A new defined medium for cultivating Leishmania promastigotes

A chemically defined medium to grow the promastigotes of 19 stocks of Leishmania is described. The medium was developed by making qualitative and quantitative modifications of the medium AR-103 devised for Trypanosoma cruzi. The new medium, designated as MD-29, can be used in studies on the nutritional requirements of promastigotes.     

利什曼原虫前鞭毛体体外生长动力学研究

目的观察不同种株利什曼原虫前鞭毛体在体外培养条件下的生长增殖情况，确定其最适体外培养条件。方法分别使用RPMI1640和M199复合培养液，观察温度、pH及新生小牛血清对硕大利什曼原虫、热带利什曼原虫、婴儿利什曼原虫、墨西哥利什曼原虫、杜氏利什曼原虫前鞭毛体体外增殖速度与增殖周期的影响。结果当培养温度为26℃时，杜氏利什曼原虫前鞭毛体在含和不含新生小牛血清、pH中性、偏碱和偏酸的PMI1640或M199复合培养基中，早期均能生长，且生长周期相对较长，其他种株利什曼原虫前鞭毛体在无血清或偏碱培养基中生长缓慢，增殖周期缩短；培养温度为37℃时，各种株利什曼原虫前鞭毛体均发生沉积，增殖停滞，不同程度地向无鞭毛体转化并发生死亡。结论使用复合培养液培养利什曼原虫前鞭毛体，温度、pH和新生小牛血清均可显著影响增殖速度和生长周期。各种株利什曼原虫前鞭毛体在相同培养条件下增殖速度和生长周期存在差异，可能与其遗传背景不同有关。     

Effects of long-term in vitro cultivation on the virulence of cloned lines of Leishmania major promastigotes.

The virulences of several clones from a single Leishmania major strain were studied in BALB/c mice. Clones showed the same pattern of infectivity and virulence two months after cloning as the parental population. After prolonged in vitro culture, however, it was apparent that two types of virulent clones existed: although the level of virulence remained stable in some clones, in others, such as C-11, it progressively decreased, as in the parental population. The progressive loss in virulence in a continuously cultured mixed population was probably due to selection, as the initial mixture of a stable virulent clone and a stable avirulent clone eventually yielded a totally avirulent promastigote population. After cultivation for 12 months, neither clone C-11 nor the parental population produced lesions in inoculated mice but virulent parasites were recovered from the inguinal nodes of the mice, possibly as a result of selection in vivo for virulent parasites.     

Effect of ethidium bromide on growth and morphology of Leishmania tarentolae promastigotes in vitro.

Incubation of Leishmania tarentolae promastigotes in 0.01 microgram/ml ethidium bromide in Neo Ye medium for 96 h resulted in 60% inhibition of cell growth and 91% dyskinetoplasty. After 48 h incubation in ethidium bromide over 50% of the cells were scored dyskinetoplastic by light microscopy although the electron microscopical examination revealed that most promastigotes contained at least a small amount of kDNA. A few of the treated cells undergoing division contained two kinetoplasts--one devoid of kDNA and the other with a reduced amount of kDNA as seen in the electron micrographs.     

The interaction of Leishmania donovani promastigotes and human fibroblasts in vitro

Leishmania donovani promastigotes derived from infected hamster spleens, in either log phase or stationary phase growth, associated with human foreskin fibroblasts in vitro and assumed the morphological characteristics of amastigotes. This apparent conversion was noted within hours at 26 degrees C, 32 degrees C or 37 degrees C; in the continued presence of promastigotes, increasing numbers of amastigote-like forms were seen for 2 weeks at 26 degrees C or 32 degrees C. At 37 degrees C amastigote-like forms declined sharply after 6 days. Multiplication of amastigote-like forms was not observed at any temperature, this was also true of freshly isolated amastigotes from hamster spleens which associated with fibroblasts but did not multiply. Approximately 0.1% of promastigotes appeared to convert per day. Amastigote-like forms were seen within fibroblasts by transmission electron microscopy, surrounded by a closely applied host membrane. Scanning electron microscopy showed promastigotes with their flagellae under or within fibroblasts, but phagocytosis was not observed. These experiments suggest that the conditions required for promastigote-to-amastigote conversion may be different than those required for amastigote multiplication, and the mammalian core body temperature may not be required for promastigote conversion.     

