The effect of UV radiation in the presence of TiO2-NPs on Leishmania major promastigotes.

BACKGROUND: Cutaneous leishmaniasis is a parasitic disease, which is difficult to treat due to high drug resistance and adverse side effects. Photodynamic therapy by ultraviolet radiation using materials with high photocatalytic features like titanium dioxide nanoparticles (TiO2-NPs) is an emerging treatment for this disease. In this study, TiO2-NPs with ultraviolet (UV) radiation were administered as photodynamic therapy against Leishmania Major (LM) promastigotes. METHODS: Two forms of TiO2 viz. including Anatase and Rutile were administered in two UV ranges< UVA and UVB for different time periods (30 and 60 min). Finally, 24 and 48 h after incubation, the MTS test was performed and cell survival percentage was calculated. RESULTS: The mean size of Anatase and Rutile-NPs is approximately 32.5 and 50.9 nm respectively by DLS and FE-SEM, and crystal phase is emphasized by XRD. The combined treatment of LM with TiO2-NPs and UV has significant effects on LM promastigotes, which vary depending on NP and UV types. The synergistic effect was anticipated in the groups irradiated by UV-B in the presence of Rutile NPs. CONCLUSION: The combined treatment with UV- radiation and TiO2-NPs can be effective in killing the promastigotes of Leishmania major. The proper concentration of NPs and the type of UV-radiation must be taken into consideration. The results suggest improved treatment methods, after proper in vivo studies.

Evaluation of methylene blue as photosensitizer in promastigotes of Leishmania major and Leishmania braziliensis.

The cutaneous leishmaniasis is caused by the protozoan of the genus Leishmania. It is considered by WHO as a public health issue and a neglected disease, which affects rural workers and it is also a risk to travelers in endemic areas. The conventional treatment is toxic and leads to severe side effects. The photodynamic therapy has been studied as an alternative treatment to cutaneous leishmaniasis. This study aimed to evaluate the methylene blue internalization and the impact of the PDT in the viability and morphology of Leishmania major and Leishmania braziliensis promastigote in culture medium. The fluorescence microscopy was used to determine the MB localization. To evaluate the mitochondrial activity (MTT), viability (Trypan blue test) and the morphological alterations both species were incubated with the MB in concentrations starting in 500µg/ml, in serial dilution, until 7,8µg/ml. The fluorescence microscopy demonstrated that the MB is internalized by both species after one hour of incubation. The MB presented low toxicity at the dark and the PDT was capable of decreasing the viability in more than 70% in the higher concentrations tested. The PDT also triggered significant morphological alterations in the Leishmania promastigotes. The results presented in this study are an indicative that the MB is a photosensitizer with promising potential to clinical application, besides its low cost.

Leishmania major and Trypanosoma cruzi present distinct DNA damage responses.

Leishmania major and Trypanosoma cruzi are medically relevant parasites and interesting model organisms, as they present unique biological processes. Despite increasing data regarding the mechanisms of gene expression regulation, there is little information on how the DNA damage response (DDR) occurs in trypanosomatids. We found that L. major presented a higher radiosensitivity than T. cruzi. L. major showed G1 arrest and displayed high mortality in response to ionizing radiation as a result of the inefficient repair of double-strand breaks (DSBs). Conversely, T. cruzi exhibited arrest in the S/G2 cell cycle phase, was able to efficiently repair DSBs and did not display high rates of cell death after exposure to gamma irradiation. L. major showed higher resistance to alkylating DNA damage, and only L. major was able to promote DNA repair and growth recovery in the presence of MMS. ASF1c overexpression did not interfere with the efficiency of DNA repair in either of the parasites but did accentuate the DNA damage checkpoint response, thereby delaying cell fate after damage. The observed differences in the DNA damage responses of T. cruzi and L. major may originate from the distinct preferred routes of genetic plasticity of the two parasites, i.e., DNA recombination versus amplification.

Antiparasitic effects of gold nanoparticles with microwave radiation on promastigots and amastigotes of Leishmania major.

PURPOSE: This study aimed to determine the efficacy of thermotherapy in the presence of gold nanoparticles (GNPs) and microwave (MW) radiation at a frequency of 2450 MHz on the survival of Leishmania major promastigotes and amastigotes. MATERIALS AND METHODS: L. major promastigotes (strain MRHO/IR/75/ER) were cultured in RPMI-1640 medium supplemented with foetal bovine serum and antibiotic. The promastigotes were incubated with GNPs for 2 h. After washing, thermotherapy was performed by MW irradiation. After 48 h the promastigote survival rate was assessed using Alamar Blue assay. In the second part of the study, after culture and proliferation of J744 cells, the infected macrophages were incubated with the GNPs and were inserted under MW irradiation. After 24 h, the number of amastigotes in the macrophages was determined after Giemsa staining by a light microscope. RESULT: Increased exposure time of the microwave to the parasites in the presence of GNPs induced a significant decline in promastigotes survival rate in comparison to similar samples without GNPs. The least survival of amastigotes was also recorded in the groups containing GNPs. The presence of GNPs during MW irradiation was more lethal for promastigotes and amastigotes in comparison to MW alone. CONCLUSION: Thermotherapy using MW radiation in the presence of GNPs may be proposed as a new approach to treat leishmaniasis in future studies.

Calcineurin is required for Leishmania major stress response pathways and for virulence in the mammalian host.

Leishmania parasites must adapt to elevated temperatures and other environmental stresses during infection of their mammalian hosts. How these environmental cues are sensed is poorly understood. In this study we show that calcium uptake is required for parasite thermotolerance at 34-37°C. To identify potential downstream targets of calcium influx, a Leishmania major mutant lacking the essential regulatory subunit (CnB) of the Ca(2+) /calmodulin-dependent serine/threonine-specific phosphatase, calcineurin, was generated. The Δcnb mutant grew as well as wild-type parasites at 27°C and differentiated normally to infective metacyclic promastigotes. However, Δcnb parasites lost viability when exposed to increased temperature (34°C) and were hypersensitive to endoplasmic reticulum and membrane stress, induced by tunicamycin and inhibitors of sterol and sphingolipid biosynthesis respectively. Δcnb promastigotes were internalized by macrophages, but their differentiation to the heat adapted amastigote stage was delayed and the resulting parasites failed to proliferate. Strikingly, the Δcnb parasites were completely cleared by susceptible BALB/c mice. Complementation of Δcnb parasites with CnB restored thermotolerance and infectivity in both macrophages and animal models. Our results suggest that Ca(2+) influx and calcineurin signalling are required for both early and long-term adaptive parasite responses to environmental stresses encountered in the mammalian host.

A randomized controlled trial of local heat therapy versus intravenous sodium stibogluconate for the treatment of cutaneous Leishmania major infection.

BACKGROUND: Cutaneous Leishmania major has affected many travelers including military personnel in Iraq and Afghanistan. Optimal treatment for this localized infection has not been defined, but interestingly the parasite is thermosensitive. METHODOLOGY/PRINCIPAL FINDINGS: Participants with parasitologically confirmed L. major infection were randomized to receive intravenous sodium stibogluconate (SSG) 20mg/kg/day for ten doses or localized ThermoMed (TM) device heat treatment (applied at 50 degrees C for 30 seconds) in one session. Those with facial lesions, infection with other species of Leishmania, or more than 20 lesions were excluded. Primary outcome was complete re-epithelialization or visual healing at two months without relapse over 12 months. Fifty-four/56 enrolled participants received intervention, 27 SSG and 27 TM. In an intent to treat analysis the per subject efficacy at two months with 12 months follow-up was 54% SSG and 48% TM (p = 0.78), and the per lesion efficacy was 59% SSG and 73% TM (p = 0.053). Reversible abdominal pain/pancreatitis, arthralgias, myalgias, headache, fatigue, mild cytopenias, and elevated transaminases were more commonly present in the SSG treated participants, whereas blistering, oozing, and erythema were more common in the TM arm. CONCLUSIONS/SIGNIFICANCE: Skin lesions due to L. major treated with heat delivered by the ThermoMed device healed at a similar rate and with less associated systemic toxicity than lesions treated with intravenous SSG. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT 00884377.

A heat-activated and thermoresistant telomerase activity in Leishmania major Friedlin.

