Dataset Information

Non-heat inactivated autologous serum increases accuracy of in vitro CFSE lymphocyte proliferation test (LPT) for nickel.

ABSTRACT:

Background

Skin patch testing is still seen as the gold standard for the diagnosis of allergic hypersensitivity. For several metals and for patients with a suspected adverse reaction to their medical device implant material, patch testing can be unreliable. The current alternative to metal allergy patch testing is the in vitro lymphocyte proliferation test (LPT) using tritiated thymidine. This method is well-established but requires handling of radioactive material, often uses heat-inactivated allogenic human pooled serum and cannot determine T cell subsets.

Objective

To develop a radioactive free LPT by using carboxyfluorescein succinimidyl ester (CFSE) and to evaluate the influence of serum source (heat-inactivated human pooled serum [HI HPS] vs autologous serum) on the sensitivity and specificity of the nickel-specific LPT.

Methods

Peripheral blood mononuclear cells derived from nickel-allergic patients and healthy controls were collected, labelled with CFSE and cultured in medium containing 10% HI HPS or 10% autologous serum with or without additional T cell skewing cytokine cocktails (Th1: IL-7/IL-12, Th2: IL-7/IL-4 or Th17: IL-7/IL-23/IL-1?) in the absence or presence of NiSO₄ . The stimulation index (SI) was calculated as the ratio of divided cells, that is the percentage of CFSE^low/neg CD3⁺ CD4⁺ T-lymphocytes upon nickel stimulation compared to the percentage of CFSE^low/neg CD3⁺ CD4⁺ T-lymphocytes without antigen. These results were compared with the history of Ni allergy, patch test results and the MELISA test.

Results

Autologous serum positively influenced Ni-specific proliferation while HI HPS negatively influenced Ni-specific proliferation. The test protocol analysing CD4⁺ cells and autologous serum without skewing cytokines scored the best diagnostic values (sensitivity 95%; specificity 93%; and overall accuracy 94%) compared to the parallel test using HI HPS (accuracy 60%). Cytokine supplements did not further improve the test protocol which used autologous serum. The protocol using HI HPS could be further improved by addition of the cytokine skewing cocktails.

Conclusions

Here, we describe an optimized and highly accurate flow cytometric LPT which comprises of CFSE-labelled cells cultured in autologous serum (not heat inactivated) and without the presence of T cell skewing cytokines.

Clinical relevance

The sensitivity and specificity of LPT is enhanced, compared to HI HPS, when autologous serum without skewing cytokines is used.

SUBMITTER: de Graaf NPJ

PROVIDER: S-EPMC7317482 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Non-heat inactivated autologous serum increases accuracy of in vitro CFSE lymphocyte proliferation test (LPT) for nickel.

de Graaf Niels P J NPJ Bontkes Hetty J HJ Roffel Sanne S Kleverlaan Cornelis J CJ Rustemeyer Thomas T Gibbs Sue S Feilzer Albert J AJ

Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 20200414 6

<h4>Background</h4>Skin patch testing is still seen as the gold standard for the diagnosis of allergic hypersensitivity. For several metals and for patients with a suspected adverse reaction to their medical device implant material, patch testing can be unreliable. The current alternative to metal allergy patch testing is the in vitro lymphocyte proliferation test (LPT) using tritiated thymidine. This method is well-established but requires handling of radioactive material, often uses heat-inact ...[more]

PMID: 32215995

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Project description:Transcriptional profiling of the digestive gland tissue of female mussel Mytilus galloprovincialis exposed to nickel along with a temperature gradient Background: The exposure of marine organisms to stressing agents may affect the level and pattern of gene expression. Although many studies have examined the ecological effects of heat stress on mussels, little is known about the physiological mechanisms that might be affected by co-exposure to heat stress and environmental contaminants such as nickel (Ni). In the present work we investigated the effects of simultaneous changes in temperature and Ni supply on lysosomal membrane stability (LMS) and malondialdehyde accumulation (MDA) in the digestive gland (DG) of the blue mussel Mytilus galloprovincialis (Lam.). To shed some light into how the molecular response to environmental stressors is modulated, we employed a cDNA microarray with 1,673 sequences to measure the relative transcript abundances in the DG of mussels exposed to Ni along with the temperature increase. Temperature and Ni rendered additive effects on LMS and MDA accumulation, increasing the toxic effects of metal cations. Ni loads in DG tissues was also affected by co-exposure to 26°C. In animals exposed only to heat stress, functional genomics analysis of the microarray data (171 DEGs) revealed 7 biological processes, largely dominated by the up-regulation of folding protein-related genes, and the down regulation of genes involved in cell migration and cellular component assembly. Exposure to Ni at 18°C and 26°C rendered respectively 188 and 262 DEGs showing distinct pattern in term of biological processes. In particular, the response of mussels exposed to Ni at 26°C was characterized by the up regulation of proteolysis, ribosome biogenesis, response to unfolded proteins and catabolic-related genes as well as the down-regulation of genes encoding cellular metabolic processes. Our data provide new insights on the transcriptomic response in mussels challenging temperature increases and Ni exposure and should be carefully considered in view of the biological effects of heat stress and particularly in polluted areas.

