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1.
Clin Immunol ; 183: 325-335, 2017 10.
Article in English | MEDLINE | ID: mdl-28943400

ABSTRACT

Functional immune responses are increasingly important for clinical studies, providing in depth biomarker information to assess immunotherapy or vaccination. Incorporating functional immune assays into routine clinical practice has remained limited due to challenges in standardizing sample preparation. We recently described the use of a whole blood syringe-based system, TruCulture®, which permits point-of-care standardized immune stimulation. Here, we report on a multi-center clinical study in seven FOCIS Centers of Excellence to directly compare TruCulture to conventional PBMC methods. Whole blood and PBMCs from healthy donors were exposed to LPS, anti-CD3 anti-CD28 antibodies, or media alone. 55 protein analytes were analyzed centrally by Luminex multi-analyte profiling in a CLIA-certified laboratory. TruCulture responses showed greater reproducibility and improved the statistical power for monitoring differential immune response activation. The use of TruCulture addresses a major unmet need through a robust and flexible method for immunomonitoring that can be reproducibly applied in multi-center clinical studies. ONE SENTENCE SUMMARY: A multi-center study revealed greater reproducibility from whole blood stimulation systems as compared to PBMC stimulation for studying induced immune responses.


Subject(s)
Antibodies/immunology , Cytokines/metabolism , Gene Expression Regulation/immunology , Immunologic Tests/instrumentation , Immunologic Tests/methods , Biomarkers/blood , Blood Donors , CD3 Complex/immunology , CD8 Antigens/immunology , Cytokines/genetics , Humans , Lipopolysaccharides/toxicity , Point-of-Care Systems
2.
Clin Oral Investig ; 21(7): 2157-2164, 2017 Sep.
Article in English | MEDLINE | ID: mdl-27834029

ABSTRACT

OBJECTIVES: Idiopathic Burning mouth syndrome (iBMS) is a poorly understood affection characterized by persistent pain in the oral cavity without any clinical or biological abnormality. Opiorphin is a natural inhibitor of enkephalin-inactivating ectopeptidases, mainly produced by salivary glands, that has demonstrated analgesic properties. The objective of the present case-control study was to test the hypothesis of a decrease in opiorphin levels in iBMS patients. MATERIALS AND METHODS: Twenty-one iBMS patients and 21 matched controls subjects were included between 2011 and 2013. Submandibular and sublingual salivary, blood, and urinary opiorphin levels of iBMS patients were compared to controls. RESULTS: Results are expressed as mean values ± SD and compared using the Wilcoxon Signed Rank test. Correlations were analyzed with Spearman coefficient. The level of significance was fixed at p < 0.05. Opiorphin levels in iBMS and controls were respectively (in ng/ml) in basal saliva: 37.8 ± 42.5 and 67.6 ± 188.9 (p = NS); stimulated saliva: 28.8 ± 25.3 and 31.1 ± 29.1 (p = NS); blood: 4.6 ± 5.4 and 1.9 ± 1.4 (p < 0.05); and urines: 68.5 ± 259.8 and 8.9 ± 6.2 (p = NS). CLINICAL RELEVANCE: In conclusion, the lack of significative difference in salivary opiorphin levels between iBMS and controls does not favor a direct local role for opiorphin in the etiopathogeny of iBMS. However, higher blood opiorphin levels may reflect a systemic dysregulation in iBMS. Trial registration NCT02686359 https://clinicaltrials.gov/ct2/show/NCT02686359.


