Treg/Th17 cell balance and phytohaemagglutinin activation of T lymphocytes in peripheral blood of systemic sclerosis patients.

AIM: To investigate T-cell activation, the percentage of peripheral T regulatory cells (Tregs), Th17 cells and the circulating cytokine profile in systemic sclerosis (SSc). METHODS: We enrolled a total of 24 SSc patients and 16 healthy controls in the study and divided the patients as having diffuse cutaneous SSc (dcSSc, n = 13) or limited cutaneous SSc (lcSSc, n = 11). We performed a further subdivision of the patients regarding the stage of the disease - early, intermediate or late. Peripheral venous blood samples were collected from all subjects. We performed flow cytometric analysis of the activation capacity of T-lymphocytes upon stimulation with PHA-M and of the percentage of peripheral Tregs and Th17 cells in both patients and healthy controls. We used ELISA to quantitate serum levels of human interleukin (IL)-6, IL-10, tissue growth factor-ß1 (TGF-ß1), and IL-17A. RESULTS: We identified a decreased percentage of CD3+CD69+ cells in PHA-stimulated samples from SSc patients in comparison with healthy controls (13.35% ± 2.90% vs 37.03% ± 2.33%, P < 0.001). However, we did not establish a correlation between the down-regulated CD3+CD69+ cells and the clinical subset, nor regarding the stage of the disease. The activated CD4+CD25+ peripheral lymphocytes were represented in decreased percentage in patients when compared to controls (6.30% ± 0.68% vs 9.36% ± 1.08%, P = 0.016). Regarding the forms of the disease, dcSSc patients demonstrated lower frequency of CD4+CD25+ T cells against healthy subjects (5.95% ± 0.89% vs 9.36% ± 1.08%, P = 0.025). With regard to Th17 cells, our patients demonstrated increased percentage in comparison with controls (18.13% ± 1.55% vs 13.73% ± 1.21%, P = 0.031). We detected up-regulated Th17 cells within the lcSSc subset against controls (20.46% ± 2.41% vs 13.73% ± 1.21%, P = 0.025), nevertheless no difference was found between dcSSc and lcSSc patients. Flow cytometric analysis revealed an increased percentage of CD4+CD25-Foxp3+ in dcSSc patients compared to controls (10.94% ± 1.65% vs 6.88% ± 0.91, P = 0.032). Regarding the peripheral cytokine profile, we detected raised levels of IL-6 [2.10 (1.05-4.60) pg/mL vs 0.00 pg/mL, P < 0.001], TGF-ß1 (19.94 ± 3.35 ng/mL vs 10.03 ± 2.25 ng/mL, P = 0.02), IL-10 (2.83 ± 0.44 pg/mL vs 0.68 ± 0.51 pg/mL, P = 0.008), and IL-17A [6.30 (2.50-15.60) pg/mL vs 0 (0.00-0.05) pg/mL, P < 0.001] in patients when compared to healthy controls. Furthermore, we found increased circulating IL-10, TGF-ß, IL-6 and IL-17A in the lcSSc subset vs control subjects, as it follows: IL-10 (3.32 ± 0.59 pg/mL vs 0.68 ± 0.51 pg/mL, P = 0.003), TGF-ß1 (22.82 ± 4.99 ng/mL vs 10.03 ± 2.25 ng/mL, P = 0.031), IL-6 [2.08 (1.51-4.69) pg/mL vs 0.00 pg/mL, P < 0.001], and IL-17A [14.50 (8.55-41.65) pg/mL vs 0.00 (0.00-0.05) pg/mL, P < 0.001]. Furthermore, circulating IL-17A was higher in lcSSc as opposed to dcSSc subset (31.99 ± 13.29 pg/mL vs 7.14 ± 3.01 pg/mL, P = 0.008). Within the dcSSc subset, raised levels of IL-17A and IL-6 were detected vs healthy controls: IL-17A [2.60 (0.45-9.80) pg/mL vs 0.00 (0.00-0.05) pg/mL, P < 0.001], IL-6 [2.80 (1.03-7.23) pg/mL vs 0.00 pg/mL, P < 0.001]. Regarding the stages of the disease, TGF-ß1 serum levels were increased in early stage against late stage, independently from the SSc phenotype (30.03 ± 4.59 ng/mL vs 13.08 ± 4.50 ng/mL, P = 0.017). CONCLUSION: It is likely that the altered percentage of Th17 and CD4+CD25-FoxP3+ cells along with the peripheral cytokine profile in patients with SSc may play a key role in the pathogenesis of the disease.