Insulin-like growth factor I is a growth-promoting factor for Leishmania promastigotes and amastigotes

Leishmaniases are diseases caused by protozoa of the genus Leishmania that affect more than 20 million people in the world. The initial phase of the infection is fundamental for either the progression or control of the disease. The Leishmania parasites are injected in the skin as promastigotes and then, after been phagocytized by the host macrophages, rapidly transform into amastigotes. In this phase different nonspecific cellular and humoral elements participate. We have shown previously that insulin-like growth factor (IGF)-I that is constitutively present in the skin induces growth of Leishmania promastigotes. In the present paper we show further evidence for the importance of this factor: (i) IGF-I also can induce a growth response in Leishmania (Leishmania) mexicana amastigotes; (ii) IGF-I binds specifically to a putative single-site receptor on both promastigotes and amastigotes; (iii) IGF-I induces a rapid tyrosine phosphorylation of parasite proteins with different molecular mass in promastigotes and amastigotes of L. (L.) mexicana; and, finally, (iv) the cutaneous lesion in the mice when challenged by IGF-I-preactivated Leishmania (Viannia) panamensis is increased significantly because of inflammatory process and growth of parasites. We thus suggest that IGF-I is another important host factor participating in the Leishmania–host interplay in the early stage during the establishment of the infection and presumably also in the later stages.     

Preservation of Leishmania donovani promastigotes in blood agar slants

Long-term cultivation of Leishmania promastigotes by weekly passage to fresh medium was reported to be disadvantageous because needs labor, risk of contamination, lowering in infectivity and virulence pattern. Cryopreservation and Lyophilization require expensive facilities which could be a burden and unaffordable to most laboratories of developing countries where the disease is endemic. These problems could be minimized by simple preservation of Leishmania donovani promastigotes in blood agar slants at 7-8 degrees C for 6-7 months. The preserved promastigotes were examined for viability up to one year at a regular interval of one month. Viable promastigotes were found and revived successfully from all the slants stored up to 7 months after that, the viability of promastigotes was found to be decreased in the slants of 8-9 month storage. No viable promastigotes were recovered from the slants stored up to 11-12 months. By this method, the promastigotes can easily be stored up to 7 months without loss of biological activity. The number of passage of promastigotes to fresh medium has been greatly reduced by this method from 30 times to 01 when compared with weekly passage in liquid medium. This simple and economical method can be recommended for short storage of Leishmania culture without loss of any activity.     

In vitro cultivation and characterization of Leishmania chagasi amastigote-like forms

For the first time, we have reported the establishment and serial propagation of an axenic culture of Leishmania chagasi amastigote-like forms. Parasites were characterized by microscopic evaluation and by the expression of two stage-specific genes, A2 and Ldccys2 amastigote-specific cysteine protease. The differentiated amastigote-like forms were maintained by serial cultivation.     

pH homeostasis in Leishmania donovani amastigotes and promastigotes.

Intracellular pH and pH gradients of Leishmania donovani amastigotes and promastigotes were determined over a broad range of extracellular pH values. Intracellular pH was determined by 31P NMR and by equilibrium distribution studies with 5,5-dimethyloxazolidine-2,4-dione or methylamine. Promastigotes maintain intracellular pH values close to neutral between extracellular pH values of 5.0 and 7.4. Amastigote intracellular pH is maintained close to neutral at external pH values as low as 4.0. Both life stages maintain a positive pH gradient to an extracellular pH of 7.4, which is important for active transport of substrates. Treatment with ionophores, such as nigericin and carbonyl cyanide m-chlorophenylhydrazone and the ATPase inhibitor dicyclohexylcarbodiimide, reduced pH gradients in both stages. Maintenance of intracellular pH in the physiologic range is especially relevant for the survival of the amastigote in its acidic in vivo environment.     

The Leishmania chagasi proteasome: role in promastigotes growth and amastigotes survival within murine macrophages

Proteasomes are multisubunit proteases that exist universally among eukaryotes. They have multiple proteolytic activities and are believed to have important roles in regulating cell cycle, selective intracellular proteolysis, and antigen presentation. Here we have partially purified Leishmania chagasi proteasome. The L. chagasi proteasome rich fraction displayed the typical features of eukaryotic 20S proteasome complexes, being active towards peptidyl substrates with hydrophobic and acidic residues, and sensitive to the proteasome-specific inhibitor lactacystin. We have shown that lactacystin, or its active form clasto-lactacystin beta-lactone, but not E-64, blocks the in vitro growth of L. chagasi promastigotes, demonstrating that the interference with parasite growth is due to the lack of proteasome activity. Furthermore, pre-treatment of L. chagasi promastigotes with lactacystin did not prevent parasite entry in host cells, but markedly restricted its intracellular survival. These results demonstrate that intact parasite proteasome function is required for replication of L. chagasi and for amastigotes survival inside the vertebrate host cell.     

In vivo and in vitro phagocytosis of Leishmania (Leishmania) amazonensis promastigotes by B‐1 cells

Leishmaniasis is caused by Leishmania parasites that infect several cell types. The promastigote stage of Leishmania is internalized by phagocytic cells and transformed into the obligate intracellular amastigote form. B‐1 cells are a subpopulation of B cells that are able to differentiate in vitro and in vivo into mononuclear phagocyte‐like cells with phagocytic properties. B‐1 cells use several receptors for phagocytosis, such as the mannose receptor and third complement receptor. Leishmania binds to the same receptors on macrophages. In this study, we demonstrated that phagocytes derived from B‐1 cells (B‐1 CDP) were able to internalize promastigotes of L. (L.) amazonensis in vitro. The internalized promastigotes differentiated into amastigotes. Our results showed that the phagocytic index was higher in B‐1 CDP compared to peritoneal macrophages and bone marrow‐derived macrophages. The in vivo phagocytic ability of B‐1 cells was also demonstrated. Parasites were detected inside purified B‐1 cells after intraperitoneal infection with L. (L.) amazonensis promastigotes. Intraperitoneal stimulation with the parasites led to an increase in both IL‐10 and TNF‐α. These results highlight the importance of studying B‐1 CDP cells as phagocytic cells that can participate and contribute to immunity to parasites.     

