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The Primary Lymphocyte Culture in the Diagnosis of Drug- and Metal-Induced Allergy
Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Drugs and metals are examples of xenobiotics that can induce hypersensitivity in humans. These adverse reactions are classified as allergy if repeated exposure leads to the same type of clinical manifestation. Together with the clinical history, the skin test is the most commonly used test for the diagnosis of allergic disease. However, in vivo testing per se has drawbacks such as the risk of potentiation of the allergy or even sensitisation to a given test substance. For this reason in vitro testing is an attractive diagnostic alternative since it does not involve any exposure of the test subject to the allergen.

The lymphocyte transformation test (LTT) has been used to complement the diagnosis of allergy to drugs and metals for more than thirty years. The principle behind this test is to show the presence of allergen-specific memory lymphocytes in peripheral blood, which is a sine qua non of a true allergy. LTT reveals the proliferation of such cells by showing DNA synthesis as the uptake of 3H-thymidine in primary PBMC (peripheral blood mononuclear cell) cultures treated with the allergen. However, LIT has not yet been generally accepted as a stand-alone test in the diagnosis of allergy. One reason for this is that different chemical properties of the allergens may lead to either false positive or false negative LTT responses.

In the present study we investigated allergy to the drug bacampicillin and to the metals Au, Pd, Ni and Hg. Three different protocols for LTT: LIT in micro cultures (LTT-micro), LTT in macro cultures (LTT-macro) and memory lymphocyte immunostimulation assay (MELISA) were compared using a skin test or clinical history as reference methods. LTI showed a sensitivity of 87% and a specificity of 90% when used in the diagnosis of allergy to bacampicillin. When allergy to Au, Pd, Ni and Hg was investigated, the sensitivity was 33- 95% and the specificity 0-79%. There were no significant differences between the test protocols, except that MELISA showed a significantly higher specificity than LTT-micro and LTT-macro when Hg2+ was used as antigen. Even so, this specificity was only 70%, which would result in 30 of 100 healthy subjects receiving a false diagnosis of Hg allergy when using the MELISA protocol. Ni2+ also induced high numbers of false-positive LTI responses, 77-85% patch-test negative subjects showed positive results to these metals. However, group comparisons showed a significantly higher proliferation intensity in allergic than in nonallergic groups for all allergens except Hg2+. Furthermore, only 56% of patients with verified allergy to mercury showed a positive MELISA, a sensitivity that is unacceptably low.

Following these findings, we investigated whether other endpoints than DNA synthesis could be used to discriminate allergic from healthy subjects, using primary PBMC cultures with Hg2+ or Ni2+ as a model system. Analysis of the T-cell receptor Vß profiles of lymphoblasts induced by these metal ions showed individual patterns, and there was no difference between healthy and allergic groups. However, the fraction of CD4+/Vß2+ cells correlated significantly with the proliferation intensity induced by Hg2+ in patients with a verified Hg allergy but not in non-allergic controls. Interestingly, such a correlation was not seen with CD8+/Nß2+ cells. This indicates that Hg2+ does not function as a superantigen, since classical superantigens also stimulate CD8+ lymphocytes. When Ni2+ was used as antigen we found significantly higher IL-10 production in allergic than in non-allergic subjects, despite no significant difference in proliferation intensity between these two groups.

In conclusion, the LTT test is useful for the diagnosis of allergy to bacampicillin. Regarding Au, Pd and Ni the LIT has low validity and can only be used to discriminate groups of allergic from non-allergic individuals. LTT with Hg2+ and Ni2+ is not useful for the diagnosis of allergy to these metals since a high fraction of non-allergic individuals show positive results, irrespective of the test protocol used. This thesis calls for further studies on the usefulness of in vitro IL-10 production for the diagnosis of Ni allergy as well as on the specificity of in vitro induced CD4+N~2+ lymphoblasts from Hg allergic subjects.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2000. , 69 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 635
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28555Local ID: 13708ISBN: 91-7219-736-6 (print)OAI: oai:DiVA.org:liu-28555DiVA: diva2:249366
Public defence
2000-03-31, Berzeliussalen, Universitetssjukhuset, Linköping, 09:30 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-08-09Bibliographically approved
List of papers
1. Characterization of primary recall in vitro lymphocyte responses to bacampicillin in allergic subjects
Open this publication in new window or tab >>Characterization of primary recall in vitro lymphocyte responses to bacampicillin in allergic subjects
2000 (English)In: Clinical and Experimental Allergy, ISSN 0954-7894, E-ISSN 1365-2222, Vol. 30, no 10, 1450-1459 p.Article in journal (Refereed) Published
Abstract [en]

