The effects of Annual SZ drug on dynamic changes in lymphocytes and cytokines of COVID-19 patients: A randomized clinical trial.

BACKGROUND: The search for a potent anti-coronavirus therapy has remained an overwhelming task since the outbreak of COVID-19. Annual SZ is a novel formulation of artemisinin and its derivatives. We aim to investigate the effect of Annual SZ on clinical outcomes, cellular immune responses, and cytokine changes in COVID-19 patients. METHODS: This study included 80 COVID-19 hospitalized patients, which were randomly allocated into two groups (intervention and control). Both groups received standard supportive treatment. In addition, the intervention group (n = 40) received Annual SZ syrup, and the control group (n = 40) received a placebo. Dynamic changes in lymphocytes, cytokines, and clinical status were evaluated since hospital admission to 7 and 14 days after treatment. RESULTS: The dynamic count of total T lymphocytes and T lymphocyte subsets (CD4+ and CD8+) in the Annual SZ group was significantly higher than the placebo group (p < 0.05). In addition, Programmed Death 1 (PD-1) was significantly increased in the CD4+ and CD8+ T cells in the placebo group compared with the Annual SZ group (p < 0.05). Also, the CD4+/CD8+ ratio was not significantly different between the groups (p > 0.9). Moreover, IL-6 levels were significantly reduced (p < 0.05), while IL-4 and IFN-γ levels were not statistically different between the two groups (p > 0.05). CONCLUSION: This research indicated that the Annual SZ syrup significantly improved clinical status and lymphocyte frequency with less exhaustion of T lymphocytes and a reduction of inflammatory responses, which seems to be beneficial in the treatment process of COVID-19 patients.

TLR4 Polymorphisms (896A>G and 1196C>T) Affect the Predisposition to Diabetic Nephropathy in Type 2 Diabetes Mellitus.

PURPOSE: Type 2 diabetes mellitus (T2DM) is a disease with a steadily increasing incidence throughout the world. Some molecules regulating the innate immune responses such as toll-like receptor 4 (TLR4) have shown to be involved in late diabetic complications. This study aimed to investigate the association of TLR4 gene polymorphisms with clinicopathological aspects of T2DM in the Iranian population. PATIENTS AND METHODS: Two TLR4 896A>G and 1196C>T polymorphisms were assessed in 100 T2DM patients and 100 healthy controls using sequence-specific primers PCR. Demographic, anthropometric, and biochemical parameters were obtained from the participants. RESULTS: After logistic regression, in 1196C>T, a significant association was shown between diabetic nephropathy (DN) and CT genotype (P= 0.04, OR= 4.35, CI= (1.04-18.1)). TG level has increased significantly in both T2DM and control subjects with CT genotype (P= 0.027, OR= 1.005, 95% CI= (1.001-1.01)). For 896A>G variant, a significant association was also detected between AG genotype and increased oral glucose tolerance test (OGTT) level (P= 0.048, OR= 1.003, 95% CI= (1.00-1.005)). CONCLUSION: Although minor alleles of 1196C>T and 896A>G variants have not directly been associated with type 2 diabetes, by involving in the dysregulation of serum TG and blood sugar levels, they might increase the risk of DN.

Insulin-like growth factor-I promotes hepatic differentiation of human adipose tissue-derived stem cells.

There are numerous studies which provide support for the use of human adipose tissue-derived stem cells (hASCs) to generate hepatocyte-like cells. However, the produced cells exhibit only a certain level of differentiation, mainly due to inefficient induction conditions. Therefore, based on the important role of insulin-like growth factor (IGF-I) in hepatic function and development, in the current study we evaluated the differentiation efficacy of the mentioned factor to induce hASCs into functional hepatocyte-like cells. To investigate this, using a two-step protocol, hASCs were treated with a combination of HGF, Dex, and OSM in the presence or absence of IGF-I up to 21 days. Hepatic differentiation was evaluated by analyzing specific hepatocyte markers at different time points of differentiation induction. Increased expression of hepatocyte-specific genes including ALB, AFP, CK18, and HNF4a, downregulation of bile duct cells marker (CK19), the higher number of ALB positive cells, increased urea production together with higher glycogen deposit was observed upon the treatment of hASCs with the induction medium containing IGF-I compared to the other treatment. In conclusion, our findings suggest IGF-I as a potent inducer of hepatic differentiation of hASCs and its potential to generate more functional hepatocyte-like cells.

