Differential tissue distribution of diverse clones of Trypanosoma cruzi in infected mice.

Chagas disease, caused by the protozoan Trypanosoma cruzi, presents variable clinical course but the phenomena underlying this variability remain largely unknown. T. cruzi has a clonal population structure and infecting strains are often multiclonal. T. cruzi genetic variability could be a determinant of differential tissue tropism or distribution and consequently of the clinical forms of the disease. We tested this hypothesis by using low-stringency single specific primer polymerase chain reaction (LSSP-PCR) to type genetically the parasites in tissues of experimental infected mice. BALB/c mice were simultaneously inoculated with two different T. cruzi populations (JG strain and Col1.7G2 clone). Doubly infected animals showed clear differential tissue distribution for the two populations (chronic phase). Our results indicate a significant influence of the genetic polymorphism of infecting T. cruzi populations in the pathogenesis of chronic Chagas disease.     

Structural bases that underline Trypanosoma cruzi calreticulin proinfective,antiangiogenic and antitumor properties

Microbes have developed mechanisms to resist the host immune defenses and some elicit antitumor immune responses. About 6 million people are infected with Trypanosoma cruzi, the protozoan agent of Chagas’ disease, the sixth neglected tropical disease worldwide. Eighty years ago, G. Roskin and N. Klyuyeva proposed that T. cruzi infection mediates an anti-cancer activity. This observation has been reproduced by several other laboratories, but no molecular basis has been proposed. We have shown that the highly pleiotropic chaperone calreticulin (TcCalr, formerly known as TcCRT), translocates from the parasite ER to the exterior, where it mediates infection. Similar to its human counterpart HuCALR (formerly known as HuCRT), TcCalr inhibits C1 in its capacity to initiate the classical pathway of complement activation. We have also proposed that TcCalr inhibits angiogenesis and it is a likely mediator of antitumor effects. We have generated several in silico structural TcCalr models to delimit a peptide (VC-TcCalr) at the TcCalr N-domain. Chemically synthesized VC-TcCalr did bind to C1q and was anti-angiogenic in Gallus gallus chorioallantoic membrane assays. These properties were associated with structural features, as determined in silico. VC-TcCalr, a strong dipole, interacts with charged proteins such as collagen-like tails and scavenger receptors. Comparatively, HuCALR has less polarity and spatial stability, probably due to at least substitutions of Gln for Gly, Arg for Lys, Arg for Asp and Ser for Arg that hinder protein-protein interactions. These differences can explain, at least in part, how TcCalr inhibits the complement activation pathway and has higher efficiency as an antiangiogenic and antitumor agent than HuCALR.     

N,N′-Thiophene-substituted polyamine analogs inhibit mammalian host cell invasion and intracellular multiplication of Trypahosoma cruzi

We studied the effects of two, N,N′-thiophene-substituted polyamine analogs (MDL 28302 and MDL 29431) on the capacities of Trypanosoma cruzi, the etiologic agent of Chagas' disease, to invade and multiply within a mammalian host cell. Both compounds inhibited infectivity significantly in a time- and concentration-dependent manner. This inhibition resulted from a selective effect on the parasite, because pretreatment of T. cruzi but not host cell cultures with either MDL 28302 or MDL 29431 reduced infectivity. The parasite gradually recovered its infective capacity after removal of unincorporated polyamine analog, denoting the reversible nature of the inhibitory effect. Some biochemical modification of MDL 28302 and MDL 29431 appeared to be required for their inhibitory activities to be exerted, since the effects of these drugs on T. cruzi infectivity were abrogated by MDL 72527, a drug known to inhibit polyamine oxidase (PAO) activity specifically. Supporting the notion of that products of MDL 28302 and MDL 29431 oxidation by PAO were involved in the activity of these compounds was the finding that PAO competitive substrates (N¹Lacetylspermine and N¹-acetylspermidine) also abolished the inhibition of T. cruzi infectivity mediated by MDL 28302 or MDL 29431. However, we can not rule out that MDL 72527 and the PAO competitive substrates might have altered an alternative mechanism because no significant polyamine oxidase activity could be demonstrated in preparations of lysed or intact T. cruzi in assays monitoring conversion of [¹⁴C]spermine to [¹⁴C]spermidine. When either MDL 28302 or MDL 29431 was added to infected cell cultures, a marked reduction in the rate of intracellular parasite growth ensued. The significance of the finding that N,N′-thiophene-substituted polyamine analogs inhibit cell invasion and cytoplasmic replication by T. cruzi resides in the fact that this pathogenic parasite requires a cytoplasmic localization to replicate in mammalian hosts.     

