metacercariae, hosted by Nassarius reticulatus and Cerastoderma edule, respectively. No intraspecific polymorphism was detected in the 18S partial region, since there was 100% homology among all the sequences analysed. The same comparison was made for the ITS1, and we observed intraspecific polymorphism in this region. To our knowledge, this is the first report of D. brusinae metacercariae infecting the mussel M. galloprovincialis with support from morphological and molecular data.
    Journal of Helminthology 08/2010; 85(2):1-6. DOI:10.1017/S0022149X10000428 · 1.30 Impact Factor
  • J.V. Neves, I.A.S. Olsson, G. Porto, P.N. Rodrigues
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    ABSTRACT: Export Date: 18 November 2011, Source: Scopus
  • João V Neves, Jonathan M Wilson, Pedro N S Rodrigues
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    ABSTRACT: Iron is essential for growth and survival, but it is also toxic when in excess. Thus, there is a tight regulation of iron that is accomplished by the interaction of several genes including the iron transporter transferrin and iron storage protein ferritin. These genes are also known to be involved in response to infection. The aim of this study was to understand the role of transferrin and ferritin in infection and iron metabolism in fish. Thus, sea bass transferrin and ferritin H cDNAs were isolated from liver, cloned and characterized. Transferrin constitutive expression was found to be highest in the liver, but also with significant expression in the brain, particularly in the highly vascularized region connecting the inferior lobe of the hypothalamus and the saccus vasculosus. Ferritin, on the other hand, was expressed in all tested organs, but also significantly higher in the liver. Fish were subjected to either experimental bacterial infection or iron modulation and transferrin and ferritin mRNA expression levels were analyzed, along with several iron regulatory parameters. Transferrin expression was found to decrease in the liver and increase in the brain in response to infection and to increase in the liver in iron deficiency. Ferritin expression was found to inversely reflect transferrin in the liver, increasing in infection and iron overload and decreasing in iron deficiency, whereas in the brain, ferritin expression was also increased in infection. These findings demonstrate the evolutionary conservation of transferrin and ferritin dual functions in vertebrates, being involved in both the immune response and iron metabolism.
    Developmental and comparative immunology 08/2009; 33(7):848-57. DOI:10.1016/j.dci.2009.02.001 · 3.71 Impact Factor
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    ABSTRACT: Diphtherostomum brusinae was first recorded by the present study in the north of Portugal. Sporocysts, containing cercariae and encysted metacercariae, were observed in the gonads and digestive gland of the gastropod Nassarius reticulatus. Metacercariae were also found infecting the foot, mantle border and gills of the cockle Cerastoderma edule. The adult form was lodged in the rectum of the definitive host Diplodus sargus. The morphology of the three parasitic stages was studied by light (LM) and scanning electron microscopy (SEM). Despite the close similarity between cercaria and metacercaria, SEM data provided information that allowed their differentiation, namely the presence of a dense crown of microvilli around the oral cavity of the cercariae, which was absent in the metacercariae. In addition, the metacercariae presented a specific pre-acetabular rectangular band with conspicuous triangular spines. The adult showed characteristics of D. brusinae species, in particular the presence of acetabular lips, compact vitellaria and large elliptical eggs. Sequenced ITS1 data clearly demonstrated that the cercariae and metacercarial cysts from N. reticulatus, the cysts from C. edule and the adult isolated from D. sargus were life cycle stages that belonged to the same species, i.e. D. brusinae. Two transmission strategies in the life cycle of this species were observed: (1) cercariae encyst within the sporocysts of N. reticulatus and await ingestion by the definitive host; and (2) N. reticulatus naturally emits cercariae; they encyst in C. edule or the environment and are ingested by the definitive host.
