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1.
Micromachines (Basel) ; 15(5)2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38793225

RESUMO

Microfluidic technology provides a solution to the challenge of continuous CaCO3 particle synthesis. In this study, we utilized a 3D-printed microfluidic chip to synthesize CaCO3 micro- and nanoparticles in vaterite form. Our primary focus was on investigating a continuous one-phase synthesis method tailored for the crystallization of these particles. By employing a combination of confocal and scanning electron microscopy, along with Raman spectroscopy, we were able to thoroughly evaluate the synthesis efficiency. This evaluation included aspects such as particle size distribution, morphology, and polymorph composition. The results unveiled the existence of two distinct synthesis regimes within the 3D-printed microfluidic chips, which featured a channel cross-section of 2 mm2. In the first regime, which was characterized by chaotic advection, particles with an average diameter of around 2 µm were produced, thereby displaying a broad size distribution. Conversely, the second regime, marked by diffusion mixing, led to the synthesis of submicron particles (approximately 800-900 nm in diameter) and even nanosized particles (70-80 nm). This research significantly contributes valuable insights to both the understanding and optimization of microfluidic synthesis processes, particularly in achieving the controlled production of submicron and nanoscale particles.

2.
Vaccines (Basel) ; 12(2)2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38400113

RESUMO

The emergence of SARS-CoV-2 mutant variants has posed a significant challenge to both the prevention and treatment of COVID-19 with anti-coronaviral neutralizing antibodies. The latest viral variants demonstrate pronounced resistance to the vast majority of human monoclonal antibodies raised against the ancestral Wuhan variant. Less is known about the susceptibility of the evolved virus to camelid nanobodies developed at the start of the pandemic. In this study, we compared nanobody repertoires raised in the same llama after immunization with Wuhan's RBD variant and after subsequent serial immunization with a variety of RBD variants, including that of SARS-CoV-1. We show that initial immunization induced highly potent nanobodies, which efficiently protected Syrian hamsters from infection with the ancestral Wuhan virus. These nanobodies, however, mostly lacked the activity against SARS-CoV-2 omicron-pseudotyped viruses. In contrast, serial immunization with different RBD variants resulted in the generation of nanobodies demonstrating a higher degree of somatic mutagenesis and a broad range of neutralization. Four nanobodies recognizing distinct epitopes were shown to potently neutralize a spectrum of omicron variants, including those of the XBB sublineage. Our data show that nanobodies broadly neutralizing SARS-CoV-2 variants may be readily induced by a serial variant RBD immunization.

3.
J Pers Med ; 12(6)2022 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-35743680

RESUMO

Immune evasion of SARS-CoV-2 undermines current strategies tocounteract the pandemic, with the efficacy of therapeutic virus-neutralizing monoclonal antibodies (nAbs) being affected the most. In this work, we asked whether two previously identified human cross-neutralizing nAbs, iB14 (class VH1-58) and iB20 (class VH3-53/66), are capable of neutralizing the recently emerged Omicron (BA.1) variant. Both nAbs were found to bind the Omicron RBD with a nanomolar affinity, yet they displayed contrasting functional features. When tested against Omicron, the neutralizing activity of iB14 was reduced 50-fold, whereas iB20 displayed a surprising increase in activity. Thus, iB20 is a unique representative of the VH3-53/66-class of nAbs in terms of breadth of neutralization, which establishes it as a candidate for COVID-19 therapy and prophylactics.

4.
Int J Mol Sci ; 23(4)2022 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-35216301

RESUMO

Despite the fact that a range of vaccines against COVID-19 have already been created and are used for mass vaccination, the development of effective, safe, technological, and affordable vaccines continues. We have designed a vaccine that combines the recombinant protein and DNA vaccine approaches in a self-assembled particle. The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 was conjugated to polyglucin:spermidine and mixed with DNA vaccine (pVAXrbd), which led to the formation of particles of combined coronavirus vaccine (CCV-RBD) that contain the DNA vaccine inside and RBD protein on the surface. CCV-RBD particles were characterized with gel filtration, electron microscopy, and biolayer interferometry. To investigate the immunogenicity of the combined vaccine and its components, mice were immunized with the DNA vaccine pVAXrbd or RBD protein as well as CCV-RBD particles. The highest antigen-specific IgG and neutralizing activity were induced by CCV-RBD, and the level of antibodies induced by DNA or RBD alone was significantly lower. The cellular immune response was detected only in the case of DNA or CCV-RBD vaccination. These results demonstrate that a combination of DNA vaccine and RBD protein in one construct synergistically increases the humoral response to RBD protein in mice.