Density gradient purification of human lymphocytes from contaminating trypomastigotes of Trypanosoma cruzi

Mixtures of normal human lymphocytes and T. cruzi trypomastigotes obtained from infected mice were centrifuged over Ficoll-Hypaque (FH) continuous and discontinuous gradients. Trypomastigotes were confined to the range 1.051–1.057 g/ml while lymphocytes ranged between 1.046 and 1.080 g/ml. Over 80% of the lymphocytes were found at 1.060 g/ml or higher densities. A discontinuous gradient of FH with 2 layers of 1.060 and 1.077 g/ml of density respectively was selected to obtain trypomastigotes-free white blood cells from blood samples. The functional capacity of lymphocytes recovered from the lower interface, where no parasites were found, was assessed. The response to phytohaemagglutinin of these high density lymphocytes was as good as of total lymphocytes, suggesting that low density lymphocytes are not necessary for proliferative responses. It is postulated that high density lymphoid populations, free from T. cruzi forms, may be used to study the presence of T cell-mediated immune response in Chagas' disease patients.     

Pfaffia paniculata (Brazilian ginseng) methanolic extract reduces angiogenesis in mice

Pfaffia paniculata (Brazilian ginseng) roots have been indicated for the treatment of several diseases. Our studies have shown that P. paniculata roots present antineoplastic effects and cancer chemopreventive activity in a mouse hepatocarcinogenesis model. The purpose of this study was to investigate the effects of the Brazilian ginseng on corneal angiogenesis in mice. We first conducted a toxicological study employing 250, 500, or 1000 mg/kg/day of the methanolic extract of P. paniculata roots by gavage to BALB/c mice. Animals did not lose weight during the treatment nor presented histopathological alterations. The effect of this root on angiogenesis in the cornea of BALB/c mice was then assessed. Male mice were treated, by gavage, once a day, with doses of 250, 500, or 1000 mg/kg of methanolic extract of P. paniculata powdered root for 10 days; filtered water was used as control. Corneal cauterization was accomplished by the contact of a silver nitrate crystal on the central area of the cornea, in the 5th day of treatment with P. paniculata, which continued thereafter; the animals were euthanized on the 6th day after cauterization. Newly formed blood vessels were filled with India ink, and the corneas were routinely processed. Blood vessels were quantified in an image analysis system. A smaller total area of neovascularization in the mouse cornea was observed in animals treated with 1000 mg/kg of the methanolic extract of P. paniculata. These results indicate an antiangiogenic effect of this extract. The mechanisms of this antiangiogenic activity of P. paniculata should be further investigated.     

Bone sialoprotein promotes tumor cell migration in both in vitro and in vivo models

The present study was conducted to determine the effects of bone sialoprotein (BSP) in promoting vascular invasion of tumor cells in metastasis. We used a Matrigel system and the MDA-231 human breast cancer cells transfected with human BSP cDNA (MDA-231/BSP). Quantative analysis indicated an average of 1.7-fold increase in cell numbers that migrated through the endothelial cells in MDA-231/BSP cells compared with empty vector-transfected MDA-231 cells (MDA-231/EV). In an in vivo assay, the MDA-231 cells were incubated with or without BSP antibodies and were then inoculated onto the upper chorioallantoic membrane (CAM) of chicken embryos, in which the only route for the tumor cells to reach the lower CAM was to migrate through the embryonic vasculature. PCR amplification using human Alu primers and genomic DNA from harvested lower CAM showed an average reduction of 67% in the samples treated with BSP antibodies. These preliminary data suggest that, in metastasis, BSP may enhance the penetrating ability of tumor cells through endothelial cells and basement membrane into blood vessels. BSP antibodies can specifically hinder this effect in an in vivo system.     