Subject(s)
Burning Mouth Syndrome/metabolism , Oligopeptides/metabolism , Salivary Proteins and Peptides/metabolism , Biomarkers/metabolism , Burning Mouth Syndrome/psychology , Case-Control Studies , Chromatography, High Pressure Liquid , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged , Pain Measurement
3.
PLoS One ; 8(11): e80267, 2013.
Article in English | MEDLINE | ID: mdl-24278267

ABSTRACT

BACKGROUND: We evaluated a dipstick test for rapid detection of Shigella sonnei on bacterial colonies, directly on stools and from rectal swabs because in actual field situations, most pathologic specimens for diagnosis correspond to stool samples or rectal swabs. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. sonnei lipopolysaccharide (LPS) O-side chains using phase I-specific monoclonal antibodies coupled to gold particles, and displayed on a one-step immunochromatographic dipstick. A concentration as low as 5 ng/ml of LPS was detected in distilled water and in reconstituted stools in 6 minutes. This is the optimal time for lecture to avoid errors of interpretation. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 4 x 10(6) CFU/ml of S. sonnei. The specificity was 100% when tested with a battery of Shigella and different unrelated strains. When tested on 342 rectal swabs in Chile, specificity (281/295) was 95.3% (95% CI: 92.9% - 97.7%) and sensitivity (47/47) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 95.5 % of cases (328/342) in comparative studies. Positive and negative predictive values were 77% (95% CI: 65% - 86.5%) and 100% respectively. When tested on 219 stools in Chile, Vietnam, India and France, specificity (190/198) was 96% (95% CI 92%-98%) and sensitivity (21/21) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 96.3 % of cases (211/219) in comparative studies. Positive and negative predictive values were 72.4% (95% CI 56.1%-88.6%) and 100 %, respectively. CONCLUSION: This one-step dipstick test performed well for diagnosis of S. sonnei both on stools and on rectal swabs. These data confirm a preliminary study done in Chile.


Subject(s)
Diarrhea/diagnosis , Feces/microbiology , Rectum/microbiology , Shigella sonnei/pathogenicity , Diarrhea/microbiology , Humans , Reproducibility of Results , Sensitivity and Specificity , Shigella sonnei/isolation & purification
4.
Biopreserv Biobank ; 10(5): 454-61, 2012 Oct.
Article in English | MEDLINE | ID: mdl-24845047

ABSTRACT

BACKGROUND: The extended role of vascular endothelial growth factor (VEGF) in human pathophysiology led us to evaluate pre-analytical parameters possibly influencing its levels in peripheral blood and tissues. The effects on VEGF protein levels and mRNA expression were measured after storage delay (blood and tissue), use of different types of anticoagulants (blood), and after different numbers of freeze-thaw cycles (blood). METHODS: Blood from healthy donors was sampled simultaneously in ethylene diamine tetraacetic acid (EDTA), acid citrate dextrose (ACD-A), hirudin, and serum separation tubes. For each anticoagulant, VEGF was measured by enzyme-linked immunosorbent assay (ELISA) with different conditions of delay at 4°C before centrifugation (2 h, 4 h, or 48 h) and of different numbers of freeze-thaw cycles (1, 2, and 10). The transcripts coding for the VEGF165 isoform were quantified in peripheral blood mononuclear cells by RT-PCR. Muscle biopsy samples were frozen with delays of 15, 30, or 60 min after surgery. VEGF expression was quantified on immunofluorescence stained slides. RESULTS: The period of storage and the number of freeze-thaw cycles correlated with an increase in the levels of circulating VEGF (for each anticoagulant but not for serum) and its expression in PBMCs. VEGF expression measured from muscle biopsy sections was higher with freezing delays, with a peak at 30 and 60 min as compared to 15 min. CONCLUSIONS: The most reliable conditions for measuring both circulating VEGF and its gene expression are to reduce time between blood collection and centrifugation, and to avoid multiple freeze-thaw cycles. Serum collection tubes with no additive and no separator were less sensitive to the pre-analytical variations analyzed in this study. Freezing delay had a significant influence on VEGF protein expression in tissue samples.