    Journal of Helminthology 04/2009; 83(4):321-31. DOI:10.1017/S0022149X09250796 · 1.30 Impact Factor
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    ABSTRACT: PPAR isotypes have been previously identified in the teleost brown trout (Salmo trutta f. fario) and their organ distribution pattern established. Being that the liver is a vital metabolic organ presenting expression of all isotypes and also knowing that estrogens/estrogen receptors seem to interact with PPARs, we hypothesized that the latter may very well change seasonally. So, we studied the expression of these receptors in the liver, along the annual reproductive cycle and in both genders. According to real-time RT-PCR, PPARalpha mRNA expression in females was significantly higher in May and lower in September than in other seasons. No significant variation was observed along the year in males. A significant difference between genders occurred in May, when PPARalpha expression was higher for females. PPARbeta expression showed little variation along the reproductive cycle in females, but in males it was significantly higher in December than in the other seasons. No significant differences existed between genders. PPARgamma was more expressed in February than in September and December, for females. As to males, it was more expressed in February than in all other seasons. No significant differences were observed between genders. The study proved our hypothesis that PPARs gene expression varies along the year. Moreover, PPARalpha expression in females followed the same annual variation pattern as peroxisome volumes and enzyme activities, and an inverse pattern relatively to the salmonid type annual plasma estradiol levels. The data agrees with the idea that PPARalpha is under estradiol modulation and that cross-talk between this receptor and the estrogen receptor possibly exists.
    General and Comparative Endocrinology 03/2009; 161(1):146-52. DOI:10.1016/j.ygcen.2008.11.019 · 2.67 Impact Factor
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    ABSTRACT: The cercaria of Bucephalus minimus infects the digestive gland and gonads of its first intermediate host, the edible cockle, Cerastoderma edule. Light microscopy (LM) and scanning electron microscopy (SEM) of the cercaria showed a tail formed by a central stem, with 2 long contractile arms presenting distinct morphological surfaces. The encysted metacercaria naturally infected the flathead grey mullet, Mugil cephalus. The cysts found in the heart, liver, and spleen were shown to be identical by the internal transcribed spacer (ITS 1) sequence and morphological features and were associated with encapsulation, recruitment of cell infiltrates, and presence of melanomacrophages and adipose tissue. To establish the life cycle, we compared the ITS1 sequence in an adult from the known definitive host, Dicentrarchus labrax; encysted metacercariae from the liver, heart, and spleen of M. cephalus; and a cercaria from C. edule. With this comparison, we determined that they had a 100% similarity. Therefore, the ITSI sequence data clearly indicate that these 3 parasitic stages belong to the same species, i.e., B. minimus.
    Journal of Parasitology 09/2008; 95(2):353-9. DOI:10.1645/GE-1719.1 · 1.26 Impact Factor
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    ABSTRACT: Cercaria sevillana is the cercaria larval stage that infects the gonads and the digestive gland of its first intermediate host, Nassarius reticulatus. In this study the decapodous crustacean Carcinus maenas was used to determine if it would serve as second intermediate host in the parasite's life cycle. The latter hypothesis was based on the knowledge that C. maenas is the second intermediate host of several other digenean species. After dissection, it was possible to observe encysted metacercariae in the antennal glands of the green crab. After biochemical excystment, the metacercariae were processed for light and scanning electron microscopy. The morphological features observed led us to conclude that this species was a microphallid fluke, and it was identified as Gynaecotyla longiintestinata. To establish a possible relationship between C. sevillana and this metacercariae, the ITS1 region was analyzed. Thus, DNA was extracted from C. sevillana and from the cysts isolated from the antennal glands. The ITS1 region was amplified and sequenced, and the alignment clearly demonstrated that the cercaria and the metacercariae belonged to the same species, G. longiintestinata.
    Journal of Parasitology 05/2007; 93(2):318-22. DOI:10.1645/GE-836R1.1 · 1.26 Impact Factor
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    ABSTRACT: During the years 2004 and 2005 high mortalities in turbot Scophthalmus maximus (L.) from a fish farm in the north of Portugal were observed. Moribund fish showed darkening of the ventral skin, reddening of the fin bases and distended abdominal cavities caused by the accumulation of ascitic fluid. Ciliates were detected in fresh mounts from skin, gill and ascitic fluid. Histological examination revealed hyperplasia and necrosis of the gills, epidermis, dermis and muscular tissue. An inflammatory response was never observed. The ciliates were not identified to species level, but the morphological characteristics revealed by light and electronic scanning microscopes indicated that these ciliates belonged to the order Philasterida. To our knowledge this is the first report of the occurrence of epizootic disease outbreaks caused by scuticociliates in marine fish farms in Portugal.
