14694
2016
eng
105
4
8
article
1
2017-04-07
--
--
The Correlation between the Virus- and Brain Antigen-Specific B Cell Response in the Blood of Patients with Multiple Sclerosis
There is a largely divergent body of literature regarding the relationship between Epstein-Barr virus (EBV) infection and brain inflammation in multiple sclerosis (MS). Here, we tested MS patients during relapse (n = 11) and in remission (n = 19) in addition to n = 22 healthy controls to study the correlation between the EBV- and brain-specific B cell response in the blood by enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA). Cytomegalovirus (CMV) was used as a control antigen tested in n = 16 MS patients during relapse and in n = 35 patients in remission. Over the course of the study, n = 16 patients were untreated, while n = 33 patients received immunomodulatory therapy. The data show that there was a moderate correlation between the frequencies of EBV- and brain-reactive B cells in MS patients in remission. In addition we could detect a correlation between the B cell response to EBV and disease activity. There was no evidence of an EBV reactivation. Interestingly, there was also a correlation between the frequencies of CMV- and brain-specific B cells in MS patients experiencing an acute relapse and an elevated B cell response to CMV was associated with higher disease activity. The trend remained when excluding seronegative subjects but was non-significant. These data underline that viral infections might impact the immunopathology of MS, but the exact link between the two entities remains subject of controversy.
Viruses
10.3390/v8040105
urn:nbn:de:bvb:20-opus-146946
Viruses 2016, 8, 105; doi:10.3390/v8040105
Marie Wunsch
Christopher Hohmann
Bianca Milles
Christina Rostermund
Paul V. Lehmann
Michael Schroeter
Antonios Bayas
Jochen Ulzheimer
Mathias Mäurer
Süleyman Ergün
Stefanie Kuerten
eng
uncontrolled
B cells
eng
uncontrolled
CMV
eng
uncontrolled
EBV
eng
uncontrolled
ELISPOT
eng
uncontrolled
MS
Krankheiten
open_access
Institut für Anatomie und Zellbiologie
Förderzeitraum 2016
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14694/088_Wunsch_viruses-08-00105.pdf
14996
2015
eng
96-111
1
4
article
1
2017-06-08
--
--
Normal distribution of CD8+ T-cell-derived ELISPOT counts within replicates justifies the reliance on parametric statistics for identifying positive responses
Accurate assessment of positive ELISPOT responses for low frequencies of antigen-specific T-cells is controversial. In particular, it is still unknown whether ELISPOT counts within replicate wells follow a theoretical distribution function, and thus whether high power parametric statistics can be used to discriminate between positive and negative wells. We studied experimental distributions of spot counts for up to 120 replicate wells of IFN-γ production by CD8+ T-cell responding to EBV LMP2A (426 – 434) peptide in human PBMC. The cells were tested in serial dilutions covering a wide range of average spot counts per condition, from just a few to hundreds of spots per well. Statistical analysis of the data using diagnostic Q-Q plots and the Shapiro-Wilk normality test showed that in the entire dynamic range of ELISPOT spot counts within replicate wells followed a normal distribution. This result implies that the Student t-Test and ANOVA are suited to identify positive responses. We also show experimentally that borderline responses can be reliably detected by involving more replicate wells, plating higher numbers of PBMC, addition of IL-7, or a combination of these. Furthermore, we have experimentally verified that the number of replicates needed for detection of weak responses can be calculated using parametric statistics.
