Immunocytochemical analysis of valproic acid induced histone H3 and H4 acetylation during differentiation of rat adipose derived stem cells into neuron-like cells.

Valproic acid (VPA) is an inhibitor of histone deacetylases (HDACs) that can regulate differentiation and proliferation of stem cells by epigenetic mechanisms. We investigated VPA induced histone H3 and H4 acetylation in adipose derived stem cells (ADSCs) transdifferentiated into neuron-like cells (NLCs). Rat ADSCs were transdifferentiated into neural stem cells (NSCs) that had been generated from neurospheres. The NSCs were differentiated into NLCs by induction with different concentrations of VPA at 24, 48 and 72 h. The NLCs were evaluated using anti-H3 and -H4 antibodies, and ADSCs, NSCs and NLCs were evaluated using immunofluorescence. The ADSCs were immunoreactive to CD90 and CD49d, but not to CD45 and CD31. Both the neurospheres and NSCs were immunostained with nestin and neurofilament 68. The neurospheres expressed Musashi1, Sox2 and Neu N genes as determined by RT-PCR. Our dose-response study indicated that the optimal concentration of VPA was 1 mM at 72 h. Histone acetylation levels of H3 and H4 immunostaining intensities in NLCs were significantly greater than for ADSCs and NSCs. VPA alters H4 and H3 acetylation immunoreactivities of ADSCs transdifferentiated into NLCs.

Leishmania tarentolae expressing CXCL-10 as an efficient immunotherapy approach against Leishmania major-infected BALB/c mice.

Recent findings have demonstrated the suitability of interferon-gamma-induced protein 10 (IP-10) or CXCL-10 as an immunotherapy tool in treatment of leishmaniasis. This chemokine can overcome Leishmania (L.) infection through inducing nitric oxide (NO) production for parasite elimination. This study was undertaken to investigate the therapeutic effects of recombinant Leishmania tarentolae expressing CXCL-10 and an expression vector encoding CXCL-10 (pcDNA-CXCL-10-EGFP) in a model of BALB/c mice susceptible to infection by Leishmania major. The outcome of intervention was examined at 3 weeks post-treatment by evaluating the parameters of parasite burden (PB), arginase activity, NO and various cytokines such as IFN-γ, IL-4, IL-6 and IL-10. The results have shown that despite the efficacy of CXCL-10 expression vector as gene therapy, the live therapy strategy using L. tarentolae expressing CXCL-10 was more effective in terms of decreasing PB. Nitric oxide production increased, especially in the live therapy approaches. Arginase activity also decreased in all regimens, which demonstrates the potency of the treatment. The overall cytokine production shifted in favour of Th1 responses in the treated mice. Altogether, recombinant L. tarentolae expressing CXCL-10 represents a promising therapeutic strategy to improve treatment of cutaneous leishmaniasis.

Arginase activity of Leishmania isolated from patients with cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is one of the most important vector-borne parasitic diseases, highly endemic in Iran, and its prevalence is increasing all over the country. Arginase (ARG) activity in isolated Leishmania parasites from CL patients is yet to be explored. This study aimed to compare the ARG activity of isolated Leishmania promastigotes from CL patients with a standard strain of Leishmania major and its influences on the disease pathogenesis. We recruited 16 confirmed CL patients from Qom Province, in central Iran; after detection of Leishmania species using PCR-RFLP, we assessed the levels of ARG in the isolated promastigotes and determined the parasites' growth rate. Only L. major was identified from CL patients. The level of ARG activity in the isolated Leishmania promastigotes from CL patients was significantly higher than that obtained from the standard strain of L. major. No significant correlations between ARG activity and lesion size, number or duration were observed; in contrast, a significant negative correlation was seen between ARG level and Leishmania' growth rate. The obtained results suggest that increased ARG expression and activity in the isolated Leishmania promastigotes might contribute to the higher parasite infectivity and play a major role in the pathogenicity of the CL.

Enhanced paromomycin efficacy by solid lipid nanoparticle formulation against Leishmania in mice model.

Leishmaniasis caused by protozoan parasites of the genus Leishmania. Intracellular infections treatment such as leishmaniasis is frequently hampered by limited access of drugs to infected cells. Moreover, most of the current drugs are confined to some toxic compounds, and there are increasing incidences of development of drug resistance. Hence, production of a new antileishmanial compound is crucial. Paromomycin sulphate (PM) is a promising antileishmanial drug. One strategy to improve drug effectiveness is to use appropriate delivery systems. Solid lipid nanoparticle (SLN) is as an excellent substitute delivery system to other colloidal carrier. In the present study, PM was loaded in solid lipid nanoparticles (PM-SLN) and the in vivo efficacy was studied against Leishmania (L.) major-infected BALB/c mice. For this reason, the footpad swelling was measured and real-time PCR was performed to quantify the parasite load after infectious challenge. The level of cytokines including interleukin-4 (IL-4) and gamma interferon (IFN-γ) and nitric oxide was evaluated. Altogether, this study showed that the PM-SLN formulation is a safe compound and SLN in PM-SLN compound is effective for treatment of leishmaniasis by improving the effectiveness of PM in killing the parasite and switching towards Th1 response.

Leishmania tarentolae secreting the sand fly salivary antigen PpSP15 confers protection against Leishmania major infection in a susceptible BALB/c mice model.

Cutaneous leishmaniasis is a zoonotic, vector-borne disease causing a major health problem in several countries. No vaccine is available and there are limitations associated with the current therapeutic regimens. Immune responses to sand fly saliva have been shown to protect against Leishmania infection. A cellular immune response to PpSP15, a protein from the sand fly Phlebotomus papatasi, was sufficient to control Leishmania major infection in mice. This work presents data supporting the vaccine potency of recombinant live non-pathogenic Leishmania (L.) tarentolae secreting PpSP15 in mice and its potential as a new vaccine strategy against L. major. We generated a recombinant L. tarentolae-PpSP15 strain delivered in the presence of CpG ODN and evaluated its immunogenicity and protective immunity against L. major infection in BALB/c mice. In parallel, different vaccination modalities using PpSP15 as the target antigen were compared. Humoral and cellular immune responses were evaluated before and at three and eight weeks after challenge. Footpad swelling and parasite load were assessed at eight and eleven weeks post-challenge. Our results show that vaccination with L. tarentolae-PpSP15 in combination with CpG as a prime-boost modality confers strong protection against L. major infection that was superior to other vaccination modalities used in this study. This approach represents a novel and promising vaccination strategy against Old World cutaneous leishmaniasis.

Bone structure investigation using X-ray and neutron radiography techniques.

In this paper we report a study of the periodic variation of bone tissue humidity immediately after death using both neutron and X-ray radiography techniques. After death, bone tissue experiences sequential change over time. This change consists of organic and inorganic phase variations of the bone structure, as well as gradual reduction of the bone's water content. These variations are investigated by periodically imaging dead bone using X-ray and neutron radiography. Chemical separation techniques such as calcification and decalcification were used to separate the organic and inorganic phases of the bone. Comparison between X-ray and neutron radiographs of bone following phase separation can be potentially used to investigate the bone disease or to determine a cause of death. In our experiments, we use adult rat femur bones, and the interpretations of these results are presented based on our understanding of bone structure and images produced by neutron and X-ray photon interactions.

A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major.

The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-gamma production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective.