Early effects of low dose C-12(6+) ion or X-ray irradiation on human peripheral blood lymphocytes

The aim of this study was to estimate the acute effects of low dose C-12(6+) ions or X-ray radiation on human immune function. The human peripheral blood lymphocytes (HPBL) of seven healthy donors were exposed to 0.05 Gy C-12(6+) ions or X-ray radiation and cell responses were measured at 24 h after exposure. The cytotoxic activities of HPBL were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT); the percentages of T and NK cells subsets were detected by flow cytometry; mRNA expression of interleukin (IL)-2, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were examined by real time quantitative RT-PCR (qRT-PCR); and these cytokines protein levels in supematant of cultured cells were assayed by enzyme-linked immunosorbent assays (ELISA). The results showed that the cytotoxic activity of HPBL, mRNA expression of IL-2, IFN-gamma and TNF-alpha in HPBL and their protein levels in supernatant were significantly increased at 24 h after exposure to 0.05 Gy C-12(6+) ions radiation and the effects were stronger than observed for X-ray exposure. However, there was no significant change in the percentage of T and NK cells subsets of HPBL. These results suggested that 0.05 Gy high linear energy transfer (LET) C-12(6+) radiation was a more effective approach to host immune enhancement than that of low LET X-ray. We conclude that cytokines production might be used as sensitive indicators of acute response to LDL (C) 2009 COSPAR. Published by Elsevier Ltd. All rights reserved.

低剂量12C6+辐射对小鼠免疫系统损伤和抗氧化剂氮乙酰半胱氨酸的保护作用研究

实验目的：随着科技的发展，人类活动范围已经逐渐向外太空扩展，对于人类太空探索的最大威胁是太空中的各种粒子辐射。这些辐射包括太阳辐射（质子和电子）和银河辐射（质子占85%，氦离子占14%，重离子占1%）。众所周知，重离子与常规X和γ射线相比有较高的传能线密度（linear energy transfer, LET）和相对生物学效应（relative biological effectiveness, RBE），对机体组织和器官有较强的影响。放射治疗是肿瘤治疗的重要手段之一，由于肿瘤细胞的异质性，其对放、化疗的反应相差悬殊。本研究的目的是： 1评估辐射对健康机体产生的生物学风险； 2研究抗氧化剂氮乙酰半胱氨酸（NAC）对机体辐射损伤的保护作用 3不同肿瘤细胞辐射敏感性的差异。实验方法： 1 X射线或12C6+离子对小鼠进行不同剂量的全身辐射。NAC处理组小鼠在照射前1小时腹腔注射200mg/kg的NAC，对照组注射等体积的生理盐水。照射后不同时间点取样，利用流式细胞仪检测小鼠免疫细胞周期和凋亡情况，单细胞电泳检测淋巴细胞DNA损伤，MTT法（3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide）检测脾脏NK（natural killer，NK）细胞活性，微核法检测淋巴细胞染色体损伤情况，小鼠体内干扰素－γ（Interferon-γ，IFN-γ）由ELISA方法得到，小鼠血清中超氧化物岐化酶(Surperoxide dismutase SOD）由分光光度法测定，并观察胸腺和脾脏指数变化。 2 不同剂量X射线和12C6+离子辐射人肺腺癌细胞H1299和A549，用细胞克隆法检测照射后细胞存活曲线，流式细胞仪检测细胞周期和凋亡，Western-blot 检测A549 细胞P53蛋白表达。 结果： 1小鼠外周血淋巴细胞、胸腺细胞和脾脏淋巴细胞周期随着X射线照射剂量的增大而被阻滞在了G0/G1期，相同剂量的12C6+离子辐射时外周血淋巴细胞周期被阻滞在S期，分次连续X射线照射时，外周血淋巴细胞周期随着累积剂量的增加被阻滞在G2/M期；细胞凋亡比例随着照射剂量的增加而增加。小鼠血清中IFN-γ水平和脾脏中NK细胞活性在重离子照射剂量为0.05Gy时有显著增加，脾脏NK细胞活性随着照射剂量的增加而减弱。 2重离子照射后，小鼠淋巴细胞DNA和染色体的损伤随辐射剂量和照射后时间的延长而加剧。脾脏NK细胞活性在照射后各个时间点减弱，血清中IFN-γ水平和SOD酶活性随着重离子照射剂量的增加而降低。预防性给予NAC，12C6+离子辐射对淋巴细胞DNA和染色体所致损伤，胸腺细胞周期和凋亡，脾脏NK细胞活性，血清中IFN-γ的水平和SOD酶的活性的损伤与盐水组比较均有显著改善。 3 X射线照射对肺腺癌H1299细胞周期和凋亡率未产生明显影响，重离子照射后随着照射剂量的增加细胞周期被阻滞在G2/M期，细胞凋亡率也呈剂量依赖性；X射线和12C6+离子照射A549细胞后，细胞周期均被阻滞在G2/M期，凋亡率剂量依赖性增加。A549细胞P53蛋白的表达水平随着重离子照射剂量的增加而增加。结论： 1重离子辐射造成细胞DNA和染色体损伤随着照射剂量的增加和照射后时间的延长而增加，比X射线辐射损伤复杂和难以修复，产生这种现象的机理为辐射导致活性氧分子簇的产生，细胞因子和与细胞氧化反应有关的酶活性的变化，同时这种损伤对胸腺细胞周期、凋亡和胸腺、脾脏指数以及机体免疫系统都有影响；低剂量重离子辐射（0.05Gy）对小鼠机体的免疫力有刺激作用，机体免疫能力随着照射剂量增加和照射后时间的推移而减弱，不同的免疫器官对辐射的敏感性也不同； 2 200mg/kg 的NAC对辐射所致小鼠免疫系统损伤有很好的保护作用； 3 肺腺癌细胞H1299比同系A549具有较强的辐射敏感性，A549细胞凋亡的增加与P53蛋白表达水平升高有关