Subject(s)
Enzyme-Linked Immunosorbent Assay , Leukocytes, Mononuclear/metabolism , Vascular Endothelial Growth Factor A/analysis , Anticoagulants/chemistry , Citric Acid/chemistry , Edetic Acid/chemistry , Freezing , Gene Expression , Glucose/analogs & derivatives , Glucose/chemistry , Hirudins/chemistry , Humans , Leukocytes, Mononuclear/cytology , Muscle, Skeletal/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Specimen Handling/instrumentation , Specimen Handling/methods , Temperature , Time Factors , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism
5.
PLoS One ; 6(10): e24830, 2011.
Article in English | MEDLINE | ID: mdl-21984895

ABSTRACT

BACKGROUND: We describe a test for rapid detection of S. dysenteriae 1 in bacterial cultures and in stools, at the bedside of patients. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. dysenteriae 1 lipopolysaccharide (LPS) using serotype 1-specific monoclonal antibodies coupled to gold particles and displayed on a one-step immunochromatographic dipstick. A concentration as low as 15 ng/ml of LPS was detected in distilled water and in reconstituted stools in 10 minutes. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 1.6×106 CFU/ml and 4.9×106 CFU/ml of S. dysenteriae 1, respectively. Optimal conditions to read the test have been determined to limit the risk of ambiguous results due to appearance of a faint yellow test band in some negative samples. The specificity was 100% when tested with a battery of Shigella and unrelated strains in culture. When tested on 328 clinical samples in India, Vietnam, Senegal and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the specificity (312/316) was 98.7% (95% CI:96.6-99.6%) and the sensitivity (11/12) was 91.7% (95% CI:59.8-99.6%). Stool cultures and the immunochromatographic test showed concordant results in 98.4 % of cases (323/328) in comparative studies. Positive and negative predictive values were 73.3% (95% CI:44.8-91.1%) and 99.7% (95% CI:98-100%). CONCLUSION: The initial findings presented here for a simple dipstick-based test to diagnose S. dysenteriae 1 demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys.


Subject(s)
Bacteriological Techniques/methods , Dysentery, Bacillary/diagnosis , Dysentery, Bacillary/microbiology , Feces/microbiology , Reagent Kits, Diagnostic , Shigella dysenteriae/isolation & purification , Adolescent , Adult , Animals , Child , Child, Preschool , Humans , India , Mice , Mice, Inbred BALB C , Middle Aged , Predictive Value of Tests , Reproducibility of Results , Time Factors , Young Adult
6.
Blood ; 112(6): 2520-8, 2008 Sep 15.
Article in English | MEDLINE | ID: mdl-18579796

ABSTRACT

The current paradigm in Plasmodium falciparum malaria pathogenesis states that young, ring-infected erythrocytes (rings) circulate in peripheral blood and that mature stages are sequestered in the vasculature, avoiding clearance by the spleen. Through ex vivo perfusion of human spleens, we examined the interaction of this unique blood-filtering organ with P falciparum-infected erythrocytes. As predicted, mature stages were retained. However, more than 50% of rings were also retained and accumulated upstream from endothelial sinus wall slits of the open, slow red pulp microcirculation. Ten percent of rings were retained at each spleen passage, a rate matching the proportion of blood flowing through the slow circulatory compartment established in parallel using spleen contrast-enhanced ultrasonography in healthy volunteers. Rings displayed a mildly but significantly reduced elongation index, consistent with a retention process, due to their altered mechanical properties. This raises the new paradigm of a heterogeneous ring population, the less deformable subset being retained in the spleen, thereby reducing the parasite biomass that will sequester in vital organs, influencing the risk of severe complications, such as cerebral malaria or severe anemia. Cryptic ring retention uncovers a new role for the spleen in the control of parasite density, opening novel intervention opportunities.


Subject(s)
Erythrocytes/parasitology , Microcirculation/parasitology , Plasmodium falciparum , Spleen/blood supply , Animals , Blood Flow Velocity , Humans , In Vitro Techniques , Perfusion , Regional Blood Flow , Spleen/parasitology
7.
J Gen Virol ; 87(Pt 12): 3587-3598, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17098974