Differential growth requirements of several Leishmania spp. in chemically defined culture media

17 strains of Leishmania from 4 species: brasiliensis, mexicana, donovani and garnhami have been continually cultured at 26 degrees C, in the absence of proteins, in a medium containing salts, glucose, D-ribose, 2-deoxyribose, hemin, tricine, HEPES, 34 amino acids and intermediates of amino acid metabolism, 23 vitamins, 6 nucleotides and tetrahydrofolic acid. A wide variation in growth requirements was observed among leishmaniae which permitted the preparation of different minimum culture media for each Leishmania spp. Virulence of parasites was maintained after 30 passages in these chemically defined media. The requirements for differentiation to amastigotes also varied among the species as a function of the temperature of incubation and the protein content of the culture medium. Bovine serum albumin tryptic peptides substituted fetal bovine serum as growth factors at 30-34 degrees C.     

A method for the purification of Leishmania promastigotes from infected phlebotomine sandflies

We describe a method for the purification of Leishmania promastigotes, isolated from infected sandflies (Lutzomyia longipalpis) using a discontinuous density centrifugation gradient (Percoll/Homem). The sandflies, infected seven days previously with Leishmania donovani chagasi or Leishmania mexicana mexicana from culture, were homogenized and centrifuged on a Percoll discontinuous gradient. Five interface bands were formed, and most of the promastigotes settled out at the interface between the (30% and 40%) Percoll concentrations. An extraction of 3.5 x 10(4) promastigotes from 90 female flies was achieved using this technique.     

Catabolism of adenosine 5'-monophosphate in promastigotes of Leishmania tropica.

The specific activities of enzymes participating in AMP-catabolizing reactions in promastigotes of Leishmania tropica were determined. The following sequence of reactions is suggested: AMP leads to adenosine leads to adenine leads to hypoxanthine. The initial enzyme of this sequence, AMP nucleotidase, is apparently membrane-bound. The significance of AMP catabolism with respect to adenylate energy charge and a signal transfering mechanism is discussed. Adenine deaminase has been partially purified and characterized. Several purine analogues, inter alia allopurinol, proved to be inhibitors of the adenine deaminase catalyzed reaction.     

Antileishmanial activities of caffeic acid phenethyl ester loaded PLGA nanoparticles against Leishmania infantum promastigotes and amastigotes in vitro

Objective: To investigate and compare the antileishmanial effects of CAPE and(CAPE)PLGA NPs on Leishmania infantum(L.infantum) promastigotes and amastigotes in vitro,Methods: Efficacies of CAPE,(CAPE)PLGA NPs and free PLGA nanoparticles(NPs) on promastigotes were evaluated using MTT and promastigote count assays,and their anti-amastigote effects were determined via infection index analysis,Griess reaction was also performed to calculate nitric oxide production of macrophages exposed to investigated molecules,Results: It was determined that CAPE and(CAPE)PLGA NPs demonstrated significant inhibitory effects on L.infantum promastigotes and amastigotes,while free NPs did not exhibit any meaningful antileishmanial effectiveness,The IC50 values of CAPE for L.infantum promastigotes and amastigotes were assessed as(51.0±0.8) and(19.0±1.4) μg/m L,respectively(P0.05),On the other side,it was revealed that(CAPE)PLGA NPs had superior antileishmanial activity on both forms of parasites since its IC50 values for L.infantum promastigotes and amastigotes were(32.0±1.3) and(8.0±0.9) μg/m L,respectively(P0.05),It was also determined that both agents strongly stimulated nitric oxide production of macrophages,Conclusions: The obtained results show that(CAPE)PLGA NPs have a great potential to be especially used in treatment of visceral leishmaniasis; however,in vivo antileishmanial screening of these molecules should be performed in the near future.     

Destruction of Leishmania mexicana amazonensis promastigotes by normal human serum

Fresh normal human serum was observed to have a lethal effect on Leishmania mexicana amazonensis promastigotes obtained from laboratory-bred Lutzomyia longipalpis or on promastigotes grown in liquid culture medium, inoculated with the same isolates. Heat inactivation abolished the Leishmania lytic activity from the sera. Resistance of culture promastigotes to lysis by normal human serum was investigated in three isolates of L. m. amazonensis. Development of resistance (up to 7%) was found in only one isolate, obtained from the bone marrow in a human case of visceral leishmaniasis.