Assuntos
Vacinas contra COVID-19/química , Vacinas contra COVID-19/farmacologia , Imunidade Humoral/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/química , Animais , Sítios de Ligação , Vacinas contra COVID-19/imunologia , Chlorocebus aethiops , Dextranos/química , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espermidina/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas de DNA/farmacologia , Células Vero
5.
Cells ; 10(12)2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34943946

RESUMO

FGF21 is a promising candidate for treating obesity, diabetes, and NAFLD; however, some of its pharmacological effects are sex-specific in mice with the Ay mutation that evokes melanocortin receptor 4 blockade, obesity, and hepatosteatosis. This suggests that the ability of FGF21 to correct melanocortin obesity may depend on sex. This study compares FGF21 action on food intake, locomotor activity, gene expression, metabolic characteristics, and liver state in obese Ay males and females. Ay mice were administered FGF21 for seven days, and metabolic parameters and gene expression in different tissues were assessed. Placebo-treated females were more obese than males and had lower levels of blood insulin and liver triglycerides, and higher expression of genes for insulin signaling in the liver, white adipose tissue (WAT) and muscles, and pro-inflammatory cytokines in the liver. FGF21 administration did not affect body weight, and increased food intake, locomotor activity, expression of Fgf21 and Ucp1 in brown fat and genes related to lipolysis and insulin action in WAT regardless of sex; however, it decreased hyperinsulinemia and hepatic lipid accumulation and increased muscle expression of Cpt1 and Irs1 only in males. Thus, FGF21's beneficial effects on metabolic disorders associated with melanocortin obesity are more pronounced in males.


Assuntos
Fígado Gorduroso/tratamento farmacológico , Fatores de Crescimento de Fibroblastos/farmacologia , Insulina/sangue , Fígado/metabolismo , Obesidade/tratamento farmacológico , Animais , Dieta Hiperlipídica , Metabolismo Energético/genética , Fígado Gorduroso/sangue , Feminino , Fatores de Crescimento de Fibroblastos/genética , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Resistência à Insulina/genética , Fígado/patologia , Masculino , Melanocortinas/toxicidade , Camundongos , Camundongos Obesos , Obesidade/sangue , Obesidade/induzido quimicamente , Obesidade/genética , Caracteres Sexuais , Triglicerídeos/metabolismo , Proteína Desacopladora 1/genética
6.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638898

RESUMO

The preference for high-calorie foods depends on sex and contributes to obesity development. Fibroblast growth factor 21 (FGF21) beneficially affects taste preferences and obesity, but its action has mainly been studied in males. The aim of this study was to compare the effects of FGF21 on food preferences and glucose and lipid metabolism in C57Bl/6J male and female mice with diet-induced obesity. Mice were injected with FGF21 or vehicle for 7 days. Body weight, choice between standard (SD) and high-fat (HFD) diets, blood parameters, and gene expression in white (WAT) and brown (BAT) adipose tissues, liver, muscles, and the hypothalamus were assessed. Compared to males, females had a greater preference for HFD; less WAT; lower levels of cholesterol, glucose, and insulin; and higher expression of Fgf21, Insr, Ppara, Pgc1, Acca and Accb in the liver and Dio2 in BAT. FGF21 administration decreased adiposity; blood levels of cholesterol, glucose, and insulin; hypothalamic Agrp expression, increased SD intake, decreased HFD intake independently of sex, and increased WAT expression of Pparg, Lpl and Lipe only in females. Thus, FGF21 administration beneficially affected mice of both sexes despite obesity-associated sex differences in metabolic characteristics, and it induced female-specific activation of gene expression in WAT.


