Hydrogel nanoparticle encapsulated plasmid as a suitable gene delivery system.

To facilitate the delivery of genetic material, the use of appropriate carriers such as polymers is necessary. Nanoparticles comprising of chitosan-alginate polymers were formed through pregel preparation method. Chi/Alg nanoparticles had a mean Z-Average diameter of 161.8 nm and mean zeta 29.3 mV, respectively. The ability of plasmidcomplex in preventing DNA migration showed Chi/Alg nanoparticles have great capacity to maintain plasmid. The efficiency of nanoparticles for transfection of pEGFP-N1 plasmid in the cultured HEK 293 cells was measured by flow cytometry. Cell viability assays indicated that nanoparticles had no toxic effect on HEK 293 cells after 4 or 24 h. Our suitable candidate for gene delivery would be Alg/Chi nanoparticles.

Distinct strains of Leishmania major induce different cytokine mRNA expression in draining lymph node of BALB/c mice.

Four genotypically distinct strains of L. major collected from persons residing in different endemic areas of cutaneous leishmaniasis in Iran were evaluated in BALB/c mice. Parasite virulence was evaluated by measuring the parasite burden in the lymph nodes. Immunogenicity of the strains was assessed by analysis of cytokines mRNA expression levels in popliteal lymph nodes of the mice in early (3, 16, 40 h) and late (week 1, W3, W5 and W8) time periods after infection. The expression of cytokines mRNA, namely Ifng, Il2,Il4,Il10 and Il12, was quantitated by real-time PCR. The lowest and the highest parasite loads were induced by Damghan (2·15 × 107) and Shiraz (9·59 × 109) strains, respectively. Moreover, Damghan strain elicited higher expression levels of Ifng and Il2 mRNA and the highest ratio of Ifng/Il4 mRNA expression compared with the other strains at 40 h and 8 weeks post-infection. The results indicate that the inoculation of BALB/c mice with different strains induced high diversity in parasite burden and cytokines gene expression. Amongst the four strains, Damghan strain showed the lowest parasite load and the highest tendency to induce expression of Th1 cytokines gene and might be considered as a safe and immunogenic strain.

Complete conservation of an immunogenic gene (lcr1) in Leishmania infantum and Leishmania chagasi isolated from Iran, Spain and Brazil.

BACKGROUND & OBJECTIVES: Kala-azar is the visceral and most severe form of leishmaniasis that leads to death if untreated. The causative agents of visceral leishmaniasis (VL) are members of Leishmania (L.) donovani complex which includes L. chagasi and L. infantum. Genome sequences have raised the question whether L. chagasi and L. infantum are synonymous or different. This question has important implications for clinical and epidemiological studies, evaluation of vaccines and drugs, and disease control. LCR1 is an immunogenic molecule discovered from L. chagasi with potential as a component of a Leishmania subunit vaccine. If this protein has potentials for being used in a vaccine or diagnostic testing, there should be little variability in this molecule between L. infantum isolates from diverse geographic regions. The aim of this study was to determine whether lcr1 of an Iranian strain of L. infantum was identical to lcr1 of both L. infantum strain from a different geographic region (Spain) and that of an L. chagasi isolate from Brazil. METHODS: L. infantum isolated from an Iranian kala-azar patient was studied. Lcr1 from this isolate was PCR amplified, cloned, and studied by restriction digest analysis and sequencing. RESULTS: The sequences of lcr1 of the Iranian L. infantum were completely identical at nucleotide level to lcr1 sequences of both the Spanish L. infantum and the Brazilian L. chagasi strains. CONCLUSION: Complete conservation of the DNA sequence encoding for LCR1 molecule between geographically distinct Leishmania species adds credibility to the potential for LCR1 as a component of a subunit vaccine and diagnostic test for kala-azar.

Dynamics of Leishmania infection rates in Rhombomys opimus (Rodentia: Gerbillinae) population of an endemic focus of zoonotic cutaneous leishmaniasis in Iran.

Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major is a great public health problem in the Old World. Leishmania major is widely distributed in populations of rodents in arid and savannah regions. In this study, seasonal variation of natural infection with Leishmania parasites in Rhombomys opimus (Rodentia: Gerbillinae) population of an endemic focus of ZCL in Iran was monitored. The study was conducted from October 2007 to October 2008 in the central part of the country. Nested polymerase chain reaction (PCR) assay was used for the detection and identification of Leishmania parasites, and the results were confirmed by PCR-restriction fragment length polymorphism (RFLP). The results showed that Leishmania infection rate was 55.8% (29 out of 52 gerbils) using nested PCR. The highest and lowest Leishmania infection rates were observed in fall and summer, respectively. Gerbils that were found to be infected only with L. major were 5.8%, and that with Leishmania turanica were 23.1%. A mixed natural infection was seen in the rodents with L. major and L. turanica (21.2%), with L. major and L. gerbilli (1.9%), and with all the three species (3.9%). Leishmania major infection alone was seen in fall and winter whereas mixed infection of L. major and L. turanica was observed in all seasons except in summer. Leishmania turanica infection was observed throughout the year. It is concluded that L. major, L. gerbilli, and L. turanica circulate in the population of R. opimus in central part of Iran. Leishmania major infection is usually accompanied by L. turanica in naturally infected gerbils with the highest rate in fall. It is recommended that the role of L. turanica in the epidemiology and transmission of ZCL should be reconsidered.

Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran.

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96.8%), whereas 85 sera were positive with DAT (sensitivity=91.4%) and 89 sera were positive with whole cell ELISA (sensitivity=95.7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89.2%) and whole cell ELISA was negative in 114 cases (specificity=87.7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.

Leishmanin skin test in guinea pig with a single purified protein of Leishmania major.

A series of hybridomas was produced by fusion of SP2/0 myeloma cells with spleen cells of mice immunized with Leishmania major (L. major). The reactivity of secreted monoclonal antibodies (mAbs) was evaluated against available leishmanin antigen by enzyme linked immunosorbent assay. Only one hybridoma designated as 7F9 secreted IgG1 mAb which was shown to be reactive with leishmanin. This mAb was further tested against four species of Leishmania (L. donovani, L. tropica, L. infantum, L. major) and a recombinant gp63. Among the four species tested it was shown to be only reactive with promastigotes of L. major. The antigen recognized by this mAb was purified and analyzed from both sonicated and supernatant cultures of L. major by immunoaffinity chromatography and reverse phase high performance liquid chromatography. The purified antigen, which gave a single band of 56kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis elicited a strong delayed-type hypersensitivity (DTH) reaction in guinea pigs sensitized with L. major. It was almost of the same degree as that produced by leishmanin. These results suggest that an L. major-specific antigen is an alternative as a specific diagnostic skin test reagent, which could lead to a better understanding of the mechanism of DTH in L. major.

Mononuclear cells from patients recovered from cutaneous leishmaniasis respond to Leishmania major amastigote class I nuclease with a predominant Th1-like response.

The Leishmania major amastigote class I nuclease (LmaCIN) is a developmentally regulated protein that is highly expressed in the amastigote stage of L. major. This protein is homologous to the P4 nuclease of L. pifanoi, which has been shown to induce protective immune response in a murine model. To evaluate LmaCIN as a potential human vaccine candidate, cellular immune responses to recombinant LmaCIN were examined in individuals recovered from Old World cutaneous leishmaniasis. Peripheral blood mononuclear cells (PBMC) from patients recovered from L. major infection were cultured either with recombinant LmaCIN or autoclaved L. major (ALM) as control. rLmaCIN induced significant proliferation of PBMC from 90% of recovered patients. Phenotypic analysis of proliferating cells showed that CD8(+) cells were the predominant cell type proliferating in response to rLmaC1N. Screening of culture supernatants for cytokines showed that rLmaCIN induced high levels of interferon (IFN)-gamma (mean +/- s.e.m.: 1398 +/- 179 pg/ml) associated with little interleukin (IL)-10 and little or no IL-5 production. These findings show that LmaCIN is immunogenic in humans during L. major infection and that it can elicit immunological responses relevant to immunoprophylaxis of leishmaniasis.

