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1.
Exp Parasitol ; 130(1): 63-8, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22019410

ABSTRACT

Toxoplasma gondii is an ubiquitous intracellular parasite, causative agent of toxoplasmosis, and a worldwide zoonosis for which an effective vaccine is needed. A group of proteins secreted by tachyzoites during host-cell invasion was isolated from the interaction medium. It induced the permeability of the cells as assessed by alpha-sarcin and consequently facilitated the entry of the parasite into the cells. SDS-PAGE of the purified proteins showed a pattern of four proteins of 67, 42, 32 and 27 kDa. MRC-5 cells incubated with the total protein and the different electroeluted bands endured a high cellular death in presence of alpha-sarcin. BALb/C mice immunized with the group of proteins had a mixed Th1/Th2 response and were protected upon challenge with the parasites.


Subject(s)
Antibodies, Protozoan/biosynthesis , Protozoan Proteins/immunology , Toxoplasma/immunology , Toxoplasma/isolation & purification , Animals , Antibodies, Protozoan/blood , CD4-CD8 Ratio , Cell Line , Cytokines/biosynthesis , Cytokines/blood , Electrophoresis, Polyacrylamide Gel , Female , Humans , Immunoglobulin G/biosynthesis , Immunoglobulin G/blood , Mice , Mice, Inbred BALB C
2.
PLoS One ; 6(3): e17818, 2011 Mar 11.
Article in English | MEDLINE | ID: mdl-21412439

ABSTRACT

Each year, during winter months, human Metapneumovirus (hMPV) is associated with epidemics of bronchiolitis resulting in the hospitalization of many infants. Bronchiolitis is an acute illness of the lower respiratory tract with a consequent inflammation of the bronchioles. The rapid onset of inflammation suggests the innate immune response may have a role to play in the pathogenesis of this hMPV infection. Since, the matrix protein is one of the most abundant proteins in the Paramyxoviridae family virion, we hypothesized that the inflammatory modulation observed in hMPV infected patients may be partly associated with the matrix protein (M-hMPV) response. By western blot analysis, we detected a soluble form of M-hMPV released from hMPV infected cell as well as from M-hMPV transfected HEK 293T cells suggesting that M-hMPV may be directly in contact with antigen presenting cells (APCs) during the course of infection. Moreover, flow cytometry and confocal microscopy allowed determining that M-hMPV was taken up by dendritic cells (moDCs) and macrophages inducing their activation. Furthermore, these moDCs enter into a maturation process inducing the secretion of a broad range of inflammatory cytokines when exposed to M-hMPV. Additionally, M-hMPV activated DCs were shown to stimulate IL-2 and IFN-γ production by allogeneic T lymphocytes. This M-hMPV-mediated activation and antigen presentation of APCs may in part explain the marked inflammatory immune response observed in pathology induced by hMPV in patients.


Subject(s)
Immunity/immunology , Inflammation/immunology , Metapneumovirus/immunology , Viral Matrix Proteins/immunology , Antigen Presentation/immunology , Antigen-Presenting Cells/metabolism , Antigens, CD1/metabolism , Apoptosis , Cell Differentiation/immunology , Dendritic Cells/cytology , Dendritic Cells/immunology , Dendritic Cells/virology , Endocytosis , HEK293 Cells , Humans , Inflammation Mediators/metabolism , Interferons/biosynthesis , Lipopolysaccharide Receptors/metabolism , Lymphocyte Activation/immunology , Monocytes/cytology , Protein Binding , Subcellular Fractions/immunology , Subcellular Fractions/virology , T-Lymphocytes/immunology , Transfection
3.
Clin Vaccine Immunol ; 13(3): 409-14, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16522785

ABSTRACT

To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any cross-reactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV.


Subject(s)
Antibodies, Viral/analysis , Blotting, Western/methods , Membrane Glycoproteins/immunology , Nucleocapsid Proteins/immunology , Protein Subunits/immunology , Recombinant Proteins/immunology , Severe acute respiratory syndrome-related coronavirus/immunology , Viral Envelope Proteins/immunology , Antibodies, Viral/biosynthesis , Coronavirus Nucleocapsid Proteins , False Positive Reactions , Humans , Nucleocapsid Proteins/biosynthesis , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/isolation & purification , Protein Subunits/biosynthesis , Protein Subunits/genetics , Protein Subunits/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus
4.
Basic Clin Pharmacol Toxicol ; 96(1): 60-5, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15667597

ABSTRACT

The anti-proliferative action of three alkyl-lysophospholipid derivatives, edelfosine (ET-OCH), miltefosine (Hexadecylphosphocholine), and ilmofosine (BM 14.440) has been studied on the promastigotes and amastigotes of Leishmania donovani. The effect of the three drugs has previously been studied, but the action mode was not clearly elucidated. In this study the effect on the intracellular amastigote forms was evaluated by two different methods: the traditional method, counting the amastigotes within the macrophages stained with Giemsa; and by a new method, staining the nuclear macrophages and amastigotes with ethidium bromide and counting the different population by flow cytometry. This new method, based on the flow cytometry, shows an advantage for evaluating the anti-proliferative effects in intracellular parasites. The ED50 were calculated for the drug activity after 72 hr, and for the three alkyl-lysophospholipid derivatives it were in the range of 26.73-33.31 microM against promastigotes and in the range of 16.46-23.16 against amastigotes. Also, studying the effect against macrophages J774A1, the ED50 were in the range of 24.28-26.38 microM. The effect of the alkyl-lysophospholipids in the macromolecular biosynthesis of the Leishmania donovani, was studied comparing the incorporation of labelled analogues ([3H] thymidine, [3H] uridine and [3H] leucine), respectively, in the DNA, RNA, and proteins of the flagellates treated. Miltefosine was the most active of the alkyl-lysophospholipids, especially in the inhibition of the RNA synthesis. The three compounds studied show high in vitro activity against L. donovani promastigotes and amastigotes.


Subject(s)
Antiprotozoal Agents/pharmacology , Leishmania donovani/drug effects , Phospholipid Ethers/pharmacology , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/pharmacology , Animals , Cell Line , DNA, Protozoan/biosynthesis , Flow Cytometry , Mice , Protozoan Proteins/biosynthesis , RNA, Protozoan/biosynthesis
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