Cloning and functional expression of Rpn1, a regulatory-particle non-ATPase subunit 1, of proteasome from Trypanosoma cruzi

Non-lysosomal protein degradation in eukaryotic cells involves a proteolytic complex referred to as 26S proteasome that consists of a 20S core particle and one or two 19S regulatory particles. We have cloned the gene RPN1 encoding Rpn1 (regulatory-particle non-ATPase subunit 1), one of the largest subunits of proteasome, from Trypanosoma cruzi. It contains 2712 bp and encodes 904 amino acid residues with a calculated molecular mass of 98.2 kDa and an isoelectric point of 5.2. The predicted amino acid sequence of the trypanosomatid Rpn1 shares 39.0 and 32.0% overall identities with human Rpn1 and Saccharomyces cerevisiae Nas1 (non-ATPase subunit 1), an Rpn1 homolog, respectively, while the sequence identities among T. cruzi, Plasmodium falciparum, and Entamoeba histolytica Rpn1 are approximately 30%. T. cruzi Rpn1 contains nine repeats of about 36 amino acid residues conserved in Rpn1s from various organisms. T. cruzi RPN1 is located on the 2300- and 1900-kb chromosomal DNA, displays a putative allelic variation as RPN1-1 and RPN1-2 with 98.8% identity between these two putative gene products, and is transcribed from both alleles at a comparable level throughout the three developmental stages of the parasite, epimastigotes, trypomastigotes, and amastigotes. The expression of the trypanosomatid Rpn1 in the temperature-sensitive nas1 yeast mutant rescued the growth defect at the restrictive temperature, indicating that Rpn1 functions as a Nas1 and probably assembles into the 19S regulatory particle of the yeast 26S proteasome.     

Inhibitory action of the antitumor agent lonidamine on mitochondrial respiration of Trypanosoma cruzi and T. brucei

The antitumor and antispermatogenic agent lonidamine inhibits Trypanosoma cruzi epimastigotes growth in culture with an ID₅₀ around 80 μM. The main site of action appears to be the mitochondria, where the rate of uncoupled respiration was inhibited in 50% at a similar lonidamine concentration (50 μM). Hexokinase (the other point where lonidamine inhibits tumor energy metabolism) was not sensitive to this drug. Lonidamine also inhibited uncoupled respiration in T. brucei procyclic trypomastigotes, suggesting a common mechanism of action with T. cruzi. When lonidamine was added to T. brucei trypomastigotes, there was little effect on the CN-insensitive respiration, demonstrating that at least in T. brucei glycolysis is not affected by the drug.     

Comparative analysis of genomic sequences suggests that Trypanosoma cruzi CL Brener contains two sets of non-intercalated repeats of satellite DNA that correspond to T. cruzi I and T. cruzi II types

Approximately 10% of the Trypanosoma cruzi genome is formed by a satellite DNA, composed by 195-bp repeats organized in 30 ± 10 kb clusters in some, but not all chromosomes. Here, the satellite DNA of six representative T. cruzi strains was sequenced and used for phylogenetic inference. The results show that CL Brener contains satellite repeats from T. cruzi I and T. cruzi II strains, although type II sequences are more abundant. The presence of types I and II sequences extends previous propositions that genetic exchange between the two major T. cruzi lineages have occurred in CL Brener, although our data accommodate alternative scenarios of hybridization within T. cruzi II, as proposed by others. Altogether, present data suggest a complex origin for CL Brener. Sequence analysis of satellites isolated from chromosomal bands indicates that satellite DNA sequences are not chromosome specific. Neighbor analysis of in tandem satellite DNAs containing up to five repeats shows that each cluster contains only one type of sequence. Consequently, clusters with intercalated types I and II repeats were not found. We propose that the CL Brener genome contains large pieces of satellite DNA originated mainly from chromosomes of T. cruzi II with introgression of T. cruzi I lineage.     

A comparative study on the effects of tumor necrosis factor-alpha (TNF-alpha), human angiogenic factor (h-AF) and basic fibroblast growth factor (bFGF) on the chorioallantoic membrane of the chick embryo.

The chorioallantoic membrane (CAM) assay is a widely used bioassay for testing angiogenic activities. In the present study we compared the gross and micromorphological effects of three angiogenic factors applied in Elvax carriers on the CAM: Tumor necrosis factor-alpha (TNF-alpha), human angiogenic factor (h-AF), and basic fibroblast growth factor (bFGF). Our question was whether the CAM responds to these factors which have very different actions with a stereotype or with a factor specific reaction. By microangiography and light microscopy, all positive reactions appeared as a spoke-wheel vascular pattern with a bundle of small capillary blood vessels in the center. These vessels were predominantly of a distended type in h-AF and TNF experiments, while narrower capillary vessels followed bFGF application. Chorioallantoic ectoderm and endoderm were thickened by cell accumulation and the mesenchymal stroma of the CAM was edematous and infiltrated with leucocytes in all three reactions. Additionally, bFGF experiments showed areas of densely arranged fibroblasts. Observations in vivo showed chorioallantoic tissue movements as a possible mechanism for the spokewheel vascular pattern. As compared with our results from studies of cytokinetics with bromodeoxyuridine, these current findings indicate that chemotaxis is responsible for the chorioallantoic angiogenic reaction rather than cellular proliferation.     