The role of LPD-nanoparticles containing recombinant major surface glycoprotein of Leishmania (rgp63) in protection against leishmaniasis in murine model.

CONTEXT: Leishmaniasis is a major public health problem. Despite numerous attempts, yet there is no effective vaccine against human leishmaniasis, mainly due to a lack of an effective vaccine delivery system as well as adjuvant. OBJECTIVE(S): The aim of this study was to evaluate the ability of recombinant glycoprotein 63 (rgp63) as a model of Leishmania antigen, entrapped in liposome-polycation-DNA (LPD) complexes nanoparticles in inducing cell mediated immune (CMI) response and protecting against L. major in BALB/c mice. MATERIALS AND METHODS: To this end, the abundant leishmania promastigote cell surface glycoprotein, gp63, was entrapped in nano-sized LPD (CpG) particles, (LPD (CpG)-rgp63), and BALB/c mice were immunized three times with either (LPD (CpG)-rgp63) or rgp63-CpG DNA or LPD (CpG) or free rgp63 and dextrose 5%. Various parameters including footpad thickness, splenic load of L. major parasites, rgp63-binding IgGs and also cytokine levels of rgp63-reactive T lymphocytes were then compared among different vaccinated animals. RESULTS: The lowest number of parasites in spleen, the higher levels of IgG2a after challenge infection, the minimal footpad swelling and high level of IFN-γ secretion, all indicated that adjuvants and antigen-delivery systems are essential in modifying immune responses; as mice received LPD (CpG)-rgp63 induced immune response stronger than the other groups. CONCLUSIONS: This study demonstrates that LPD nanoparticle is a promising and adaptable delivery system which could be modified towards specific vaccine targets to induce a more potent immune response in combination with rgp63.

Umbelliprenin induced both anti-inflammatory and regulatory cytokines in C57/BL6 mice.

OBJECTIVES: Umbelliprenin is a prenyloxy-coumarin with pharmacologically polyvalent activity. Several studies have shown Several studies have been shown its anti-inflammatory, anti-tumor, antioxidant, and antigenotoxic activities. However, the exact mechanism of action of this compound on the immune response has not yet been shown. Here, we investigated umbelliprenin effects on the predominance of Th1 and Th2 responses in normal C57/BL6 mice. MATERIALS AND METHODS: Umbelliprenin (2.5 mg/200 µl IP) were administered to six C57/BL6 mice every other day for 8 days. Paraffin and PBS-injected mice were enrolled as solvent and control groups, respectively (n=6 mice/group). IL-10, IFN-γ, and IL-4 levels were determined in sera and also in splenocytes culture supernatants in the presence of Con A (3 µg/ml) after 72 hr. H&E staining of paraffin embedded blocks was performed for lung and liver tissues of mice. RESULTS: Umbelliprenin could significantly increase the secretion of IFN-γ and IL-4 in sera and IL-10 in splenocytes cultures. Comparison of IFN-γ/IL-4 in the sera and splenocytes culture supernatants showed lower ratios in umbelliprenin treated mice than in solvent and untreated groups. CONCLUSION: The in vivo study showed that umbelliprenin could induce anti-inflammatory responses via the predominance of Th2 cells and some regulatory responses in C57/BL6 mice.

Interleukin-23 Receptor Gene Variants in Acute Lymphoblastic Leukemia and Their Relation to Prognostic Factors.