Background

Antigen-specific cell lines or clones are often used as models of drug-specific allergy. However, cloning procedures are time consuming, and the repeated antigen stimulation cycles as well as the addition of various growth enhancers may affect the in vivo relevance of these systems.

Objective

Using bacampicillin-allergic subjects, we wanted to investigate the applicability of primary recall in vitro lymphocyte responses to characterize type I and type IV allergy. The sensitivity and specificity of LTT (Lymphocyte transformation test), when used as an in vitro diagnostic tool, were also assessed.

Methods

A total of 39 patients with symptoms of type I (rhinitis) or type IV (allergic contact dermatitis, ACD) allergy following occupational exposure to bacampicillin, were included. Ten individuals without penicillin allergy or occupational exposure to bacampicillin served as controls. All subjects were LTT tested. Four patients with rhinitis and two patients with ACD were available for studying the immunophenotype and the TCR-Vβ repertoire of bacampicillin induced lymphoblasts as well as the cytokine profiles and expression of the activation markers CD23 and CD134 in primary PBMC cultures.

Results

LTT was positive in 87% and at least one of the skin tests was positive in 85% of the patients with allergic symptoms. 69% of the patients with type I allergies were patch test-positive. Results from LTT and skin test correlated in 87% of the cases. The combined sensitivity of LTT and skin tests was 92%. The specificity of LTT was 90% in healthy controls. Bacampicillin induced lymphoblasts were mainly CD4 + in both ACD and rhinitis patients. The TCR-Vβ profiles of the predominant CD4 + lymphoblasts were heterogeneous with individual skewing towards Vβ2, Vβ3, Vβ5.1 and/or Vβ14. An increased expression of IFNγ was detected in bacampicillin treated PBMC cultures from the ACD but not from rhinitis patients. IL-5 was detected in bacampicillin exposed PBMC cultures from all patients but not from healthy controls. This Th2 environment could also be verified by CD23 and CD134 expression.

Conclusion

LTT and skin tests are equally sensitive in identifying bacampicillin allergic subjects. When the two tests are combined, the sensitivity increases. The patch test is useful not only for detection of type IV but also for the identification of type I allergies. When using primary PBMC cultures, IFNγ is the most suitable cytokine to discriminate between type I and type IV allergy. IL-5 can possibly be used as a general marker for bacampicillin induced allergy. Thus, primary cell cultures may be considered as an alternative to T-cell lines or clones for the study of drug induced allergy.

Keyword
activation markers, allergic contact dermatitis, bacampicillin, cytokines, flow cytometry, human lymphocytes, lymphocyte transformation test, rhinitis, skin test, T-cell receptor Vβ
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25872 (URN)10.1046/j.1365-2222.2000.00905.x (DOI)10309 (Local ID)10309 (Archive number)10309 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
2. In vitro Lymphocyte Proliferation as Compared to Patch Test Using Gold, Palladium and Nickel
Open this publication in new window or tab >>In vitro Lymphocyte Proliferation as Compared to Patch Test Using Gold, Palladium and Nickel
1997 (English)In: International Archives of Allergy and Immunology, ISSN 1018-2438, E-ISSN 1423-0097, Vol. 112, no 3, 212-217 p.Article in journal (Refereed) Published
Abstract [en]

Background: A conventional lymphocyte transformation test (LTT) was compared to the commercially available MELISA® (memory lymphocyte immuno-stimulation assay), a lymphoproliferative assay that has been suggested to be a valuable instrument for the diagnosis of metal allergy. Sensitivity and specificity of the two assays were calculated using a patch test as a reference method.