Bone Sialoprotein Promotes Tumor Cell Migration in Both In Vitro and In Vivo Models

The present study was conducted to determine the effects of bone sialoprotein (BSP) in promoting vascular invasion of tumor cells in metastasis. We used a Matrigel system and the MDA-231 human breast cancer cells transfected with human BSP cDNA (MDA-231/BSP). Quantative analysis indicated an average of 1.7-fold increase in cell numbers that migrated through the endothelial cells in MDA-231/BSP cells compared with empty vector-transfected MDA-231 cells (MDA-231/EV). In an in vivo assay, the MDA-231 cells were incubated with or without BSP antibodies and were then inoculated onto the upper chorioallantoic membrane (CAM) of chicken embryos, in which the only route for the tumor cells to reach the lower CAM was to migrate through the embryonic vasculature. PCR amplification using human Alu primers and genomic DNA from harvested lower CAM showed an average reduction of 67% in the samples treated with BSP antibodies. These preliminary data suggest that, in metastasis, BSP may enhance the penetrating ability of tumor cells through endothelial cells and basement membrane into blood vessels. BSP antibodies can specifically hinder this effect in an in vivo system.     

Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi

A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The K_m values of the enzyme for ATP, GDP and dTDP were 0.2 ± 0.008 mM, 0.125 ± 0.012 mM and 0.4 ± 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65°C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [γ-³²P]ATP or thiophosphorylated with [³⁵S]GTPγS. The incubation of the ³²P-labelled phosphoenzyme with unlabelled nucleoside 5′-diphosphates resulted in the formation of ³²P-labelled nucleoside 5′-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5′-diphosphates, GTP was preferentially formed.     

基因重组纤溶酶原 Kringle 5 对血管生成抑制作用的研究

目的 研究基因重组纤溶酶原 (Kringle 5, K5) 对小鼠的血管生成的抑制作用。 方法 以体外 培养的小鼠内皮细胞为研究对象, 利用划痕实验和黏附能力测定实验观察重组 K5 对内皮细胞迁移、 黏附 能力的影响; 利用体外血管生成体系、 创伤愈合和鸡胚尿囊膜 ( chicken embryo allantoic membrane, CAM) 血管生成能力实验, 检测 K5 对血管生成作用的影响。 结果 200 nmol / L K5 显著抑制内皮细胞的迁移、 黏 附以及新生血管的生长长度。 并且 K5 在 2 ~ 18 mg / kg 浓度范围内对小鼠创伤愈合具有明显的抑制作用。 此 外, 6、 12 与 25 mg / L K5 显著抑制 CAM 血管生成, 25 mg / L K5 的抑制作用最强。 结论 K5 能明显抑制血 管生成, 为临床抑制血管生成治疗提供理论依据。     

A comparative study on the effects of tumor necrosis factor-α (TNF-α), human angiogenic factor (h-AF) and basic fibroblast growth factor (bFGF) on the chorioallantoic membrane of the chick embryo

The chorioallantoic membrane (CAM) assay is a widely used bioassay for testing angiogenic activities. In the present study we compared the gross and micromorphological effects of three angiogenic factors applied in Elvax? carriers on the CAM: Tumor necrosis factor-α (TNF-α), human angiogenic factor (h-AF), and basic fibroblast growth factor (bFGF). Our question was whether the CAM responds to these factors which have very different actions with a stereotype or with a factor specific reaction. By microangiography and light microscopy, all positive reactions appeared as a spoke-wheel vascular pattern with a bundle of small capillary blood vessels in the center. These vessels were predominatly of a distended type in h-AF and TNF experiments, while narrower capillary vessels followed bFGF application. Chorioallantoic ectoderm and endoderm were thickened by cell accumulation and the mesenchymal stroma of the CAM was edematous and infiltrated with leucocytes in all three reactions. Additionally, bFGF experiments showed areas of densely arranged fibroblasts. Observations in vivo showed chorioallantoic tissue movements as a possible mechanism for the spokewheel vascular pattern. As compared with our results from studies of cytokinetics with bromodeoxyuridine, these current findings indicate that chemotaxis is responsible for the chorioallantoic angiogenic reaction rather than cellular proliferation. © 1992 Wiley-Liss, Inc.     