<b>A Japanese Herbal Medicine, Shigyakusan, Induces Prolonged Survival of Fully MHC-Mismatched Murine Cardiac A</b><b>llografts</b> / 日本東洋医学雑誌

<p><b>Background </b>: Shigyakusan, a 4-component Japanese herbal medicine (Paeoniae radix, Aurantii fructus immaturus, Glycyrrhizae radix and Bupleuri radix), is used not only for cholecystitis and gastritis as an antiinflammatory agent, but also for anxiety neurosis and insomnia as an anti-anxiety agent.<br><b>Methods </b>: We investigated the effects of shigyakusan on alloimmune responses in fully MHC-mismatched murine cardiac allograft transplantation. CBA mice underwent transplantation of a C57BL/6 heart and received shigyakusan or one component of shigyakusan administered orally from the day of transplantation until 7 days afterward. Histologic studies, cytokine measurements, and flow cytometry assessments were performed.<br><b>Results </b>: Untreated CBA recipients acutely rejected C57BL/6 cardiac grafts (median survival times [MST], 7 days). On the other hand, CBA transplant recipients given shigyakusan had significantly prolonged C57BL/6 allograft survival (MST, 22.5 days). MSTs for C57BL/6 transplant recipients given Paeoniae radix, Aurantii fructus immaturus, Glycyrrhizae radix and Bupleuri radix were 11, 9.5, 18.5 and 8 days, respectively. Additionally, flow cytometry studies showed that the percentage of CD25+Foxp3+ cell populations in CD4+ cells was increased in transplant recipients given shigyakusan.<br><b>Conclusion </b>: Shigyakusan induced hyporesponsiveness to fully MHC-mismatched allogeneic cardiac allografts and may generate CD4+CD25+Foxp3+ cells in our model.</p>

T-regulatory lymphocytes in peripheral blood of gastric and colorectal cancer patients.

AIM: To assess the absolute number of T-regulatory cells (Tregs; CD4+CD25+Foxp3+) in the peripheral blood of gastric and colorectal cancer patients. METHODS: We enrolled 70 cancer patients (33 gastric cancer, 37 colorectal cancer) and 17 healthy volunteers. The CD3+CD4+ lymphocytes and CD4+CD25+Foxp3+ Tregs in the peripheral blood were analyzed with flow cytometry. The absolute numbers of Tregs were calculated based on the CD4+CD25+Foxp3+ cells percentage of CD3+CD4+ cells and the absolute numbers of CD3+CD4+ cells per microliter. RESULTS: The mean number of CD4+CD25+Foxp3+ cells per microliter in colorectal cancer patients was 15.7 (SD: 21.8), for gastric cancer patients 12.2 (SD: 14.3), and for controls 17.5 (SD: 11.4). The absolute number of Tregs was significantly lower in gastric cancer patients than in controls (P = 0.026). There was no statistically significant difference for gastric vs colorectal cancer or colorectal cancer vs controls. The absolute number of Tregs was also significantly depressed in N+ vs N⁻ cancer patients [22.0 (27.7) vs 10.1 (9.0), P = 0.013], and in the subgroup of gastric cancer patients [30.3 (27.6) vs 9.6 (8.0), P = 0.003]. No statistical difference was observed in the proportion of Tregs in the CD4+ population between the groups. CONCLUSION: The absolute number of Tregs in peripheral blood of gastric cancer but not colorectal cancer patients was significantly decreased in comparison with that in healthy controls.