Effects of thymulin 5cH in granuloma evolution and B1 cell differentiation: an experimental model to understand its biological mechanisms

In previous studies, we found that thymulin (a thymic hormone), when prepared in homeopathic 5cH potency, had the property to improve the productive performance of broiler chickens infected with reovirus, as well as modulate the development of Ehrlich tumor and granuloma inflammatory lesions in mice by immune-mediated mechanisms. The aim of the present work was to study the immunomodulatory mechanisms of thymulin 5cH in a granuloma experimental model, by subcutaneous inoculation of BCG in mice, focusing the B-1 cells and zinc involvement in this process. Three groups of male Balb/c SPF mice (group A treated with thymulin 5cH, group B treated with thymulin 5cH incubated in Chelex ® - a zinc chelant - and group C, control, treated with vehicle) were inoculated with BCG in the left footpad and subcutaneous granuloma and spleen were harvested for histomorphometry analysis, after 7, 14 and 21 days. Ziehl-Neelsen, HE and Prussia Blue staining methods were used. Flow cytometry was also used in the same times to characterize and quantify peritoneal cells. Positive cells for CD11b (activated phagocytes, B-1 cells), CD19 (B-1 and B-2 cells), CD23 (negative B-1 cells, positive B2 cells) and CD5 (B-1a cells) were analyzed in a FACS Calibur (BD) device. Statistical analysis was performed using Kruskal - Wallis / Dunn for nonparametric evaluations and ANOVA / Tuckey-Krammer for the parametric ones. The X² method was used to evaluate the cell count in flow cytometry. P values ≤ 0.05 were considered statistically significant. Mice treated with thymulin 5cH presented higher macrophage activity and increase in the follicular area were seen in spleen after 7 days. Increase in gross lesion diameter and decrease in local BCG infection were seen after 21 days. At this time, the flow cytometry demonstrated the increase in peritoneal phagocytes derived from B-1 cells in thymulin 5cH treated mice, independently of Chelex ® incubation. The incubation of thymulin 5cH with Chelex ® blocked its effects only upon the number of B2 cells in the peritoneum and reduces Mn levels in the medicine solution. We conclude that thymulin 5CH modulates the BCG-induced granuloma through more than one mechanism, especially by peritoneal B1 cell differentiation into phagocytes.

Differentiation and modulation of phagocyte activity in murine granuloma after treatment with thymulin 5cH

In previous studies, it was found that thymulin (thymic hormone), when prepared in homeopathic 5CH dilution, can modulate diseases progress and immune processes, such as virus infection in poultry and experimental tumor development. This project aims to study the immune modulatory mechanisms of thymulin 5CH in granulomatous experimental model induced by BCG in mice, with special focus on the phagocytes differentiation from peritoneal B1 stem cells and on the activity of these cells and lymphocyte cooperation at the injury site. Male adult Balb/c mice were inoculated with BCG into the footpad to induce granuloma and its cells were evaluated by histomorphometric methods (Ziehl-Neelsen staining of macrophages) and flow cytometry. The phenotypic characterization of phagocytes, T and B lymphocytes in the peritoneum and regional lymph node was done. Thus, CD11b (activated phagocytes and B1 cells), CD19 (B1 cells and B2), CD23 (B2 cells), CD5 (B1 cell subtypes and T cells), CD4, CD8 (effective T lymphocytes) and CD25 (regulatory lymphocytes) positive cells were quantified. After 21 days of infection, the differentiation of B1 peritoneal stem cells into phagocytes reached the peak, being higher in thymulin-treated mice (X2, p=0.0001). Simultaneously, the number of infected phagocytes in the lesion decreased (KW, p=0.001), indicating remission of the infection. The number of B1 derived phagocytes, CD4+ and CD8+ T lymphocytes in the local lymph node increased in thymulin-treated mice (X2, p=0.0001), at the same time. No difference was seen regarding to CD25+ cells. The results show that thymulin 5CH treatment is able to improve the granuloma inflammatory process by modulating local and systemic phagocyte differentiation, as well as T cell migration into the local lymph node.

Chronic inflammatory response modulation against Leishmania (L.) amazonensis by homeopathic thymulin and antimonium crudum inBalb/c mice

In previous studies it was found that thymulin 5cH (thymic hormone) can modulate immune processes in several diseases. Additionally, the Antimonium crudum has used in dogs bearing leishmaniosis, according to the similia principle. We studied the inflammatory and immune modulation by thymulin 5CH and Antimonium crudum 30CH treatment in mice experimentally inoculated with Leishmania (L.) amazonensis. Male adult Balb/c mice were inoculated with Leishmania (2x105 promastigotes) into the footpad to induce inflammatory response and peritoneum and spleen cells were evaluated by flow cytometry after 60 days. Animals were divided in 3 groups (n=10): thymulin 5cH, Antimonium crudum 30cH and vehicle /control. Treatment was made in blind, daily, in water/alcohol 30% diluted 1:2500 in drinking water, during all experimental period. CD11b (activated phagocytes and B1 cells), CD19 (B1 cells and B2), CD4 and CD8 (effective T lymphocytes) markers were used to identify immune cells subsets in peritoneal washing fluid and spleen cell suspension. Mice treated with thymulin 5cH presented increase in peritoneal and spleen B1 stem cells (X2=0.0001) and higher CD8+/CD4+ ratio in spleen, regarding to the control. Also, Antimonium crudum 30CH induced a mild increase in B1 cells in peritoneum and spleen ( both X2, p=0.0001). Further histological analysis of the primary lesion will be done in the next step, to elucidate the impact of these findings in the disease evolution. The results show that both treatments stimulate B1 stem cell proliferation and suggest the cooperation of T spleen lymphocytes in the process.

The Hidden Function of Vitamin D

AIM: There are no reports regarding the influence of vitamin D on thymosin ß4 and the cluster of differentiation CD4 levels which are important for maintaining a healthy immune system. Consequently, we aimed to explore this relationship through a study.MATERIAL AND METHODS: The study was carried out on 35 subjects, screened for 25-hydroxy vitamin D[25 (OH) D] using ELISA method and they were divided into two groups: Group 1 consists of 10 healthy subjects with sufficient vit. D level > 24.8 ng/ml. Group 2 consists of 25 subjects suffering, severely, from vitamin D deficiency at level < 11.325 ng/ml. Also, Thymosin ß4, CD4 and zinc levels were performed.RESULTS: There were significant differences between the two groups in the concentration levels of thymosin β4, as the group 1 has shown higher levels (P = 0.005). Whereas, CD4 and zinc levels didn’t show any significant difference between the two groups. At the same time, a significant positive correlation has been observed between vitamin D, thymosin β4, and CD4 at (r = 0.719; P = 0.001), and (r = 0.559, P = 0.001) respectively.CONCLUSION: We concluded that vitamin D may be an essential factor that influence or determine the level of thymosin β4. This study is the first that focused on demonstrating that sufficient level of vitamin D may have the ability to influence the thymic hormone thymosin β4 levels. Further studies on large scale of subjects are needed to explore the positive correlation we had found between vitamin D and thymosin β4 and CD4.