塞隆骨提取物对CFA诱导活化的小鼠腹腔巨噬细胞功能的影响

目的：研究塞隆骨提取物对弗氏完全佐剂诱导活化的腹腔巨噬细胞的免疫抑制作用。方法：小鼠腹腔注射弗氏完全佐剂诱导腹腔巨噬细胞活化并回收，体外加入LPS及IFN-γ刺激培养巨噬细胞，利用酶联免疫法测定培养上清中细胞因子（IL-1β、IL-6、IL-12p40、TNF-α水平。结果：塞隆骨水提物及90％醇沉部分体内给药均能够能显著抑制弗氏完全佐剂诱导的巨噬细胞向腹腔的浸润，能抑制巨噬细胞在LPS及IFN-γ刺激下生成细胞因子（IL-12p40、IL-1β、IL-6、TNF-α。结论：塞隆骨提取物对巨噬细胞功能有一定程度的影响，能够抑制其产生炎症因子，可能是其治疗类风湿性关节炎的作用机理之一。

塞隆骨提取物对牛Ⅱ型胶原诱导的小鼠关节炎的治疗作用及机制研究

目的 了解塞隆骨水提物（SLG—B）对胶原诱导的小鼠关节炎的治疗作用及治疗机制。方法 利用牛Ⅱ型胶原诱导DBA／1小鼠类风湿性关节炎模型即CIA。SLG—B口服给药观察小鼠关节炎指数的变化情况，体外培养关节炎小鼠脾细胞及巨噬细胞，ELISA法测定IL-12、IL-2、IFN-γ TNF-α几种细胞因子。RT-PCR测定SLG-B对关节炎小鼠巨噬细胞IL-1β、IL-6、iNOS mRNA表达的影响。结果 SLG-B能明显的抑制牛Ⅱ型胶原诱导关节炎的发生，能够减轻关节炎的各种症状。SLG-B在加抗原和不加抗原的情况下都能够明显的抑制关节炎小鼠脾细胞的增殖同时发现SLG-B能够抑制IL-2、IFN-1、L-12p40、TNF-α细胞因子的产生。还能够抑制小鼠腹腔巨噬细胞IL-1、IL-6及iNOS mRNA的表达。这可能是SLG—B在小鼠关节炎中表现治疗效果的分子基础。结论 SLG—B对小鼠关节炎有很好的治疗效果，其作用机制主要是通过抑制巨噬细胞及脾细胞产生炎性细胞因子和抑制炎性细胞因子mRNA的表达来达到治疗类风湿性关节炎的作用。

STAT1 – główny czynnik regulatorowy w zapaleniu i dysfunkcji naczyniowej

Wydział Biologii: Instytut Biologii Molekularnej i Biotechnologii

Integracja szlaków sygnalizacyjnych interferonu gamma i receptora TLR4 w procesie zapalnym naczyń krwionośnych, warunkowana czynnikami transkrypcyjnymi STAT1 i IRF