Molecular characterization of a novel amastigote stage specific Class I nuclease from Leishmania major.

Leishmania parasites like other kinetoplastids are unable to synthesize purines de novo and so are reliant on a salvage pathway for recycling ribonucleotides. A stage specific class one nuclease enzyme, 3'-Nucleotidase/nuclease, has been implicated in salvage of preformed purines in Leishmania insect stage promastigote via hydrolysis of 3'-nucleotides and nucleic acids. Although a similar activity is known to exist in amastigotes which reside in infected mammalian cells, the homologous gene and the corresponding protein responsible for carrying out this function have not been well characterized. Using primers specific for conserved regions of trypanosomatid class one nucleases, a gene encoding a novel class one nuclease from amastigotes of Leishmania major (LmaC1N) was cloned and sequenced. The coding sequence consists of 951 bp encoding a 316 amino acid protein with a predicted molecular mass of 35,300 Da. Analysis of the deduced amino acid sequence showed that LmaC1N is highly homologous to other class I nucleases and contains all five conserved regions reported for promastigotes 3'-Nucleotidase/nuclease. Analysis by reverse transcriptase polymerase chain reaction and Western blotting demonstrated that expression of LmaC1N gene is regulated in a stage-specific manner. Whereas the gene appeared to be silenced in promastigotes, high level expression in amastigotes implied an important function in support of parasite survival and multiplication in the mammalian cells.

Characterization of a monoclonal antibody against neopterin using an enzyme-linked immunosorbent assay with penicillinase as label.

An active ester derivative of neopterin was prepared using 4-(N-maleimidomethyl) cyclohexan 4-carboxilic acid N-hydroxy succinimide ester (MCH-NHS), conjugated to bovine serum albumin (BSA) and injected for antibody production (for both monoclonal and polyclonal antibodies). High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. Neopterin was conjugated to the enzyme penicillinase by a one-step glutaraldehyde method, which was used as tracer. A novel enzyme immunoassay was developed using this conjugate to screen and characterize the monoclonal antibody (MAb) produced in these experiments. After limiting dilutions, it was found that antibody produced by one clone with a Ka value of 7.6 x 10-7 mol/L was specific for a number of structurally related molecules. This clone was found to be of IgG class and IgG2a subclass. The standard curvewas constructed with a sensitivity of 10 pg/well (100 pg/mL) covering up to 1 ng/mL.

Compositional changes of PBL population in patients with chronic hepatitis B virus infection.

In this report we have analysed the peripheral blood lymphocyte of several patients with chronic hepatitis B virus infection with flow cytometry. Based on the presence and absence of the HBeAb, patients were divided into two groups. In both, all the patients were HBsAg positive with normal range of serum alanine aminotranferase (23.9 +/- 17.8). We have found that the immunophenotypic profiles of patients were different from healthy donors with significant decrease in CD(3)(+) T cells, specially CD(8)(+) T cells and a significant increase in the CD(19)(+) B cells. The differences were seen in other subset of T cells (CD(4)(+)) or NK cells (CD(56)(+)/CD(16)(+)) and HLA-DR markers were not significant. When the phenotypic profiles of both groups were compared with each other, such changes were more dominant in group II, with HBeAb positive than in group I, with HBeAb negative. Also, we have seen a correlation between the increase of CD(19)(+) B cells and the decrease of CD CD(3)(+) T cells. No such correlation was observed with other cells.

Comparison of the immune profile of nonhealing cutaneous Leishmaniasis patients with those with active lesions and those who have recovered from infection.