Cells
10.3390/cells4010096
PMC4381212
urn:nbn:de:bvb:20-opus-149968
Cells 2015, 4(1), 96-111. DOI: 10.3390/cells4010096
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Alexey Y. Karulin
Richard Caspell
Marcus Dittrich
Paul V. Lehmann
eng
uncontrolled
ELISPOT
eng
uncontrolled
statistics
eng
uncontrolled
t-Test
eng
uncontrolled
ANOVA
eng
uncontrolled
T-cells
eng
uncontrolled
normal distribution
Biowissenschaften; Biologie
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14996/108_Karulin_Cells.pdf
12612
2014
eng
138
2
article
1
2016-01-28
--
--
Categorization of multiple sclerosis relapse subtypes by B cell profiling in the blood
Introduction
B cells are attracting increasing attention in the pathogenesis of multiple sclerosis (MS). B cell-targeted therapies with monoclonal antibodies or plasmapheresis have been shown to be successful in a subset of patients. Here, patients with either relapsing-remitting (n = 24) or secondary progressive (n = 6) MS presenting with an acute clinical relapse were screened for their B cell reactivity to brain antigens and were re-tested three to nine months later. Enzyme-linked immunospot technique (ELISPOT) was used to identify brain-reactive B cells in peripheral blood mononuclear cells (PBMC) directly ex vivo and after 96 h of polyclonal stimulation. Clinical severity of symptoms was determined using the Expanded Disability Status Scale (EDSS).
Results
Nine patients displayed B cells in the blood producing brain-specific antibodies directly ex vivo. Six patients were classified as B cell positive donors only after polyclonal B cell stimulation. In 15 patients a B cell response to brain antigens was absent. Based on the autoreactive B cell response we categorized MS relapses into three different patterns. Patients who displayed brain-reactive B cell responses both directly ex vivo and after polyclonal stimulation (pattern I) were significantly younger than patients in whom only memory B cell responses were detectable or entirely absent (patterns II and III; p = 0.003). In one patient a conversion to a positive B cell response as measured directly ex vivo and subsequently also after polyclonal stimulation was associated with the development of a clinical relapse. The evaluation of the predictive value of a brain antigen-specific B cell response showed that seven of eight patients (87.5%) with a pattern I response encountered a clinical relapse during the observation period of 10 months, compared to two of five patients (40%) with a pattern II and three of 14 patients (21.4%) with a pattern III response (p = 0.0005; hazard ratio 6.08 (95% confidence interval 1.87-19.77).
Conclusions
Our data indicate actively ongoing B cell-mediated immunity against brain antigens in a subset of MS patients that may be causative of clinical relapses and provide new diagnostic and therapeutic options for a subset of patients.
Acta Neuropathologica Communications
10.1186/s40478-014-0138-2
urn:nbn:de:bvb:20-opus-126124
Acta Neuropathologica Communications 2014, 2:138. doi:10.1186/s40478-014-0138-2
Christopher Hohnmann
Bianca Milles
Michael Schinke
Michael Schroeter
Jochen Ulzheimer
Peter Kraft
Christoph Kleinschnitz
Paul V. Lehmann
Stefanie Kuerten
eng
uncontrolled
predictive value
eng
uncontrolled
MS
eng
uncontrolled
ELISPOT
eng
uncontrolled
B cells
eng
uncontrolled
relapse
Medizin und Gesundheit
open_access
Institut für Anatomie und Zellbiologie
Neurologische Klinik und Poliklinik
Förderzeitraum 2015
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/12612/Kuerten_s40478-014-0138-2.pdf
12058
2014
eng
138
2
article
1
2015-10-21
--
--
Categorization of multiple sclerosis relapse subtypes by B cell profiling in the blood
INTRODUCTION:
B cells are attracting increasing attention in the pathogenesis of multiple sclerosis (MS). B cell-targeted therapies with monoclonal antibodies or plasmapheresis have been shown to be successful in a subset of patients. Here, patients with either relapsing-remitting (n = 24) or secondary progressive (n = 6) MS presenting with an acute clinical relapse were screened for their B cell reactivity to brain antigens and were re-tested three to nine months later. Enzyme-linked immunospot technique (ELISPOT) was used to identify brain-reactive B cells in peripheral blood mononuclear cells (PBMC) directly ex vivo and after 96 h of polyclonal stimulation. Clinical severity of symptoms was determined using the Expanded Disability Status Scale (EDSS).