Wydział Biologii: Instytut Biologii Molekularnej i Biotechnologii Zakład Genetyki Molekularnej Człowieka

Novel ingredients from brewers' spent grain - bioactivity in cell culture model systems and bioactivity retention in fortified food products

Functional food ingredients, with scientifically proven and validated bioactive effects, present an effective means of inferring physiological health benefits to consumers to reduce the risk of certain diseases. The search for novel bioactive compounds for incorporation into functional foods is particularly active, with brewers’ spent grain (BSG, a brewing industry co-product) representing a unique source of potentially bioactive compounds. The DNA protective, antioxidant and immunomodulatory effects of phenolic extracts from both pale (P1 - P4) and black (B1 – B4) BSG were examined. Black BSG extracts significantly (P < 0.05) protected against DNA damage induced by hydrogen peroxide (H2O2) and extracts with the highest total phenolic content (TPC) protected against 3-morpholinosydnonimine hydrochloride (SIN-1)-induced oxidative DNA damage, measured by the comet assay. Cellular antioxidant activity assays were used to measured antioxidant potential in the U937 cell line. Extracts P1 – P3 and B2 - B4 demonstrated significant (P < 0.05) antioxidant activity, measured by the superoxide dismutase (SOD) activity, catalase (CAT) activity and gluatathione (GSH) content assays. Phenolic extracts P2 and P3 from pale BSG possess anti-inflammatory activity measured in concanavalin-A (conA) stimulated Jurkat T cells by an enzyme-linked immunosorbent assay (ELISA); significantly (P < 0.05) reducing production of interleukin-2 (IL-2), interleukin-4 (IL-4, P2 only), interleukin-10 (IL-10) and interferon-γ (IFN-γ). Black BSG phenolic extracts did not exhibit anti-inflammatory effects in vitro. Hydroxycinnamic acids (HA) have previously been shown to be the phenolic acids present at highest concentration in BSG; therefore the HA profile of the phenolic extracts used in this research, the original barley (before brewing) and whole BSG was characterised and quantified using high performance liquid chromatography (HPLC). The concentration of HA present in the samples was in the order of ferulic acid (FA) > p-coumaric acid (p-CA) derivatives > FA derivatives > p-CA > caffeic acid (CA) > CA derivatives. Results suggested that brewing and roasting decreased the HA content. Protein hydrolysates from BSG were also screened for their antioxidant and anti-inflammatory potential. A total of 34 BSG protein samples were tested. Initial analyses of samples A – J found the protein samples did not exert DNA protective effects (except hydrolysate H) or antioxidant effects by the comet and SOD assays, respectively. Samples D, E, F and J selectively reduced IFN-γ production (P < 0.05) in Jurkat T cells, measured using enzyme linked immunosorbent assay (ELISA). Further testing of hydrolysates K – W, including fractionated hydrolysates with molecular weight < 3, < 5 and > 5 kDa, found that higher molecular weight (> 5 kDa) and unfractionated hydrolysates demonstrate greatest anti-inflammatory effects, while fractionated hydrolysates were also shown to have antioxidant activity, by the SOD activity assay. A commercially available yogurt drink (Actimel) and snack-bar and chocolate-drink formulations were fortified with the most bioactive phenolic and protein samples – P2, B2, W, W < 3 kDa, W < 5 kDa, W > 5 kDa. All fortified foods were subjected to a simulated gastrointestinal in vitro digestion procedure and bioactivity retention in the digestates was determined using the comet and ELISA assays. Yogurt fortified with B2 digestate significantly (P < 0.05) protected against H2O2-induced DNA damage in Caco-2 cells. Greatest immunomodulatory activity was demonstrated by the snack-bar formulation, significantly (P < 0.05) reducing IFN-γ production in con-A stimulated Jurkat T cells. Hydrolysate W significantly (P < 0.05) increased the IFN-γ reducing capacity of the snack-bar. Addition of fractionated hydrolysate W < 3 kDa and W < 5 kDa to yogurt also reduced IL-2 production to a greater extent than the unfortified yogurt (P < 0.05).

