Post challenging serum cytokine profile (Th1 & Th2) in the vaccinated mice (Balb/C) with a new formulation of Leishmania major antigen.

OBJECTIVE: The aim of this study was to carry out experiments further to our previous new formulation to modify the Leishmania major antigen that had satisfactory results previously. METHODS: In this study we made a preliminary, new vaccine with the same methodology and selected two injection doses (100&200 µg/o.1 mL), three injection Groups: Leishmania plus BCG (LB), Leishmania plus new adjuvant (Teucrium Polium) [LT], Leishmania plus BCG and Teucrium Polium (LBT), and one susceptible mouse Group (Balb/c) and measure two types of cytokines: Th1 (IFN-γ, IL-12) and Th2 (IL-4, IL-10) We prepared crude antigen combinations by five different methods using antigens from L. major parasites. Phase I was done in the animal model. In our study, Leishmania antigen was examined both with BCG and the new adjuvant (TP) in three Groups in two injection doses (100.200 µg/1 mL) and Balb/c mice. RESULTS: Our results showed that in three injection Groups (LB, LT and LBT) that received each or both BCG and TP as adjutant with injection doses of 100 and 200 µg/1 mL with two booster doses: the LBT Group had the lowest IFNγ and highest IL-12 value, LT and LB Groups have equal IL-12, but LB have more IFNγ and IL-10 but less than IL-4 in the LT Group. CONCLUSION: In this study, the LBT Group has statistical differences regarding IL-12 and IL-10 from the other Groups.

Antioxidants and proinflamatory cytokines in the sera of patients with cutaneous leishmaniasis.

BACKGROUND: Leishmania is a significant health problem in many parts of the world. Tumor necrosis factor (TNF) plays an essential role in Leishmania major infections. OBJECTIVE: To study the pro-inflammatory cytokines and antioxidants in four groups of cutaneous leishmaniasis patients. METHODS: 39 patients were divided into four groups of: 1) active (acute phase of treatment); 2) non-healing (received treatment for almost two years without recovery); 3) healing (recovered upon treatment); and 4) healed (previously received treatment and achieved complete remission) patients. Serum levels of pro-inflammatory cytokines (IL-1B, TNF-α, IL-6) and serum antioxidant levels were measured by ELISA and FRAP assays, respectively. RESULTS: While serum antioxidant levels were elevated in the non-healing group, there was no difference among other groups of patients and healthy controls in this regard. Interleukin-1ß showed the highest level in the non-healing group followed by the other groups of patients. The mean serum IL-6 level was highest in the non-healing group, but showed no significant change in the other groups. TNF-α and IL-1ß levels were non-significantly elevated in the sera of active and non-healing patients. CONCLUSION: Pro-inflammatory cytokines IL-1ß, TNF-α, IL-6 maybe related to the progression of leishmaniasis. Serum antioxidant levels maybe correlated with patient response to drug treatment.

Listeria monocytogenes activated dendritic cell based vaccine for prevention of experimental tumor in mice.

BACKGROUND: The use of dendritic cells (DCs) as a cellular adjuvant provides a promising approach in immunotherapy of cancer. It has been demonstrated that Listeria monocytogenes activated DCs pulsed ex vivo with tumor antigens trigger a systemic Th1-biased specific immune response and a single dose of this vaccine will cause a considerable anti tumor immunity. OBJECTIVE: The present study was designed to evaluate the ability of multiple doses of tumor antigen-pulsed DCs, matured in the presence of Listeria monocytogenes components in induction of a potent anti-tumor response and the prevention of tumor formation in an experimental model. METHODS: Bone-marrow derived DCs (BMDCs) were cultured in the presence of GM-CSF and IL-4. After 5 days, tumor lysates with/without Listeria monocytogenes lysate were added to the culture media for another 2 days. Mice received mature and tumor antigen pulsed dendritic cells subcutaneously in 3 groups. Tumor growth was monitored and two weeks after immunotherapy, cytotoxic activity of CD8+ T cells was evaluated in different groups. RESULTS: According to the findings, repeated doses of vaccine did not lead to a significant increase in the activity of cytotoxic T cells and decreased tumor growth of immunized animals. CONCLUSION: The current study suggests that increased doses of vaccine do not have sufficient efficiency for prevention of tumor induction. Generation of T regulatory responses upon repeated doses of such vaccines should be considered in future investigations.

Dendritic cell maturation with CpG for tumor immunotherapy.

BACKGROUND: Bacterial DNA has immunostimulatory effects on different types of immune cells such as dendritic cells (DCs). Application of DCs as a cellular adjuvant represents a promising approach in the immunotherapy of infectious disease and cancers. OBJECTIVE: To investigate the effect of tumor antigen pulsed DCs in the presence of CpG-1826 in treatment of a murine model of cancer. METHODS: WEHI-164 cells (Balb/c derived fibrosarcoma cell line) were injected subcutaneously in the right flank of mice. Bone marrow cells were cultured in the presence of GM-CSF and IL-4. After 5 days, tumor lysate, CpG-1826, and oligodeoxynucleosides, as control, were added to the culture media and incubated for 2 days. Cytokine production in DCs culture media was measured by ELISA. Then DCs were injected subcutaneously around the tumor site in the right flank of mice. Tumor growth rate was monitored in case and control groups. Two weeks after DCs immunotherapy, cytotoxic assay was conducted using various amounts of effector (splenic T cells) and target cells (WEHI-164 or CT26) for 6 h. RESULTS: Immunotherapy with DCs treated with CpG led to a significant increase in the activity of cytotoxic T cells and decreased tumor growth in immunized mice. In the control group which received DCs without CpG treatment, no change in cytotoxic activity and tumor growth rate was detected. CONCLUSION: The current study suggests that specific anti tumor immune responses can be induced by DCs matured with CpG and proposes CpG usage in DCs targeted clinical strategies.

Detection of opioid receptors on murine lymphocytes by indirect immunofluorescence: mature normal and tumor bearing mice lymphocytes.

Opioid peptides modulate immune responses via ligation to classical opioid receptors (mu, delta and kappa), expressed on immune cells, or in an indirect fashion via the central nervous system. The combination of immunofluorescent technique and flow cytometry has proven to be sensitive methods for detection of opioid receptors on leukocytes. In the current study a fluorescein isothiocyanate-conjugated naltrexone (FITC-NTX) derivative in the absence or presence of naltrexone, as a competitor, was used to detect opioid receptors on thymocytes and then on splenocytes of normal and tumor bearing Balb/c mice. Tumor bearing mice were made by intraperitoneal injection of fibrosarcoma cell line. In a two weeks interval, tumor grew and then mice splenocytes were harvested. Cells were incubated with FITC-NTX alone (direct fluorescence), or FITC-NTX followed by biotin-conjugated anti-fluorescein IgG and extravidin-R-phycoerythrin (indirect immunofluorescence). Using flow cytometry we found that, with direct fluorescence staining there is only nonspecific cell staining. In contrast, indirect staining of cells demonstrated labeling of opioid receptors. Thymocytes displayed 37.5+/-7% specific labeling by current staining procedure. However, this specific staining was 17.2+/-4% and 7.5+/-2% in splenocytes of normal and tumor bearing mice, respectively. Taken together, these results showed that, direct fluorescence staining failed to stain opioid receptors expressed on lymphocytes. These receptors can only be detected by a biotin-streptavidin amplification procedure. We also found that the level of opioid receptors on mature lymphocytes is less than that of immature ones and are even lesser in the tumor bearing mice lymphocytes.