BACKGROUND: Interleukin (IL)-23 has an important role in tumor immune regulation. OBJECTIVE: To investigate the possible association of interleukin-23 receptor (IL23R) gene variants rs1884444, rs10889677 and rs11209026 with development of acute lymphoblastic leukemia (ALL). METHODS: The IL23R variants were studied in 164 ALL patients and compared to 175 healthy controls by polymerase chain reaction-restriction fragment length polymorphism. The relationship between these variants and clinical and laboratory features of the patients and response to therapy were evaluated. RESULTS: No significant differences in genotype and allele frequencies existed between patients and controls. The rs1884444TG genotype was significantly lower in patients who relapsed (24.2%) compared to those without relapse (55.9%, p=0.006). Fewer patients who relapsed had evidence of the G allele (p=0.034). The TG genotype was associated with a longer complete remission at 1804±116 days compared to other genotypes (<1217 days, p=0.028), however, this result was not significant in multivariate analysis. The rs10889677 AA genotype and A allele were associated with age (p<0.041) and platelet number (p=0.03) in precursor-B cell ALL (B-ALL) patients. Both occurred more frequently in patients aged 2-10 years (63.6% and 66%, respectively) and in those with platelets >100×10ˆ3 µL (68.4% and 52.4%, respectively). CONCLUSION: Our findings showed a lack of association of the studied polymorphisms with the risk of ALL. The influence of the rs1884444 polymorphism on relapse rate and association of rs10889677 AA genotype with favorable prognostic factors suggest the effect of the studied polymorphisms on ALL response to therapy and prognosis.

An elevated pro-inflammatory cytokines profile in Behcet's disease: A multiplex analysis.

The serum levels of sixteen cytokines related to innate immunity, Th1, Th2 and Th17 cells in the sera of 44 patients with Behcet's Disease (BD) and 44 healthy controls have been investigated using the cytokine array technique. Among the cytokines related to innate immunity, the levels of IL-1α, IL-1 ß, IL-6, IL-12, IL-15 and TNF-α were statistically higher in BD patients than healthy controls. In the case of Th1- and Th17-related cytokines, IL-2, IFN-γ, IL-17 and IL-23 were significantly higher in patients. From Th2-related cytokines, only IL-13 showed statistically higher levels in patients than controls. Among different evaluated cytokines, the differences in IL-1 α, IL-1 ß, IL-6 and Æ©innate-related cytokines were more prominent between cases and controls. In addition, the results showed that Æ©innate- and Æ©Th17-related cytokines are better indicators of cytokines imbalances in BD than each one of the innate- and Th17-related cytokines. Moreover, disease activity score and clinical activity index can also be affected by the levels of pro-inflammatory (IL-6) and anti-inflammatory (IL-4) cytokines. In conclusion, the results revealed that imbalances in the expression of innate immunity- as well as Th1- and Th17-related cytokines may play not only a pivotal role in BD pathogenesis but also can be important in disease severity.

Altered Serum Cytokine Profiles in Relapse Phase of Relapsing-Remitting Multiple Sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system and cytokines may play a role in the development of MS lesions. OBJECTIVE: To determine levels of different cytokines in patients with relapsing-remitting MS (RR-MS) compared to healthy controls. METHODS: Profiles of pro-inflammatory, Th1-, Th2-, and Th17-related cytokines were compared by quantitative multiplexed ELISA-based chemiluminescent assay in 44 RR-MS and 44 healthy age- and sex-matched individuals from the same ethnicity. RESULTS: Among pro-inflammatory cytokines, the levels of IL-6 (p=0.003), IL-8 (p=0.05) and TNF-α (p=0.002) were higher in patients than controls, though IL-4 and IL-10 as well as ΣTh2 cytokines were lower in patients (p=0.05, p=0.02 and p=0.05, respectively). After gender classification, the higher levels of IL-4 in male patients remained significant and IL-13 also showed significantly higher levels in male patients compared to male controls (p=0.003 and p=0.05, respectively). A significant negative correlation was detected between EDSS and IL-10 or ΣTh2 levels (p=0.005). In addition, IL-1α (r=0.4, p=0.05) and IFN-γ (r=0.35, p=0.05) were also directly correlated with EDSS in female patients. CONCLUSIONS: Patients with RR MS who are in the relapse clinical phase exhibit higher levels of pro-inflammatory cytokines and reduction in protective Th2-related cytokines.