Th1-type cellular immune responses play a critical role in protection against infection with Leishmania parasites, whereas activation of Th2-type cells results in progressive disease. Cutaneous leishmaniasis caused by Leishmania major is often a self-healing disease; however, persistent nonhealing forms are also known. In the present study, we have described cell-mediated immune responses in nonhealing patients by measuring T-cell proliferation, cytokine production, and phenotypic characterization of these cells. The responses were compared with those of patients with active lesions, patients who had recovered from infection, and healthy controls. Peripheral blood mononuclear cells from patients with active lesions and recovered donors proliferated vigorously and produced Th1-type cytokine when stimulated with L. major antigens, whereas in nonhealing patients the proliferative responses were significantly lower and showed a Th2-type response to Leishmania antigens. Interleukin-10 (IL-10) production was not a feature of L. major stimulation. Flow cytometric analysis revealed that L. major antigen induced proliferation of the CD4-positive population and that these cells were the major source of gamma interferon and IL-4. These results show a distinct dichotomy in the cytokine response to L. major infection.

A randomised, double-blind, controlled trial of a killed L. major vaccine plus BCG against zoonotic cutaneous leishmaniasis in Iran.

Safety and efficacy of killed (autoclaved) L. major promastigotes, ALM, mixed with BCG against zoonotic cutaneous leishmaniasis was tested in healthy volunteers (n = 2453) in a randomized double blind trial vs. BCG as control. Side-effects were similar in both groups but tended to be slightly more frequent and prolonged in the ALM + BCG group. Leishmanin skin test conversion (induration > or =5 mm) was significantly greater in the ALM + BCG than in the BCG group (36.2% vs. 7.9% on day-80 and 33% vs. 19%, after 1 year, respectively). Cumulative incidence rates for 2 years, were similar in both groups (18.0% vs. 18.5%). However, LST responders on day 80 (> or =5 mm) had a significantly lower incidence (35%) of CL during the first year than non-responders. A single dose of ALM + BCG is not sufficiently immunogenic to provide a measurable response when compared to BCG alone. A single dose of this vaccine has been shown to be safe with no evidence of an exacerbating response following natural infection; hence, multiple doses or other adjuvants should be considered to increase its immunogenicity.

Biochemical analysis and immunogenicity of Leishmania major amastigote fractions in cutaneous leishmaniasis.

Soluble Leishmania antigen (SLA) from both developmental stages of L. major (L. major MRHO/IR/75/ER) were prepared. Three and five subfractions of SLA from amastigote and promastigote were obtained by fast protein liquid chromatography (FPLC), respectively. Biochemical analyses and comparison of amastigote and promastigote SLA were done. The biochemical analyses revealed that the first fraction of L. major amastigote possesses a distinct band on its electrophoretic mobility pattern corresponding to a position of 24 kD, and it has enzymatic activity with characteristics of a cysteine proteinase. The isolated fractions of amastigote were tested for induction of proliferation, interferon-gamma (IFN-gamma) and IL-4 production in cultures of peripheral blood mononuclear cells (PBMC) from individuals who had recovered and also chronic patients of cutaneous leishmaniasis caused by L. major. The cells of recovered individuals compared with chronic cases proliferated profoundly in response to the first fraction of amastigote SLA. In all recovered individuals, the IFN-gamma, but not IL-4, was secreted in response to stimulation with the first fraction of amastigote SLA. In chronic cutaneous leishmaniasis, IFN-gamma was infrequently observed in response to stimulation by all three fractions of amastigote SLA, but secretion of IL-4 was observed. These data indicate that first fraction of amastigote SLA is a strong inducer of primed human immune response to L. major, and may have a protective function.

Recombinant interleukin-1 promotes leishmaniasis in susceptible mice.

Interleukin-1 exacerbates leishmanial lesions in Leishmania major-infected BALB/c mice. Indomethacin can modulate the effect of IL-1, so at least part of the IL-1 effect on disease progression is due to the induction of prostaglandin synthesis.