RESULTS:
Nine patients displayed B cells in the blood producing brain-specific antibodies directly ex vivo. Six patients were classified as B cell positive donors only after polyclonal B cell stimulation. In 15 patients a B cell response to brain antigens was absent. Based on the autoreactive B cell response we categorized MS relapses into three different patterns. Patients who displayed brain-reactive B cell responses both directly ex vivo and after polyclonal stimulation (pattern I) were significantly younger than patients in whom only memory B cell responses were detectable or entirely absent (patterns II and III; p = 0.003). In one patient a conversion to a positive B cell response as measured directly ex vivo and subsequently also after polyclonal stimulation was associated with the development of a clinical relapse. The evaluation of the predictive value of a brain antigen-specific B cell response showed that seven of eight patients (87.5%) with a pattern I response encountered a clinical relapse during the observation period of 10 months, compared to two of five patients (40%) with a pattern II and three of 14 patients (21.4%) with a pattern III response (p = 0.0005; hazard ratio 6.08 (95% confidence interval 1.87-19.77).
CONCLUSIONS:
Our data indicate actively ongoing B cell-mediated immunity against brain antigens in a subset of MS patients that may be causative of clinical relapses and provide new diagnostic and therapeutic options for a subset of patients.
Acta Neuropathologica Communications
10.1186/s40478-014-0138-2
2051-5960
25597707
urn:nbn:de:bvb:20-opus-120580
Acta Neuropathologica Communications 2014, 2:138. doi:10.1186/s40478-014-0138-2
Christopher Hohmann
Bianca Milles
Michael Schinke
Michael Schroeter
Jochen Ulzheimer
Peter Kraft
Christoph Kleinschnitz
Paul V. Lehmann
Stefanie Kuerten
eng
uncontrolled
ELISPOT
eng
uncontrolled
MS
eng
uncontrolled
predictive value
eng
uncontrolled
relapse
eng
uncontrolled
B cells
Medizin und Gesundheit
open_access
Institut für Anatomie und Zellbiologie
Neurologische Klinik und Poliklinik
Institut für Klinische Epidemiologie und Biometrie
Förderzeitraum 2015
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/12058/104_Hohmann_Acta_Neuropatholica_Communications.pdf
14964
2015
eng
56-70
1
4
article
1
2017-06-02
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ELISPOTs produced by CD8 and CD4 cells follow Log Normal size distribution permitting objective counting
Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters is critical. If the spot sizes follow a known statistical distribution, precise predictions on minimal and maximal spot sizes, belonging to a given T cell population, can be made. We studied the size distributional properties of IFN-γ, IL-2, IL-4, IL-5 and IL-17 spots elicited in ELISPOT assays with PBMC from 172 healthy donors, upon stimulation with 32 individual viral peptides representing defined HLA Class I-restricted epitopes for CD8 cells, and with protein antigens of CMV and EBV activating CD4 cells. A total of 334 CD8 and 80 CD4 positive T cell responses were analyzed. In 99.7% of the test cases, spot size distributions followed Log Normal function. These data formally demonstrate that it is possible to establish objective, statistically validated parameters for counting T cell ELISPOTs.
Cells
10.3390/cells4010056
PMC4381209
urn:nbn:de:bvb:20-opus-149648
Cells 2015, 4(1), 56-70. DOI: 10.3390/cells4010056
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Alexey Y. Karulin
Kinga Karacsony
Wenji Zhang
Oleg S. Targoni
Ioana Moldova
Marcus Dittrich
Srividya Sundararaman
Paul V. Lehmann
eng
uncontrolled
ELISPOT
eng
uncontrolled
software
eng
uncontrolled
IFN-γ
eng
uncontrolled
IL-17
eng
uncontrolled
T cells
eng
uncontrolled
Normal Distribution
eng
uncontrolled
spot size
eng
uncontrolled
gating
eng
uncontrolled
cytokines
eng
uncontrolled
IL-2
eng
uncontrolled
IL-4
eng
uncontrolled
IL-5
eng
uncontrolled
CD8
eng
uncontrolled
CD4
Biowissenschaften; Biologie
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14964/094_Karulin_Cells.pdf
15021
2015
eng
40-55
1
4
article
1
2017-06-09
--
--
Serial measurements of apoptotic cell numbers provide better acceptance criterion for PBMC quality than a single measurement prior to the T cell assay
As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay.