Umbelliprenin induced production of IFN-γ and TNF-α, and reduced IL-10, IL-4, Foxp3 and TGF-ß in a mouse model of lung cancer.

Umbelliprenin is a member of the 7-prenyloxycoumarins with potential therapeutic properties such as cytotoxic effects on various cancer cells. The present study investigates the effect of umbelliprenin on predominance of Th1 and Th2 responses in Lewis lung cancer (LLC) mouse model. The cytotoxic effect of umbelliprenin was explored on LLC cells and mouse splenocytes by MTT assay. Mice into which LLC had been transplanted were treated with umbelliprenin on alternate days, at 2.5 mg/200 µl intraperitoneally. Foxp3, TNF-α and TGF-ß mRNA expressions were assessed in tumor and lung tissues of LLC mice. In addition, IL-10, IFN-γ and IL-4 levels were determined in sera and also in splenocyte culture supernatants at the presence of tumor cell lysate (10 µg/ml) and Con A (3 µg/ml) after 72 h. Results showed the cytotoxic effects of umbelliprenin on LLC cells (IC50 = 51.6 ± 5.4 µM) while no adverse effect was seen at this concentration on normal splenocytes. TNF-α mRNA expression in both lung and tumor tissues was increased. However, Foxp3 and TGF-ß expressions were decreased in tumor tissues. Serum level of IFN-γ was elevated in the umbelliprenin treated cancerous mice compared to the control group while IL-10 and IL-4 secretions were reduced. Tumor size was also decreased in umbelliprenin treated group. In summary, umbelliprenin has shown a partially Th1 bias with a reduction of regulatory immune response. Although the mechanism behind this action is not known, it is speculated that upon changing the Th1/Th2 balance in favour of Th1, umbelliprenin induces its antitumor activity.

Engraftment of plasma membrane vesicles into liposomes: A new method for designing of liposome-based vaccines.

OBJECTIVES: One of the major challenges in the field of vaccine design is choosing immunogenic antigens which can induce a proper immune response against complex targets like malignant cells or recondite diseases caused by protozoan parasites such as leishmaniasis. The aim of this study was to find a way to construct artificial liposome-based cells containing fragments of target's cell membrane. This structure not only mimics the real biological properties of proteins in the cell membrane of target cells, but also may induce the required immune responses, which culminate in eradication of target cells. MATERIALS AND METHODS: Five different techniques have been investigated to engraft the plasma membrane's vesicles (PMVs) derived from a characterized Leishmania parasite into liposomes. The most efficient method was tested again on the PMVs derived from well-known breast cancer cell line SK-BR-3. The percentage of engraftment was determined by two-color flowcytometry after staining the engrafted dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine DiI-labeled liposomes with FITC-labeled PMVs. RESULTS: Among the investigated techniques, freeze-drying method with 91±2% and 90±3% of engraftment for Leishmania and SK-BR-3 derived PMVs, respectively, showed superiority over the other methods. In addition, after 9 weeks storage in refrigerator, freeze-dried fused particles kept their original size (660±350 nm) and fusion efficiency (94±3%). CONCLUSION: Among five different engraftment techniques, freeze-drying is preferred over the other methods due to its simplicity, more fusion efficiency and stability of produced particles during storage.

The role of liposome size on the type of immune response induced in BALB/c mice against leishmaniasis: rgp63 as a model antigen.