Project description:Transcriptional profiling of the digestive gland tissue of female mussel Mytilus galloprovincialis exposed to nickel along with a temperature gradient Background: The exposure of marine organisms to stressing agents may affect the level and pattern of gene expression. Although many studies have examined the ecological effects of heat stress on mussels, little is known about the physiological mechanisms that might be affected by co-exposure to heat stress and environmental contaminants such as nickel (Ni). In the present work we investigated the effects of simultaneous changes in temperature and Ni supply on lysosomal membrane stability (LMS) and malondialdehyde accumulation (MDA) in the digestive gland (DG) of the blue mussel Mytilus galloprovincialis (Lam.). To shed some light into how the molecular response to environmental stressors is modulated, we employed a cDNA microarray with 1,673 sequences to measure the relative transcript abundances in the DG of mussels exposed to Ni along with the temperature increase. Temperature and Ni rendered additive effects on LMS and MDA accumulation, increasing the toxic effects of metal cations. Ni loads in DG tissues was also affected by co-exposure to 26M-BM-0C. In animals exposed only to heat stress, functional genomics analysis of the microarray data (171 DEGs) revealed 7 biological processes, largely dominated by the up-regulation of folding protein-related genes, and the down regulation of genes involved in cell migration and cellular component assembly. Exposure to Ni at 18M-BM-0C and 26M-BM-0C rendered respectively 188 and 262 DEGs showing distinct pattern in term of biological processes. In particular, the response of mussels exposed to Ni at 26M-BM-0C was characterized by the up regulation of proteolysis, ribosome biogenesis, response to unfolded proteins and catabolic-related genes as well as the down-regulation of genes encoding cellular metabolic processes. Our data provide new insights on the transcriptomic response in mussels challenging temperature increases and Ni exposure and should be carefully considered in view of the biological effects of heat stress and particularly in polluted areas. Digestive gland tissue from individual animals in different experimental conditions were analyzed in a complete loop design. Dual color competitive hybridizations (Control M-bM-^@M-^\16M-BM-0CM-bM-^@M-^] vs M-bM-^@M-^\20M-BM-0CM-bM-^@M-^] vs M-bM-^@M-^\24M-BM-0CM-bM-^@M-^] vs M-bM-^@M-^\16M-BM-0C+CuM-bM-^@M-^], M-bM-^@M-^\20M-BM-0CM-bM-^@M-^] vs M-bM-^@M-^\24M-BM-0CM-bM-^@M-^] vs M-bM-^@M-^\20M-BM-0C+CuM-bM-^@M-^], M-bM-^@M-^\24M-BM-0CM-bM-^@M-^] vs M-bM-^@M-^\24M-BM-0C+CuM-bM-^@M-^], M-bM-^@M-^\16M-BM-0C+CuM-bM-^@M-^] vsM-bM-^@M-^]20M-BM-0C+CuM-bM-^@M-^] vs M-bM-^@M-^\24M-BM-0C+CuM-bM-^@M-^]; M-bM-^@M-^\20M-BM-0C+CuM-bM-^@M-^] vs M-bM-^@M-^\24M-BM-0C+CuM-bM-^@M-^]) including label swap. Single individuals. Four biological replicates. One replicate per array.

Dataset Information

Non-heat inactivated autologous serum increases accuracy of in vitro CFSE lymphocyte proliferation test (LPT) for nickel.

Background

Objective

Methods

Results

Conclusions

Clinical relevance

Publications

Non-heat inactivated autologous serum increases accuracy of in vitro CFSE lymphocyte proliferation test (LPT) for nickel.

Similar Datasets

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