Assuntos
Tecido Adiposo/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/administração & dosagem , Expressão Gênica/efeitos dos fármacos , Obesidade/metabolismo , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Colesterol/sangue , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/genética , Ácido Graxo Sintase Tipo I/genética , Feminino , Fatores de Crescimento de Fibroblastos/genética , Insulina/sangue , Masculino , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/etiologia , Obesidade/genética , PPAR alfa/genética , Piruvato Quinase/genética , Fatores Sexuais
7.
Biomedicines ; 9(10)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34680410

RESUMO

There is experimental evidence that chronic social defeat stress is accompanied by the development of an anxiety, development of a depression-like state, and downregulation of serotonergic genes in midbrain raphe nuclei of male mice. Our study was aimed at investigating the effects of chronic lithium chloride (LiCl) administration on anxiety behavior and the expression of serotonergic genes in midbrain raphe nuclei of the affected mice. A pronounced anxiety-like state in male mice was induced by chronic social defeat stress in daily agonistic interactions. After 6 days of this stress, defeated mice were chronically treated with saline or LiCl (100 mg/kg, i.p., 2 weeks) during the continuing agonistic interactions. Anxiety was assessed by behavioral tests. RT-PCR was used to determine Tph2, Htr1a, Htr5b, and Slc6a4 mRNA expression. The results revealed anxiolytic-like effects of LiCl on social communication in the partition test and anxiogenic-like effects in both elevated plus-maze and social interaction tests. Chronic LiCl treatment upregulated serotonergic genes in midbrain raphe nuclei. Thus, LiCl effects depend on the treatment mode, psycho-emotional state of the animal, and experimental context (tests). It is assumed that increased expression of serotonergic genes is accompanied by serotonergic system activation and, as a side effect, by higher anxiety.

8.
Cell Discov ; 7(1): 96, 2021 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-34667147

RESUMO

In the absence of virus-targeting small-molecule drugs approved for the treatment and prevention of COVID-19, broadening the repertoire of potent SARS-CoV-2-neutralizing antibodies represents an important area of research in response to the ongoing pandemic. Systematic analysis of such antibodies and their combinations can be particularly instrumental for identification of candidates that may prove resistant to the emerging viral escape variants. Here, we isolated a panel of 23 RBD-specific human monoclonal antibodies from the B cells of convalescent patients. A surprisingly large proportion of such antibodies displayed potent virus-neutralizing activity both in vitro and in vivo. Four of the isolated nAbs can be categorized as ultrapotent with an apparent IC100 below 16 ng/mL. We show that individual nAbs as well as dual combinations thereof retain activity against currently circulating SARS-CoV-2 variants of concern (such as B.1.1.7, B.1.351, B.1.617, and C.37), as well as against other viral variants. When used as a prophylactics or therapeutics, these nAbs could potently suppress viral replication and prevent lung pathology in SARS-CoV-2-infected hamsters. Our data contribute to the rational development of oligoclonal therapeutic nAb cocktails mitigating the risk of SARS-CoV-2 escape.

9.
Clin Transl Immunology ; 10(2): e1245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33552508

RESUMO

OBJECTIVES: To predict the spread of coronavirus disease (COVID-19), information regarding the immunological memory for disease-specific antigens is necessary. The possibility of reinfection, as well as the efficacy of vaccines for COVID-19 that are currently under development, will largely depend on the quality and longevity of immunological memory in patients. To elucidate the process of humoral immunity development, we analysed the generation of plasmablasts and virus receptor-binding domain (RBD)-specific memory B (Bmem) cells in patients during the acute phase of COVID-19. METHODS: The frequencies of RBD-binding plasmablasts and RBD-specific antibody-secreting cells (ASCs) in the peripheral blood samples collected from patients with COVID-19 were measured using flow cytometry and the ELISpot assay. RESULTS: The acute phase of COVID-19 was characterised by the transient appearance of total as well as RBD-binding plasmablasts. ELISpot analysis indicated that most patients exhibited a spontaneous secretion of RBD-specific ASCs in the circulation with good correlation between the IgG and IgM subsets. IL-21/CD40L stimulation of purified B cells induced the activation and proliferation of Bmem cells, which led to the generation of plasmablast phenotypic cells as well as RBD-specific ASCs. No correlation was observed between the frequency of Bmem cell-derived and spontaneous ASCs, suggesting that the two types of ASCs were weakly associated with each other. CONCLUSION: Our findings reveal that SARS-CoV-2-specific Bmem cells are generated during the acute phase of COVID-19. These findings can serve as a basis for further studies on the longevity of SARS-CoV-2-specific B-cell memory.