To develop an efficient liposomal vaccine delivery system, the size of liposomes is critical to their adjuvant activities. In the present study, liposomes with different sizes (100, 400, 1000 nm) containing recombinant major surface glycoprotein of Leishmania (rgp63) were prepared, characterized, and inoculated subcutaneously into BALB/c mice to evaluate the rate of protection and the type of immune response generated against leishmaniasis. The lowest footpad lesion size and splenic parasite burden were seen in the mice immunized with large size (≥400 nm) liposomes after challenge with Leishmania major. The production of IFN-γ was only elevated in the spleen cells of mice immunized with large size (≥400 nm) liposomes. The highest IgG2a/IgG1 ratio was also seen in the sera of the mice immunized with large size (≥400 nm) liposomes before and 14 weeks after challenge. The results showed that immunization with small size (100 nm) liposomes induces a Th2 response, whereas immunization with large size (≥400 nm) liposomes induces a Th1 type of immune response. There was no significant difference in the type of induced immune response between the mice immunized with liposomes of 400 nm and those immunized with liposomes of 1000 nm or unextruded. The results of the current study demonstrated that the size of liposomes plays a significant role in the type of generated immune response.

Nanovaccine for leishmaniasis: preparation of chitosan nanoparticles containing Leishmania superoxide dismutase and evaluation of its immunogenicity in BALB/c mice.

BACKGROUND: Leishmaniasis is a protozoan disease, affecting 12 million people in different regions of the world with a wide spectrum of diseases. Although several chemotherapeutic agents have been used for treating the disease, long-term therapy, limited efficacy and the development of drug-resistant parasites remain the major limitations. METHODS: To develop a new nanovaccine for leishmaniasis, recombinant Leishmania superoxide dismutase (SODB1) was loaded onto chitosan nanoparticles by the ionotropic gelation method. Size and loading efficiency of the nanoparticles were evaluated and optimized, and an immunization study was undertaken on BALB/c mice. The mice received phosphate buffer saline (PBS), superoxide dismutase B1 (SODB1) in PBS and nanoparticles via subcutaneous injection. Soluble Leishmania Antigens (SLA) and complete Freund's adjuvant (CFA) were also injected subcutaneously three times every three weeks (some groups received only a single dose). Three weeks after the last injection, blood samples were collected and assessed with ELISA to detect IgG2a and IgG1. RESULTS: Immunological analysis showed that in single and triple doses of SODB1 nanoparticles, IgG2a and IgG2a/IgG1 were significantly higher than the other groups (P<0.05). CONCLUSION: The results revealed that formulations of SODB1 in biodegradable and stable chitosan nanoparticles can increase the immunogenicity toward cell-mediated immunity (T(H)1 cells producing IgG2a in mice) that is effective in Leishmania eradication and could be presented as a single dose nanovaccine for leishmaniasis.

Cloning and expression of Leishmania major superoxide dismutase B1: A potential target antigen for serodiagnosis of Leishmaniasis.

BACKGROUND: Leishmaniasis- a neglected public health problem- is a group of diseases affecting an estimated 12 million people worldwide. OBJECTIVE: In the present study, recombinant Leishmania major superoxide dismutase B1 (rLmSODB1) has been utilized as a potential antigen for the serodiagnosis of human cutaneous (CL) and visceral leishmaniasis (VL) in the endemic regions of southern part of Iran. Additionally, the sensitivity and specificity of ELISA-based serodiagnosis using rLmSODB1 and the soluble Leishmania antigen (SLA) were compared. METHODS: For the first time, rLmSODB1 has been cloned successfully and used for ELISA-based serodiagnosis. Sera from 30 CL and 24 VL cases were included in this study. Additional studies were also done for the evaluation of cross-reactivity using sera from 41 endemic controls including normal endemic donors (n=20), systemic lupus erythematosus patients (n=5), rheumatoid arthritis patients (n=5), and patients with tuberculosis (n=11). RESULTS: Analysis indicated that rLmSODB1 was recognized by 62.5% and 13.3% of sera from patients with VL and CL, showing a sensitivity of 72.7% and 53.6%, respectively. However 95.8% of VL and 30% of CL sera reacted with SLA, revealing sensitivities of 96% and 58.8%, respectively. Additionally, from 41 sera collected either from healthy subjects or patients affected with other diseases, 97.5% were negative with SLA or rLmSODB1 (specificity 97.6%). CONCLUSION: These results show that rLmSODB1 almost does not react with sera from patients with tuberculosis and autoimmune diseases and may be considered as a candidate antigen for the specific immunodiagnosis of visceral leishmaniasis.