The use of different blood sample preparations in the development of biomarkers for the environmental monitoring of domestic farm animals

The application of biological effect monitoring for the detection of environmental chemical exposure in domestic animals is still in its infancy. This study investigated blood sample preparations in vitro for their use in biological effect monitoring. When peripheral blood mononuclear cells (PBMCs), isolated following the collection of multiple blood samples from sheep in the field, were cryopreserved and subsequently cultured for 24 hours a reduction in cell viability (<80%) was attributed to delays in the processing following collection. Alternative blood sample preparations using rat and sheep blood demonstrated that 3 to 5 hour incubations can be undertaken without significant alterations in the viability of the lymphocytes; however, a substantial reduction in viability was observed after 24 hours in frozen blood. Detectable levels of early and late apoptosis as well as increased levels of ROS were detectable in frozen sheep blood samples. The addition of ascorbic acid partly reversed this effect and reduced the loss in cell viability. The response of the rat and sheep blood sample preparations to genotoxic compounds ex vivo showed that EMS caused comparable dose-dependent genotoxic effects in all sample preparations (fresh and frozen) as detected by the Comet assay. In contrast, the effects of CdCl2 were dependent on the duration of exposure as well as the sample preparation. The analysis of leukocyte subsets in frozen sheep blood showed no alterations in the percentages of T and B lymphocytes but led to a major decrease in the percentage of granulocytes compared to those in the fresh samples. The percentages of IFN-γ and IL-4 but not IL-6 positive cells were comparable between fresh and frozen sheep blood after 4 hour stimulation with phorbol 12-myrisate 13-acetate and ionomycin (PMA+I). These results show that frozen blood gives comparable responses to fresh blood samples in the toxicological and immune assays used.

Investigating the role of Fas (CD95) signalling in the modification of innate immune induced inflammation

Background/Aim: It has been demonstrated that a number of pathologies occur as a result of dysregulation of the immune system. Whilst classically associated with apoptosis, the Fas (CD95) signalling pathway plays a role in inflammation. Studies have demonstrated that Fas activation augments TLR4-mediated MyD88-dependent cytokine production. Studies have also shown that the Fas adapter protein FADD is required for RIG-I-induced IFNβ production. As a similar signalling pathway exists between RIG-I, TLR3 and the MyD88- independent of TLR4, we hypothesised that Fas activation may modulate both TLR3- and TLR4-induced cytokine production. Results: Fas activation reduced poly I:C-induced IFNβ, IL-8, IL-10 and TNFα production whilst augmenting poly I:C-, poly A:U- and Sendai virus-induced IP-10 production. TLR3-, RIG-I- and MDA5-induced IP-10 luciferase activation were inhibited by the Fas adapter protein FADD using overexpression studies. Poly I:C-induced phosphorylation of p-38 and JNK MAPK were reduced by Fas activation. Overexpression of FADD induced AP-1 luciferase activation. Point mutations in the AP-1 binding site enhanced poly I:C-induced IP- 10 production. LPS-induced IL-10, IL-12, IL-8 and TNFα production were enhanced by Fas activation, whilst reducing LPS-induced IFNβ production. Absence of FADD using FADD-/- MEFs resulted in impaired IFNβ production. Overexpression studies using FADD augmented TLR4-, MyD88- and TRIF-induced IFNβ luciferase activation. Overexpression studies also suggested that enhanced TLR4-induced IFNβ production was independent of NFκB activation. Conclusion: Viral-induced IP-10 production is augmented by Fas activation by reducing the phosphorylation of p-38 and JNK MAPKs, modulating AP-1 activation. The Fas adapterprotein FADD is required for TLR4-induced IFNβ production. Studies presented here demonstrate that the Fas signalling pathway can therefore modulate the immune response. Our data demonstrates that this modulatory effect is mediated by its adapter protein FADD, tailoring the immune response by acting as a molecular switch. This ensures the appropriate immune response is mounted, thus preventing an exacerbated immune response.

Repeated concanavalin A challenge in mice induces an interleukin 10-producing phenotype and liver fibrosis.