Methods: 34 patients were patch-tested for gold sodium thiosulfate, palladium chloride and nickel sulfate, and the lymphocyte proliferation to these metals was tested in vitro using mononuclear cells from peripheral blood.

Results: No significant differences regarding sensitivity and specificity were found between MELISA and conventional LTT. The sensitivity varied between 55 and 95% and the specificity between 17 and 79%.

Conclusions: The low specificity of the two in vitro assays suggests that they are not useful for diagnosis of contact allergy to the metals gold, palladium and nickel, since a large number of false-positive results will be obtained.

Keyword
Lymphocytes human, Skin test, Metals
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79573 (URN)10.1159/000237456 (DOI)
Available from: 2012-08-09 Created: 2012-08-09 Last updated: 2017-12-07Bibliographically approved
3. IL-10 production in primary PBMC cultures: an in vitro marker for nickel allergy?
Open this publication in new window or tab >>IL-10 production in primary PBMC cultures: an in vitro marker for nickel allergy?
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Nickel (Ni) is one of the most known contact allergens and at present, patch test and clinical history constitute the two cornerstones in the diagnostic procedure. Since the patch test is inherited with in vivo provocation and subjective interpretation of the test result, a non-invasive in vitro method with objective interpretation of the test result has long been searched for. Unfortunately, in vitro diagnosis of Ni- allergy is hampered by the fact that Ni2+ is able to trigger in vitro proliferative responses in lymphocytes from both Ni-allergic and non-allergic subjects. This constitutes a problem when LTT (lymphocyte transformation test), the most frequently used in vitro test as a complement in the diagnosis of contact allergy, is considered for Ni allergy. However, other parameters in the in vitro response might be more useful. In this study, Ni2+-stimulated primary PBMC-cultures derived from Ni-allergic and non-allergic subjects were assessed for IFN-γ, IL-4, IL-10 and IL-17. Also, Ni2+ induced lymphoblasts from such cultures were characterized by their immunophenotype and T-cell receptor Vß-affiliation.

We found a significantly higher release of IL-10 in Ni2+ treated cultures from allergic than from non-allergic subjects. The Ni2+-induced lymphoblasts from both groups were predominantly CD4+. Two of the allergic patients (n=5) showed a skewing towards TCR-Vß17, a Vß family earlier associated with Ni-allergy. A significant increase in CD134 and CD23 expression indicated that Ni2+ activates B-cells in vitro. In conclusion, IL-10 seems to be a promising marker for Ni-allergy using primary PBMC cultures. Further, flow cytometric screening of Ni2+ induced lymphoblasts can detect expanded TCR-Vß families that may be used for preparation of Ni-specific T cell clones.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79574 (URN)
Available from: 2012-08-09 Created: 2012-08-09 Last updated: 2012-08-09Bibliographically approved
4. In vitro Lymphoproliferative Assays with HgCl2 Cannot Identify Patients with Systemic Symptoms Attributed to Dental Amalgam
Open this publication in new window or tab >>In vitro Lymphoproliferative Assays with HgCl2 Cannot Identify Patients with Systemic Symptoms Attributed to Dental Amalgam
Show others...
1999 (English)In: Journal of Dental Research, ISSN 0022-0345, E-ISSN 1544-0591, Vol. 78, no 8, 1450-1458 p.Article in journal (Refereed) Published
Abstract [en]