Enhancement of immune response and protection in BALB/c mice immunized with liposomal recombinant major surface glycoprotein of Leishmania (rgp63): the role of bilayer composition.

Development of new generation vaccines requires adjuvants to elicit the type and intensity of immune response needed for protection. Liposomes have been shown to be an effective adjuvant formulation. In this study, the role of liposome bilayer composition with different phase transition temperature (T(c)) to induce a T helper 1 (Th1) type of immune response and protection against leishmaniasis in BALB/c mice was assessed. Liposome formulations with different bilayer compositions consisting of egg phosphatidylcholine (EPC, T(c)<0 degrees C), dipalmitoylphosphatidylcholine (DPPC, T(c) 41 degrees C), or distearoylphosphatidylcholine (DSPC, T(c) 54 degrees C) were prepared. All liposomes were contained rgp63 as a recombinant antigen and used to immunize mice subcutaneously 3 times in 3-week intervals. Evaluation of lesion development and splenic parasite burden after challenge with L. major, evaluation of Th1 cytokine (IFN-gamma) and Th2 cytokine (IL-4), and titration of IgG isotypes were carried out to assess the type of generated immune response and extent of protection. The results indicated the generated immune response in mice was influenced by the bilayer composition of liposomes, so that mice immunized with liposomes consisting of EPC induced a Th2 type of immune response while liposome consisting of DPPC or DSPC induced Th1 type of immune response. It seems that liposomes prepared with higher Tm phospholipids are suitable formulation to induce Th1 type of immune response and protection, and so might be used for further investigations to develop an effective vaccine against leishmaniasis.

Synthesis and immunomodulation of human lymphocyte proliferation and cytokine (interferon-gamma) production of four novel malonitrilamides.

Leflunomide is an immunomodulator drug with applications in the management of arthritis rheumatoid. In this study, four novel analogs (4a-d) of A771726, the active metabolite of leflunomide were synthesized and examined in vitro for their immunomodulation activity by examining human lymphocyte proliferation and determination of the cytokine interferon-gamma concentrations in human lymphocyte cell culture. For this purpose, 5 x 10(4) human lymphocyte cells were incubated at 37 degrees C in 5% CO(2) with phytohemagglutinin and one of the analogs (concentrations 1-100 mM), negative controls or cyclosporine (0.1 mM). Effects of the compounds on lymphocyte proliferation and interferon-gamma production were determined by MTT assay and enzyme-linked immunosorbent assay, respectively. Our results showed that all four compounds dose-dependently suppressed lymphocyte proliferation. Moreover, these compounds at some concentrations reduced interferon-gamma production which is an indicator of the immune response. Generally, the most potent analog was 4b with an amide linkage (X=NH) and the weakest analog was 4a with an ester linkage (X=O). Compound 4a has little similarity with the leflunomide active metabolite which has an amide linkage. In this study, four novel compounds were synthesized that showed considerable immunosuppressive effects that deserve further investigations.

The role of liposome charge on immune response generated in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63).