Weekly injections of Concanavalin A (Con A) were performed in BALB/c mice to evaluate the pattern of cytokine production and liver injury. High serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), IL-4, and interferon gamma (IFN-gamma) were found in the serum after the first 2 injections of Con A but rapidly decreased from the third injection. Conversely, IL-10 serum levels after repeated Con A challenge increased by 7 times from week 1 to 20. In vivo depletion studies indicated that CD4(+) T cells are essential in IL-10 production. Hepatocyte necrosis was only observed after the first injections of Con A whereas centrilobular inflammatory infiltrates persisted up to 20 weeks. Perisinusoidal liver fibrosis was also increasingly detected in BALB/c mice, whereas no fibrous change was observed in nude mice after 6 weeks of Con A challenge. The number of stellate cells, detected by immunostaining, increased after 20 weeks of Con A injections. Liver cytokine messenger RNA (mRNA) expression after 20 weeks showed expression of transforming growth factor beta1 (TGF-beta1), IL-10, and IL-4 whereas IL-2 was no more expressed. The present study shows that mice repeatedly injected with Con A develop liver fibrosis. The cytokine-release pattern observed after 1 injection of Con A is rapidly shifted towards an immunomodulatory phenotype characterized by the systemic production of large amounts of IL-10.

Diversion of intestinal flow decreases the numbers of interleukin 4 secreting and interferon gamma secreting T lymphocytes in small bowel mucosa.

BACKGROUND/AIMS: The intestinal immune system faces large amounts of antigens, and its regulation is tightly balanced by cytokines. In this study, the effect of intestinal flow diversion on spontaneous secretion of interleukin (IL)-4 and interferon (IFN)- gamma was analysed. METHODS: Eight patients (two with Crohn's disease, four with ulcerative colitis, and two with previous colon cancer) carrying a double lumen small bowel stoma after a total colectomy procedure were included in the study. For each patient, eight biopsy samples were taken endoscopically from both the diverted and non-diverted part of the small bowel. Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) were isolated separately and assayed for numbers of cells spontaneously secreting IL-4 and/or IFN-gamma by an ELISPOT technique. RESULTS: Compared with the non-diverted mucosa, a significant decrease in the number of spontaneously IFN-gamma secreting CD3 lymphocytes was observed in the diverted small bowel mucosa among both IELs (p = 0.008) and LPLs (p = 0.007). The same results, although less significant, were obtained for IL-4, especially in LPLs (p = 0.01). CONCLUSION: The intestinal content influences the spontaneous secretion of IFN-gamma and IL-4 by intestinal lymphocytes. These results could help to elucidate the anti-inflammatory role of split ileostomy in patients suffering from inflammatory bowel diseases.

Helicobacter pylori modulation of gastric and duodenal mucosal T cell cytokine secretions in children compared with adults.

BACKGROUND: In contrast to adults, ulcers are un-common in Helicobacter pylori-infected children. Since immunological determinants influence the outcome of H. pylori infection, we have investigated mucosal T cell responses in H. pylori-infected children and compared them with those of adults and negative controls. MATERIAL AND METHODS: Mucosal biopsies were obtained from 43 patients undergoing an upper GI endoscopy for dyspeptic symptoms. The concentrations of released cytokines and the density of CD3+, CD25+ and CD69+cells were evaluated by flow cytometry, and the numbers of cytokine-secreting cells were measured by ELISPOT. RESULTS: The numbers of isolated antral CD3+ lymphocytes were only significantly raised in infected adults compared with noninfected controls (p < 0.05), whereas the proportion of CD3+ cells expressing activation markers (CD25 or CD69) remained low. In the stomach, IFN-gamma concentrations increased in infected children and infected adults compared with controls (p < 0.05), but IFN-gamma concentrations were tenfold lower in children than in adults (p < 0.01). IL-2, IL-4, IL-10 and TNF-alpha concentrations were similar in infected and in uninfected children and adults. In contrast, in the duodenum, IFN-gamma, as well as IL-4 and IL-10 concentrations were only increased in infected children compared with controls (p < 0.05). The concentrations of these cytokines were similar in both groups of adults who, however, like children, displayed a higher number of duodenal IL-4-secreting cells compared to controls (p < 0.05). CONCLUSION: These results suggest that IFN-gamma secretion in the stomach of H. pylori-infected patients is lower in children than in adults. This could protect children from development of severe gastro-duodenal diseases such as ulcer disease. In addition, infected patients are characterised by a dysregulation of the mucosal cytokine secretion at distance from the infection site.

IL-12 inhibits in vitro immunoglobulin production by human lupus peripheral blood mononuclear cells (PBMC).