    Diseases of Aquatic Organisms 04/2007; 74(3):249-53. DOI:10.3354/dao074249 · 1.59 Impact Factor
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    ABSTRACT: Hepatic iron overload in hemochomatosis patients can be highly variable but in general it develops in older patients. The purpose of this study was to compare development of iron load in of beta2m-/- and Hfe-/- mice paying special attention to liver pathology in older age groups. Liver iron content of beta2m-/-, Hfe-/- and control B6 mice of different ages (varying from 3 weeks to 18 months) was examined. Additional parameters (haematology indices, histopathology, lipid content and ferritin expression) were also studied in 18-month-old mice. The beta2m-/- strain presents higher hepatic iron content, hepatocyte nuclear iron inclusions, mitochondria abnormalities. In addition, hepatic steatosis was a common observation in this strain. In the liver of Hfe-/- mice, large mononuclear infiltrates positive for ferritin staining were commonly observed. The steatosis commonly observed the beta2m-/- mice may be a reflection of its higher hepatic iron content. The large hepatic mononuclear cell infiltrates seen in Hfe-/- stained for ferritin, may point to the iron sequestration capacity of lymphocytes and contribute to the clarification of the differences found in the progression of hepatic iron overload and steatosis in older animals from the two strains.
    International Journal of Experimental Pathology 09/2006; 87(4):317-24. DOI:10.1111/j.1365-2613.2006.00491.x · 2.05 Impact Factor
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    ABSTRACT: Background: Despite heavy alcohol consumption, only a low percentage of heavy drinkers develop liver disease. Imbalances in T-cell subsets and iron metabolism parameters are common findings in heavy drinkers, yet the possible role played by discrete T-lymphocyte subsets under heavy alcohol consumption remains unclear.Methods: To gain new insights into the possible role played by T lymphocytes during alcohol consumption, characterization of CD28 expression and TcR repertoire in peripheral blood CD4+ and CD8+ T cells by two and three-color flow cytometry was performed. A group of heavy alcohol drinkers (AHD, n= 71) and a group of age-matched controls (n= 81), both HLA-phenotyped and HFE-genotyped, constituted the groups under study.Results: Marked expansions of CD28− T cells within the CD8+ but not the CD4+ T-cell pool were observed in AHD compared with controls. These CD8+CD28− expansions were paralleled by expansions of CD8+ T cells bearing specific TcR Vα/β chains, namely Vβ5.2. Moreover, AHD, but not controls, carrying the H63D mutation in the HFE gene showed significantly higher percentages of CD28− T cells within the CD8+ T-cell pool than AHD carrying the normal HFE gene. Finally, high numbers of CD8+CD28− T cells in AHD were associated with lower levels of the liver-related enzymes ALT and GGT.Conclusions: This study showed that under active ethanol consumption, expansions of discrete CD8+ T-cell subsets occur within the CD8+ T-cell pool, that molecules of the MHC-class I locus seem to influence the extent of the expansions, and that high numbers of CD8+CD28− T cells are associated with low levels of liver enzymes in AHD.
    Alcoholism Clinical and Experimental Research 04/2006; 24(4):519 - 527. DOI:10.1111/j.1530-0277.2000.tb02020.x · 3.31 Impact Factor

Publication Stats

528 Citations
123.86 Total Impact Points

Institutions

  • 2005–2014
    • University of Porto
      • • Departamento de Biologia
      • • Instituto de Ciências Biomédicas Abel Salazar (ICBAS)
      Oporto, Porto, Portugal
  • 2012
    • Centro Nacional Patagonico
      Madryn, Chubut, Argentina
  • 2000–2011
    • Institute for Molecular and Cell Biology
      Oporto, Porto, Portugal
  • 2004
    • Instituto de Ciencias
      Santa Clara de Portugal, Michoacán, Mexico