Liposomes as a lipid-based system have been shown to be an effective adjuvant formulation. In this study, the role of liposome charge in induction of a Th1 type of immune response and protection against leishmaniasis in BALB/c mice was studied. Liposomes containing rgp63 were prepared by Dehydration-Rehydration Vesicle (DRV) method. Neutral liposomes consisted of dipalmitoylphosphatidylcholine and cholesterol. Positively and negatively charged liposomes were prepared by adding dimethyldioctadecylammonium bromide (DDAB) or dicetyl phosphate (DCP) to the neutral liposome formulation, respectively. Female BALB/c mice were immunized subcutaneously with negatively, positively charged or neutral liposomes encapsulated with rgp63, rgp63 in soluble form or PBS, three times in 3week intervals. The extent of protection and type of immune response generated were studied in different groups of mice. The group of mice immunized with rgp63 encapsulated in neutral liposomes showed a significantly (P<0.01) smaller footpad swelling upon challenge with Leishmania major compared with positively or negatively charged liposomes. The mice immunized with neutral liposomes also showed a significantly (P<0.01) the lowest splenic parasite burden, the highest IgG2a/IgG1 ratio and IFN-gamma production and the lowest IL-4 level compared to the other groups. The results indicated that a Th1 type of immune response was induced in mice immunized with neutral liposomes more efficiently than positively charged liposomes and conversely negatively charged liposomes induced a Th2 type of immune response.

Effect of topical liposomes containing paromomycin sulfate in the course of Leishmania major infection in susceptible BALB/c mice.

The aim of this study was to evaluate the antileishmanial effects of topical liposomal paromomycin sulfate (PM) in Leishmania major-infected BALB/c mice. Liposomes containing 10 or 15% PM (Lip-PM-10 and Lip-PM-15, respectively) were prepared by the fusion method and were characterized for their size and encapsulation efficiency. The penetration of PM from the liposomal PM formulations (LPMFs) through and into skin was evaluated in vitro with Franz diffusion cells fitted with mouse skin at 37 degrees C for 8 h. The in vitro permeation data showed that almost 15% of the LPMFs applied penetrated the mouse skin, and the amount retained in the skin was about 60% for both formulations. The 50% effective doses of Lip-PM-10 and Lip-PM-15 against L. major promastigotes in culture were 65.32 and 59.73 microg/ml, respectively, and those against L. major amastigotes in macrophages were 24.64 and 26.44 microg/ml, respectively. Lip-PM-10 or Lip-PM-15 was used topically twice a day for 4 weeks to treat L. major lesions on BALB/c mice, and the results showed a significantly (P < 0.001) smaller lesion size in the mice in the treated groups than in the mice in the control group, which received either empty liposomes or phosphate-buffered saline (PBS). Eight weeks after the beginning of the treatment, every mouse treated with LPMFs was completely cured. The spleen parasite burden was significantly (P < 0.001) lower in mice treated with Lip-PM-10 or Lip-PM-15 than in mice treated with PBS or control liposomes, but no significant difference was seen between the two groups treated with either Lip-PM-10 or Lip-PM-15. The results suggest that topical liposomal PM may be useful for the treatment of cutaneous leishmaniasis.

The enhancement of immunosuppressive effects of cyclosporine A on human T-cells using fusogenic liposomes.