Here we studied the telomerase activity of the human parasite Leishmania major. In this organism we have detected a high activity of this enzyme once several parameters such as heat activation, sequence of extension primer, and protein concentration are adjusted. The activity was not only heat activated, but also very resistant to heat denaturation. We believe L. major telomerase is an important activity and it may provide an adequate drug therapy target.

Photodynamic therapy for cutaneous leishmaniasis: the effectiveness of topical phenothiaziniums in parasite eradication and Th1 immune response stimulation.

Photodynamic therapy (PDT) is emerging as a therapeutic modality in the clinical management of cutaneous leishmaniasis (CL). The efficacy of PDT against CL has been demonstrated previously with aminolevulinic acid, although the prolonged terms of therapy were less than ideal, and the search for new photosensitizers (PS) is ongoing. However, phenothiaziniums have demonstrated high parasiticidal effects in vitro. The subject of our investigation is the in vivo activity of two PS, 5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium chloride (EtNBSe) and (3,7-Bis(N,N-dibutylamino) phenothiazinium bromide (PPA904). The results of our comparative analysis of the efficacy of these two phenothiazinium analogues demonstrated a high antiparasitic activity of EtNBSe in vitro, and the higher efficacy of PPA904 in a mouse model of CL. The kinetics of photodestruction are different in parasite and mammalian cells, and with both dyes, the macrophages are more susceptible to photodynamic effects than L. major parasites. As the number of parasites in the lesions undergoes a biphasic change, temporarily increasing on days 2-4 and decreasing on days 5-7, more than one treatment is required within an interval of 5 to 7 days. We have also shown that PPA904-PDT can provide an immunomodulating, dose-dependent efflux on IL-12p70 production. This mechanism could be responsible for promoting a more rapid healing in PPA904-PDT treated mice. Our initial data indicate that phenothiaziniums exhibit a high parasiticidal effect in vivo against CL; this finding may be of use in establishing curative PDT regimens for future clinical trials.

Leishmania inactivation in human pheresis platelets by a psoralen (amotosalen HCl) and long-wavelength ultraviolet irradiation.

BACKGROUND: Leishmania spp. are protozoans that cause skin and visceral diseases. Leishmania are obligate intracellular parasites of mononuclear phagocytes and have been documented to be transmitted by blood transfusion. STUDY DESIGN AND METHODS: This study examines whether Leishmania can be inactivated in human platelet (PLT) concentrates by a photochemical treatment process that is applicable to blood bank use. Human PLT concentrates were contaminated with Leishmania mexicana metacyclic promastigotes or mouse-derived Leishmania major amastigotes and were exposed to long-wavelength ultraviolet (UV) A light (320-400 nm) plus the psoralen amotosalen HCl. RESULTS: Neither treatment with amotosalen nor UVA alone had an effect on Leishmania viability; however, treatment with 150 micromol per L amotosalen plus 3 J per cm(2) UVA inactivated both metacyclic promastigotes and amastigotes to undetectable levels, more than a 10,000-fold reduction in viability. CONCLUSIONS: This study demonstrates the effectiveness of photochemical treatment to inactivate Leishmania in PLT concentrates intended for transfusion. Both metacylic promastigotes, which represent the infectious form from the sand fly vector, and amastigotes, which represent the form that grows in mononuclear phagocytes, were extremely susceptible to photochemical inactivation by this process. Thus, the photochemical treatment of PLT concentrates inactivates both forms of Leishmania that would be expected to circulate in blood products collected from infected donors.

Influence of heat shock, drugs, and radiation on karyotype of Leishmania major.

Leishmaniasis is one of the important tropical diseases in the world. Although it is not prevalent in Korea, imported cases have been recorded. The karyotype of Leishmania sp. has been observed to be variable by localities or by strains, but the karyotype of a strain is known to be stable. This study was performed to observe if the karyotype of a Leishmania sp. would be changed under some stressful conditions. The karyotype, analyzed by pulsed field gradient gel electrophoresis, was not grossly changed by heat shock, chemotherapeutics, UV illumination, and gamma irradiation. Radiation destroyed the chromosomes mechanically, but subcultured organisms after irradiation showed unaffected karyotype. The present findings suggest that the karyotype of a Leishmania strain is so stable that it is not altered by temporary stimulation with heat, drugs, and radiation.