Cells
10.3390/cells4010040
urn:nbn:de:bvb:20-opus-150213
Cells 2015, 4:1, 40-55. DOI: 10.3390/cells4010040
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Marie Wunsch
Richard Caspell
Stefanie Kuerten
Paul V. Lehmann
Srividya Sundararaman
eng
uncontrolled
T cell assay
eng
uncontrolled
apoptosis
eng
uncontrolled
acceptance
eng
uncontrolled
viability
eng
uncontrolled
ELISPOT
Medizin und Gesundheit
open_access
Institut für Anatomie und Zellbiologie
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/15021/129_Wunsch_Cells.pdf
15146
2015
eng
4414
4437
7
article
1
2017-07-13
--
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Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity
Most humans become infected with human cytomegalovirus (HCMV). Typically, the immune system controls the infection, but the virus persists and can reactivate in states of immunodeficiency. While substantial information is available on the contribution of CD8 T cells and antibodies to anti-HCMV immunity, studies of the T\(_{H}\)1, T\(_{H}\)2, and T\(_{H}\)17 subsets have been limited by the low frequency of HCMV-specific CD4 T cells in peripheral blood mononuclear cell (PBMC). Using the enzyme-linked Immunospot\(^{®}\) assay (ELISPOT) that excels in low frequency measurements, we have established these in a sizable cohort of healthy HCMV controllers. Cytokine recall responses were seen in all seropositive donors. Specifically, interferon (IFN)-\({\gamma}\) and/or interleukin (IL)-17 were seen in isolation or with IL-4 in all test subjects. IL-4 recall did not occur in isolation. While the ratios of T\(_{H}\)1, T\(_{H}\)2, and T\(_{H}\)17 cells exhibited substantial variations between different individuals these ratios and the frequencies were relatively stable when tested in samples drawn up to five years apart. IFN-\({\gamma}\) and IL-2 co-expressing polyfunctional cells were seen in most subjects. Around half of the HCMV-specific CD4 cells were in a reversible state of exhaustion. The data provided here established the T\(_{H}\)1, T\(_{H}\)2, and T\(_{H}\)17 characteristic of the CD4 cells that convey immune protection for successful immune surveillance against which reactivity can be compared when the immune surveillance of HCMV fails.
Viruses
10.3390/v7082828
urn:nbn:de:bvb:20-opus-151462
Viruses 2015, 7, 4414-4437. DOI: 10.3390/v7082828
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Marie Wunsch
Wenji Zhang
Jodi Hanson
Richard Caspell
Alexey Y. Karulin
Mascha S. Recks
Stefanie Kuerten
Srividya Sundararaman
Paul V. Lehmann
eng
uncontrolled
memory cells
eng
uncontrolled
hcv infection
eng
uncontrolled
signature
eng
uncontrolled
Enzyme-Linked Immunospot assay (ELISPOT)
eng
uncontrolled
cytokine secretion kinetics
eng
uncontrolled
chronic viral infection
eng
uncontrolled
HCMV infection
eng
uncontrolled
CD4 T cells
eng
uncontrolled
exhaustion
eng
uncontrolled
activation
eng
uncontrolled
human cytomegalovirus (HCMV)
eng
uncontrolled
B cells
eng
uncontrolled
cytomegalovirus
eng
uncontrolled
elispot
Krankheiten
open_access
Institut für Anatomie und Zellbiologie
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/15146/012_Wunsch_Viruses.pdf