The aim of this study was to prepare and characterize neutral, positively charged, negatively charged and fusogenic liposomes of different sizes that contain cyclosporine A (CyA) and to evaluate their immunosuppressive activity on human T-cells. Neutral liposomes containing CyA were prepared from dipalmitoylphosphatidylcholine (DPPC) and cholesterol using the solvent evaporation method. To prepare positively charged, negatively charged and fusogenic liposomes containing CyA; stearylamine (SA), dicetylphosphate (DCP) and dioleoylphosphatidylethanolamine (DOPE) were added to the neutral liposome formulation, respectively. To reduce the size of liposomes containing CyA, extrusion through polycarbonate filters (1000, 400 and 100 nm) was used. The liposomes were characterized by their size, zeta potential and encapsulation efficiency. The in vitro immunosuppressive effects of an aqueous solution of CyA and different liposomes containing CyA were determined on human T-cells by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. The mean diameter of the various multilamellar vesicle (MLV) liposomes containing CyA was between 1.76 and 2.49 microm. The encapsulation efficiency for the different MLV and extruded liposomes containing CyA ranged from 73% to 90%. In vitro immunosuppressive evaluation by T-cell culture showed that fusogenic liposomes have the best inhibitory effects on T-cell proliferation compared to the other liposomes. Reducing the size of the liposomes did not affect the in vitro immunosuppressive activity. The average IC(50) for the aqueous solution of CyA and the neutral, positively charged, negatively charged and fusogenic liposomes containing CyA was 4.98 x 10(-2), 7.38, 1.43, 3.84 x 10(-3) and 7.93 x 10(-5) mM, respectively. The results of this study indicate that fusogenic liposomes have the strongest immunosuppressive activity and could be considered as a suitable delivery system for CyA.

Coencapsulation of CpG oligodeoxynucleotides with recombinant Leishmania major stress-inducible protein 1 in liposome enhances immune response and protection against leishmaniasis in immunized BALB/c mice.

CpG oligodeoxynucleotides (CpG ODN) have been shown to have potent adjuvant activity for a wide range of antigens. The purpose of this study was to determine the potential benefit of using liposomes as a delivery vehicle to enhance the adjuvant activity of CpG ODN with Leishmania major stress-inducible protein 1 (LmSTI1) antigen in induction of the Th1 response in a murine model of leishmaniasis. BALB/c mice were immunized subcutaneously three times in 3-week intervals with liposomal recombinant LmSTI1 (Lip-rLmSTI1), rLmSTI1 coencapsulated with CpG ODN in a liposome (Lip-rLmSTI1-CpG ODN), rLmSTI1 plus CpG ODN in phosphate-buffered saline (PBS), rLmSTI1 plus non-CpG ODN in PBS, rLmSTI1 in PBS, empty liposome, or PBS. The intensity of infection induced by L. major promastigote challenge was measured by footpad swelling. A significant (P < 0.001) inhibition of infection in mice immunized with Lip-rLmSTI1-CpG ODN was shown compared to the other groups, and no parasite was detected in the spleens of this group 14 weeks after challenge. The highest immunoglobulin G2a (IgG2a) titer and the highest IgG2a/IgG1 ratio were also shown in the sera of mice immunized with Lip-rLmSTI1-CpG ODN before and 14 weeks after challenge. The results indicated the superiority of CpG ODN in its liposomal form over its soluble form to induce the Th1 response when used in association with rLmSTI1 antigen. It seems that using a liposome delivery system carrying CpG ODN as an adjuvant coencapsulated with Leishmania antigen plays an important role in vaccine development strategies against leishmaniasis.

The role of CpG ODN in enhancement of immune response and protection in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63) encapsulated in cationic liposome.

CpG oligodeoxynucleotides (CpG ODN) are known to be a potent immunoadjuvant for a wide range of antigens. The aim of this study was to evaluate the role of CpG ODN co-encapsulated with rgp63 antigen in cationic liposomes (Lip-rgp63-CpG ODN) in immune response enhancement and protection in BALB/c mice against leishmaniasis. Lip-rgp63-CpG ODN prepared by using dehydration-rehydration vesicle (DRV) method significantly inhibited (P<0.001) Leishmania major infection in mice measured by footpad swelling compared to Lip-rgp63, rgp63 alone, rgp63 plus CpG ODN, PBS or control liposomes. The mice immunized with Lip-rgp63-CpG ODN also showed the lowest spleen parasite burden, highest IgG2a/IgG1 ratio and IFN-gamma production and the lowest IL-4 production compared to the other groups. The results indicate that co-encapsulation of CpG ODN in liposomes improves the immunogenicity of Leishmania antigen.