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by polyclonal B cell activation and by the production of anti-double-stranded (ds) DNA antibodies. Given the inhibitory effects of IL-12 on humoral immune responses, we investigated whether IL-12 displayed such an activity on in vitro immunoglobulin production by SLE PBMC. Spontaneous IgG, IgG1, IgG2, IgG3 and IgM antibody production was dramatically reduced by addition of IL-12. These results were confirmed by Elispot assays detecting IgG- and anti-dsDNA-secreting cells. While IL-6 and TNF titres measured in PBMC supernatants were not modified by addition of IL-12, interferon-gamma (IFN-gamma) titres were up-regulated and IL-10 production down-regulated. Since addition of IFN-gamma did not down-regulate immunoglobulin production and since the inhibitory activity of IL-12 on immunoglobulin synthesis was not suppressed by anti-IFN-gamma antibody, we concluded that the effect of IL-12 on immunoglobulin production was not mediated through IFN-gamma. Our data also argue against the possibility that down-regulation of endogenous IL-10 production was responsible for the effect of IL-12. Thus, inhibition of IL-10 production by IFN-gamma was not accompanied by inhibition of immunoglobulin production, and conversely, restoration of IL-10 production by anti-IFN-gamma antibody did not suppress the inhibitory activity exerted by IL-12 on immunoglobulin production. Taken together, our data indicate that reduction of excessive immunoglobulin and anti-dsDNA antibody production by lupus PBMC can be achieved in vitro by IL-12, independently of IFN-gamma and IL-10 modulation.

Spontaneous secretion of interferon gamma and interleukin 4 by human intraepithelial and lamina propria gut lymphocytes.

BACKGROUND: Cytokines secreted by intestinal T lymphocytes probably play a critical role in regulation of the gut associated immune responses. AIMS: To quantify interferon gamma (IFN-gamma) and interleukin 4 (IL-4) secreting cells (SC) among human intraepithelial (IEL) and lamina propria (LPL) lymphocytes from the duodenum and right colon in non-pathological situations and in the absence of in vitro stimulation. PATIENTS: Duodenal and right colonic biopsy specimens were obtained from patients with no inflammation of the intestinal mucosa. METHODS: Intraepithelial and lamina propria cell suspensions were assayed for numbers of cells spontaneously secreting IFN-gamma and IL-4 by a two site reverse enzyme linked immunospot technique (ELISPOT). RESULTS: The relatively high proportion of duodenal lymphocytes spontaneously secreting IFN-gamma (IEL 3.6%; LPL 1.9%) and IL-4 (IEL 1.3%; LPL 0.7%) contrasted with the very low numbers of spontaneously IFN-gamma SC and the absence of spontaneously IL-4 SC among peripheral blood mononuclear cells. In the basal state, both IFN-gamma and IL-4 were mainly produced by CD4+ cells. Within the colon, only 0.2% of IEL and LPL secreted IFN-gamma in the basal state, and 0.1% secreted IL-4. CONCLUSIONS: Compared with peripheral lymphocytes substantial proportions of intestinal epithelial and lamina propria lymphocytes spontaneously secrete IFN-gamma and/or IL-4. These cytokines are probably involved in the normal homoeostasis of the human intestinal mucosa. Disturbances in their secretion could play a role in the pathogenesis of gastrointestinal diseases.

Antigen-specific responses assessment for the evaluation of Bordetella pertussis T cell immunity in humans.

Measurement of antigen-specific T cell responses is an adjunctive parameter to evaluate protection induced by a previous Bordetella pertussis infection or vaccination. The assessment of T cell responses is technically complex and usually performed on fresh peripheral blood mononuclear cells (PBMC). The objective of this study was to identify simplified methods to assess pertussis specific T cell responses and verify if these assays could be performed using frozen/thawed (frozen) PBMC. Three read-outs to measure proliferation were compared: the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution test, the number of blast cells defined by physical parameters, and the incorporation of (3)H-thymidine. The results of pertussis-specific assays performed on fresh PBMC were compared to the results on frozen PBMC from the same donor. High concordance was obtained when the results of CFSE and blast read-outs were compared, an encouraging result since blast analysis allows the identification of proliferating cells and does not require any use of radioactive tracer as well as any staining. The results obtained using fresh and frozen PBMC from the same donor in the different T cell assays, including IFNγ and TNFα cytokine production, did not show significant differences, suggesting that a careful cryopreservation process of PBMC would not significantly influence T cell response evaluation. Adopting blast analysis and frozen PBMC, the possibility to test T cell responses is simplified and might be applied in population studies, providing for new instruments to better define correlates of protection still elusive in pertussis.