10.
Front Immunol ; 9: 1079, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29892283

RESUMO

The aim of this study was to fill important gaps in the evolutionary history of immunoglobulins by examining the structure and diversity of IgL genes in non-teleost ray-finned fish. First, based on the bioinformatic analysis of recent transcriptomic and genomic resources, we experimentally characterized the IgL genes in the chondrostean fish, Acipenser ruthenus (sterlet). We show that this species has three loci encoding IgL kappa-like chains with a translocon-type gene organization and a single VJC cluster, encoding homogeneous lambda-like light chain. In addition, sterlet possesses sigma-like VL and J-CL genes, which are transcribed separately and both encode protein products with cleavable leader peptides. The Acipenseriformes IgL dataset was extended by the sequences mined in the databases of species belonging to other non-teleost lineages of ray-finned fish: Holostei and Polypteriformes. Inclusion of these new data into phylogenetic analysis showed a clear subdivision of IgL chains into five groups. The isotype described previously as the teleostean IgL lambda turned out to be a kappa and lambda chain paralog that emerged before the radiation of ray-finned fish. We designate this isotype as lambda-2. The phylogeny also showed that sigma-2 IgL chains initially regarded as specific for cartilaginous fish are present in holosteans, polypterids, and even in turtles. We conclude that there were five ancient IgL isotypes, which evolved differentially in various lineages of jawed vertebrates.


Assuntos
Peixes/genética , Genes de Cadeia Leve de Imunoglobulina , Variação Genética , Isotipos de Imunoglobulinas/genética , Sequência de Aminoácidos , Animais , Peixes/classificação , Perfilação da Expressão Gênica , Loci Gênicos , Genoma , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Hibridização in Situ Fluorescente , Filogenia , Transcriptoma , Recombinação V(D)J
11.
Cytometry B Clin Cytom ; 94(4): 683-687, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29236355

RESUMO

BACKGROUND: Fc receptor-like A (FCRLA) is a unique member of a family of Fc receptor like-molecules that lacks a transmembrane region and is an ER-resident protein. In mice and humans, FCRLA has been known as a B cell specific protein. We report here that, in humans, FCRLA is also expressed in a subpopulation of plasmacytoid dendritic cells (pDCs). METHODS: Human peripheral blood mononuclear cells (PBMC), splenocytes, and tonsillar cells were stained for lineage markers followed by fixation/saponin permeabilization and intracellular staining for FCRLA, and then analyzed by flow cytometry with CD123 and CD303 used as pDC markers. RESULTS: We conducted an extensive flow cytometric analysis of a rare population of CD19-FCRLA+ cells found for the first time in human lymphoid tissues that we assigned to pDCs as they were lin-/CD123+/CD303+. FCRLA expression in human pDCs was further confirmed by the RT-PCR analysis of cDNA of pDCs isolated from the peripheral blood of a healthy donor. FCRLA-positive pDCs expressed a lower level of HLA-DR than their FCRLA-negative counterparts. CONCLUSIONS: FCRLA has long been viewed as a B cell specific protein, and this is the first time its expression has also been shown in human pDCs. © 2017 International Clinical Cytometry Society.


Assuntos
Células Dendríticas/imunologia , Receptores Imunológicos/biossíntese , Citometria de Fluxo , Humanos , Receptores Fc
12.
Monoclon Antib Immunodiagn Immunother ; 33(4): 209-14, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25170999

RESUMO

SLAMF9 is a member of the signaling lymphocyte-activating molecule (SLAM) immunoreceptor family. The SLAM family receptors are expressed in a broad range of immune cells and play an important role in immunity. To date, SLAMF9 is the least studied member of this family. Its ligand, signaling properties, and cells on whose surface it is expressed are unknown. We generated hybridoma clones 6E11 and 7G5 secreting monoclonal antibodies specific to human SLAMF9. BALB/c mice were immunized with Escherichia coli-expressed purified SLAMF9 protein; splenocytes from these mice were fused with mouse myeloma cell line NS-1. Based on isotyping of the MAbs, clone 6E11 was referred to the IgG1 subclass, while 7G5 to IgG2b. The specificity of these MAbs was assessed by ELISA, immunoblotting, immunohistochemistry, and flow cytometry. According to the results of epitope analysis, clone 6E11 reacts with the C2-like domain, whereas 7G5 is specific to the V-like domain of the SLAMF9 molecule. The generated MAbs were demonstrated to be applicable in various immunochemical analyses. They may be useful tools in studies clarifying the expression and function of human SLAMF9.


Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Animais , Especificidade de Anticorpos , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Terciária de Proteína/genética , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária
13.
Immunogenetics ; 63(10): 679-89, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21667045

RESUMO

We studied the evolution of the CD2 family in tetrapods by extracting and analyzing CD2-like genes from the genome of the amphibian species Silurana (Xenopus) tropicalis. An exhaustive analysis of the genomic and cDNA databases resulted in the identification of at least 70 CD2-like genes. The predicted receptors mostly maintain the typical VC2 ectodomains, but are highly diverse in their C-termini, which suggests a broad range of signaling capacities. Apart from the presumed monomeric receptors with ITSM and/or ITIM motifs, the Silurana family includes secreted proteins. Furthermore, a fraction of the receptors contain a conserved TM subtype with the NxxR motif that is known to promote an association with the FcRγ subunit and that was previously found in the members of the FcR- and KIR-related receptors. The expression analysis of a sample of the genes showed broad tissue distribution and gene-specific expression patterns. Phylogenetic analysis predicted that the CD58, CD150/SLAM, and SLAMF8 genes were maintained as single-copy genes in both mammals and amphibians, while others expanded/contracted in a lineage-specific manner.


Assuntos
Antígenos CD/genética , Antígenos CD2/genética , Receptores de Superfície Celular/genética , Xenopus/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD/classificação , Antígenos CD2/classificação , Evolução Molecular , Dados de Sequência Molecular , Filogenia , Receptores de Superfície Celular/classificação , Alinhamento de Sequência , Transdução de Sinais , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Xenopus/genética
14.
Immunol Lett ; 134(2): 174-82, 2011 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-20933011

RESUMO

FCRL6 receptor is a more recently identified representative of the FCRL family. We generated a panel of mouse mAbs to baculovirus-derived recombinant FCRL6 protein. The clone 7B2 was found to specifically recognize a 63kDa protein expressed preferentially on the surface of CD8 T and CD56 NK cells in human peripheral blood and spleen. The clone 7B2 reacts with FCRL6 in Western blotting, FACS, and immunohistochemistry. In the T cell lineage, FCRL6 functions in antigen-experienced cells. Mitogenic stimulation of PB leukocytes in vitro resulted in an abrogation of the FCRL6 gene expression. We found a significant decrease in the FCRL6 gene expression in peripheral T cells of patients with certain autoimmune and blood diseases, and its upregulation at the late stages of HIV infection. Study of the FCRL6 association with signaling molecules showed its ability to recruit SHP-1, SHP-2, SHIP-1, and SHIP-2 phosphatases, and also adaptor protein Grb2 through phosphorylated cytoplasmic tyrosines. The current results demonstrate inhibitory potential of FCRL6 and suggest its possible involvement in modulation of CTL effector functions in various immune disorders.


Assuntos
Proteínas de Transporte/imunologia , Regulação da Expressão Gênica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Processamento Alternativo , Sequência de Aminoácidos , Doenças Autoimunes/imunologia , Células Sanguíneas/citologia , Linfócitos T CD8-Positivos/imunologia , Doenças Hematológicas/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Matadoras Naturais/imunologia , Dados de Sequência Molecular , RNA Mensageiro/imunologia , Alinhamento de Sequência , Baço/citologia
15.
J Immunol Methods ; 332(1-2): 73-81, 2008 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-18241881

RESUMO

We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.


Assuntos
Fosfatase Alcalina/imunologia , Epitopos/imunologia , Receptores Fc/imunologia , Receptores Imunológicos/imunologia , Fosfatase Alcalina/genética , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Western Blotting , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Receptores Fc/genética , Receptores Imunológicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
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