ABSTRACT

As a tool for the identification and/or purification of hepatitis C virus (HCV)-infected cells, a chimeric form of the Gal4VP16 transcription factor was engineered to be activated only in the presence of the HCV NS3/4A protease and to induce different reporter genes [choramphenical acetyltransferase (CAT), green fluorescent protein (GFP) and the cell-surface marker H-2K(k)] through the (Gal4)(5)-E1b promoter. For this, the NS5A/5B trans-cleavage motif of HCV of genotype 1a was inserted between Gal4VP16 and the N terminus of the endoplasmic reticulum (ER)-resident protein PERK, and it was demonstrated that it could be cleaved specifically by NS3/4A. Accordingly, transient transfection in tetracycline-inducible UHCV-11 cells expressing the HCV polyprotein of genotype 1a revealed the migration of the Gal4VP16 moiety of the chimera from the ER to the nucleus upon HCV expression. Activation of the chimera provoked specific gene induction, as shown by CAT assay, first in UHCV-11 cells and then in Huh-7 cells expressing an HCV replicon of genotype 1b (Huh-7 Rep). In addition, the GFP reporter gene allowed rapid fluorescence monitoring of HCV expression in the Huh-7 Rep cells. Finally, the chimera was introduced into Huh-7.5 cells infected with cell culture-generated HCV JFH1 (genotype 2a), allowing the purification of the HCV-infected cells by immunomagnetic cell sorting using H-2K(k) as gene reporter. In conclusion, the Gal4VP16 chimera activation system can be used for the rapid identification and purification of HCV-infected cells.


Subject(s)
Carrier Proteins/metabolism , Hepacivirus/physiology , Immunomagnetic Separation/methods , Viral Nonstructural Proteins/metabolism , Viral Proteins/metabolism , Carrier Proteins/genetics , Cell Line , Cell Nucleus/chemistry , Chloramphenicol O-Acetyltransferase/biosynthesis , Chloramphenicol O-Acetyltransferase/genetics , Endoplasmic Reticulum/chemistry , Genes, Reporter , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , H-2 Antigens/biosynthesis , Hepacivirus/genetics , Humans , Intracellular Signaling Peptides and Proteins , Membrane Proteins/genetics , Molecular Chaperones/genetics , Recombinant Fusion Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Viral Nonstructural Proteins/genetics , Viral Proteins/genetics
8.
Cell Signal ; 18(11): 1865-75, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16600570

ABSTRACT

The interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) has been shown to activate NF-kappaB independently of its kinase function after interaction with the IKK complex. In order to investigate the mechanism of NF-kappaB activation by PKR, we identified the domain of PKR responsible for stimulating the NF-kappaB pathway in PKR-deficient fibroblasts using an NF-kappaB dependent reporter assay. The N-terminal 1-265 AA of PKR activates NF-kappaB, whereas the 1-180 AA N-terminus restricted to the two dsRNA Binding Domains (DRBD), the third basic domain alone (AA 181-265), or the C-terminus of PKR (AA 266-550) were unable to stimulate the expression of the NF-kappaB dependent reporter gene. Using confocal microscopy, we confirmed that PKR full length as well as PKR N-terminus colocalized with IKKbeta. By GST-pulldown analysis, using different PKR domains, we then revealed the specific ability of the PKR N-terminus 1-265 to bind to and activate IKK and showed that this activation requires the integrity of the IKK complex. This activation is not only due to DRBDs since the DRBD fragment 1-180 failed to inhibit PKR 1-265 induced NF-kappaB activation. Our results therefore demonstrate that the ability of PKR to mediate NF-kappaB activation resides in its full N-terminus, and requires both DRBDs and the third basic domain.


Subject(s)
I-kappa B Kinase/metabolism , NF-kappa B/metabolism , Signal Transduction , eIF-2 Kinase/metabolism , Animals , Base Sequence , Cells, Cultured , Gene Transfer Techniques , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Molecular Sequence Data , Multiprotein Complexes/metabolism , Protein Structure, Tertiary/physiology , Up-Regulation , eIF-2 Kinase/genetics
9.
Blood ; 107(9): 3745-52, 2006 May 01.
Article in English | MEDLINE | ID: mdl-16384927