Dental amalgam is suspected, by some exposed individuals, to cause various systemic psychological, sensory, and neurological symptoms. Since not all amalgam-bearers experience such reactions, an individual characteristic—for example, a susceptible immune system—might explain these conditions. In vitro lymphocyte proliferation is a valuable tool in the diagnosis of allergy. With HgCl2 as the antigen, however, the test is hampered, because Hg2+ can cause unspecific lymphocyte proliferation, optimal at 1.4 to 9.5 μg HgCl2/mL. Recently, the use of suboptimal HgCl2 concentrations (≤ 0.5 μg/mL) has been suggested to circumvent these problems. The main aim of this study was to investigate whether patients with systemic symptoms alleged to result from the presence of dental amalgam differ from healthy controls, with reference to in vitro lymphoproliferative responses to HgCl2 ≤ 0.5 μg/mL. Three different test protocols—lymphocyte transformation test (LTT) in micro- and macro-cultures, and the memory lymphocyte immunostimulation assay (MELISA®)—were used. Other immune parameters—such as a standard patch test for dental materials, the number of T- and B-lymphocytes, monocytes, granulocytes, and NK cells in peripheral blood, allergic symptoms, and predisposition-were also investigated. Twenty-three amalgam patients, 30 healthy blood donors with amalgam, ten healthy subjects without amalgam, and nine patients with oral lichen planus (OLP) adjacent to dental amalgam and a positive patch test to Hg0 were tested. None of the investigated immune parameters revealed any significant differences between amalgam patients and controls. The sensitivity of in vitro lymphocyte proliferation ranged from 33 to 67%, with the OLP patients as a positive control group, and the specificity from 0 to 70% for healthy controls with a negative patch test to Hg°. Thus, despite the use of HgCl2 ≤ 0.5 μg/mL, a high frequency of positive results was obtained among healthy subjects with or without dental amalgam. Consequently, in vitro lymphocyte proliferation with HgCl2 cannot be used as an objective marker for mercury allergy in dental amalgam-bearers.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25875 (URN)10.1177/00220345990780081101 (DOI)10312 (Local ID)10312 (Archive number)10312 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
5. Characterization of mercuric mercury (Hg2+)-induced lymphoblasts from patients with mercury allergy and from healthy subjects
Open this publication in new window or tab >>Characterization of mercuric mercury (Hg2+)-induced lymphoblasts from patients with mercury allergy and from healthy subjects
2000 (English)In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 121, no 1, 23-30 p.Article in journal (Refereed) Published
Abstract [en]

Hg2+ induces lymphocyte proliferation when added to cell cultures from both healthy and mercury-allergic subjects. Consequently, when measuring DNA synthesis a possible Hg2+-specific response, resulting from proliferating memory cells, cannot be discriminated from a non-allergic response. The mechanism behind this non-allergic response is unknown but a superantigenic effect of Hg2+ has been suggested. In this study, five mercury-allergic patients, with oral lichen planus (OLP) lesions adjacent to dental amalgam and a positive patch test to Hg0, and five healthy subjects without amalgam were examined. The immunophenotype and the T cell receptor Vβ (TCR Vβ) repertoire of Hg2+-induced lymphoblasts as well as the expression of the lymphocyte activation markers CD23 and CD134 were analysed for possible differences between healthy and allergic subjects. The mechanism of Hg2+-induced proliferation was examined by comparing the TCR Vβ expression of Hg- and staphylococcal enterotoxin B (SEB)-activated lymphoblasts, the latter used as a positive superantigen control. It was not possible to discriminate between mercury-allergic and healthy subjects using the immunophenotype or the TCR Vβ profile of the Hg2+-induced lymphoblasts or the expression of CD23 and CD134. However, Hg2+-induced CD4+ lymphoblasts showed a skewing towards Vβ2. This relative increase in Vβ2 was only detected in the CD4+ but not in the CD8+ lymphoblast population. In conclusion, Hg2+ induced a proliferation-dependent skewing towards CD4+ but not CD8+ lymphocytes expressing Vβ2. In this respect Hg2+ differs from the classical bacterial superantigen SEB, which also stimulates unique TCR Vβ families among CD8+ cells.

Keyword
Hg allergy, oral lichen planus, human lymphocytes, in vitro
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25873 (URN)10.1046/j.1365-2249.2000.01268.x (DOI)10310 (Local ID)10310 (Archive number)10310 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved

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