ABSTRACT

The spleen plays a central role in the pathophysiology of several potentially severe diseases such as inherited red cell membrane disorders, hemolytic anemias, and malaria. Research on these diseases is hampered by ethical constraints that limit human spleen tissue explorations. We identified a surgical situation--left splenopancreatectomy for benign pancreas tumors--allowing spleen retrieval at no risk for patients. Ex vivo perfusion of retrieved intact spleens for 4 to 6 hours maintained a preserved parenchymal structure, vascular flow, and metabolic activity. Function preservation was assessed by testing the ability of isolated-perfused spleens to retain Plasmodium falciparum-infected erythrocytes preexposed to the antimalarial drug artesunate (Art-iRBCs). More than 95% of Art-iRBCs were cleared from the perfusate in 2 hours. At each transit through isolated-perfused spleens, parasite remnants were removed from 0.2% to 0.23% of Art-iRBCs, a proportion consistent with the 0.02% to 1% pitting rate previously established in artesunate-treated patients. Histologic analysis showed that more than 90% of Art-iRBCs were retained and processed in the red pulp, providing the first direct evidence of a zone-dependent parasite clearance by the human spleen. Human-specific physiologic or pathophysiologic mechanisms involving clearing or processing functions of the spleen can now be experimentally explored in a human tissue context.


Subject(s)
Spleen/physiology , Animals , Antimalarials/pharmacology , Artemisinins/pharmacology , Artesunate , Erythrocytes/drug effects , Erythrocytes/parasitology , Humans , In Vitro Techniques , Malaria/parasitology , Malaria/physiopathology , Perfusion , Phagocytosis , Plasmodium falciparum/drug effects , Plasmodium falciparum/pathogenicity , Sesquiterpenes/pharmacology , Spleen/blood supply , Spleen/immunology , Spleen/parasitology
10.
J Virol ; 79(7): 3969-78, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15767399

ABSTRACT

Interferon (IFN) is one important effector of the innate immune response, induced by different viral or bacterial components through Toll-like receptor (TLR)-dependent and -independent mechanisms. As part of its pathogenic strategy, hepatitis C virus (HCV) interferes with the innate immune response and induction of IFN-beta via the HCV NS3/4A protease activity which inhibits phosphorylation of IRF-3, a key transcriptional regulator of the IFN response. In the present study, we demonstrate that inhibition by the protease occurs upstream of the noncanonical IKK-related kinases IKKepsilon and TBK-1, which phosphorylate IRF-3, through partial inhibition of the TLR adapter protein TRIF/TICAM1-dependent pathway. Use of TRIF(-/-) mouse embryo fibroblasts however revealed the presence of a TRIF-independent pathway involved in IFN induction that was also inhibited by NS3/4A. Importantly, we show that NS3/4A can strongly inhibit the ability of the recently described RIG-I protein to activate IFN, suggesting that RIG-I is a key factor in the TRIF-independent, NS3/4A-sensitive pathway. Expression of IFN signaling components including IKKepsilon, TBK-1, TRIF, and wild type or constitutively active forms of RIG-I in the HCV replicon cells resulted in IFN-beta promoter transactivation, with IKKepsilon displaying the highest efficiency. Subsequently, overexpression of IKKepsilon resulted in 80% inhibition of both the positive and negative replicative strands of the HCV replicon. The partial restoration of the capacity of the host cell to transcribe IFN-beta indicates that IKKepsilon expression is able to bypass the HCV-mediated inhibition and restore the innate antiviral response.


Subject(s)
Hepacivirus/immunology , Hepacivirus/physiology , Interferon-beta/metabolism , Protein Serine-Threonine Kinases/physiology , RNA Helicases/metabolism , Signal Transduction , Adaptor Proteins, Vesicular Transport/antagonists & inhibitors , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Cell Line , Cell Line, Tumor , DEAD Box Protein 58 , DEAD-box RNA Helicases , DNA-Binding Proteins/metabolism , Electrophoretic Mobility Shift Assay , Gene Expression Regulation , Humans , I-kappa B Kinase , Immunity, Innate/immunology , Interferon Regulatory Factor-3 , Membrane Glycoproteins/immunology , Mice , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/analysis , RNA, Messenger/isolation & purification , Receptors, Cell Surface/immunology , Receptors, Immunologic , Replicon , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptors , Transcription Factors/metabolism
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