Antiangiogenic Activity of Acer tegmentosum Maxim Water Extract in Vitro and in Vivo

Angiogenesis, the formation of new blood vessels, is critical for tumor growth and metastasis. Notably, tumors themselves can lead to angiogenesis by inducing vascular endothelial growth factor (VEGF), which is one of the most potent angiogenic factors. Inhibition of angiogenesis is currently perceived as one of the most promising strategies for the blockage of tumor growth. In this study, we investigated the effects of Acer tegmentosum maxim water extract (ATME) on angiogenesis and its underlying signal mechanism. We studied the antiangiogenic activity of ATME by using human umbilical vein endothelial cells (HUVECs). ATME strongly inhibited VEGF-induced endothelial cell proliferation, migration, invasion, and tube formation, as well as vessel sprouting in a rat aortic ring sprouting assay. Moreover, we found that the p44/42 mitogen activated protein (MAP) kinase signaling pathway is involved in the inhibition of angiogenesis by ATME. Moreover, when we performed the in vivo matrigel plug assay, VEGF-induced angiogenesis was potently reduced when compared to that for the control group. Taken together, these results suggest that ATME exhibits potent antiangiogenic activity in vivo and in vitro and that these effects are regulated by the extracellular regulated kinase (ERK) pathway.

Graphical Abstract

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Chrysobalanus icaco L. extract for antiangiogenic potential observation

Angiogenesis is an important process in several physiological situations and it is also implicated in the development of some diseases such as diabetes and cancer. This study investigated the antiangiogenic potential of Chrysobalanus icaco methanol extract in the chicken embrionary tissue. Clinical trials for cancer treatment using drugs based on this mechanism are already in progress. Chorioallantoic membrane model (CAM) of chicken embryos, with C. icaco methanol extract in plastic diskes was used. The results showed an average of 44% angiogenesis inhibition in CAM areas with the plant extract compared to the controls. The data indicate that C. icaco methanol extract reduce the formation of new blood vessels in chicken chorioallantoic membrane.     

Protective immunity induced by a Trypanosoma cruzi soluble extract antigen in experimental Chagas' disease. Role of interferon gamma

CBA/J mice can be protected against lethal infection with Trypanosoma cruzi by treatment using T. cruzi soluble extract antigen (TCSE). In vivo administration of TCSE (400 μg/mouse) into naive mice increased the cellular proliferative response to Con A and elevated the levels of IFN-γ. The production of IFN-γ was extremely important in controlling the replication of the parasite since the protective activity of TCSE was completely abrogated by in vivo treatment with an anti IFN-γ neutralizing antibody. These results suggest that depending on the level, cytokine production results in the control of replication of the parasite in experimental Chagas'disease.     

Protective immunity induced by a Trypanosoma cruzi soluble extract antigen in Experimental Chagas' Disease. Role of Interferon γ

CBA/J mice can be protected against lethal infection with Trypanosoma cruzi by treatment using T. cruzi soluble extract antigen (TCSE). In vivo administration of TCSE (400 μg/mouse) into naive mice increased the cellular proliferative response to Con A and elevated the levels of IFN-γ. The production of IFN-γ was extremely important in controlling the replication of the parasite since the protective activity of TCSE was completely abrogated by in vivo treatment with an anti IFN-γ neutralizing antibody. These results suggest that depending on the level, cytokine production results in the control of replication of the parasite in experimental Chagas'disease.     

Evaluation of pharmacological activities and assessment of intraocular penetration of an ayurvedic polyherbal eye drop (Itone™) in experimental models

Background

The polyherbal eye drop (Itone?) is a mixture of aqueous distillates of nineteen traditionally used ingredients that sum up to impart potency to the formulation and make it a useful adjunct in various ocular pathologies. However, as there have been no controlled experimental studies accounting to the above claim, therefore, the present study was designed to evaluate the polyherbal formulation (PHF) for antiangiogenic, anti-inflammatory, anticataract, antioxidant and cytotoxicity in addition to the evaluation of intraocular penetration of PHF in rabbit eyes using LC-MS/MS.

Materials and methods

Antiangiogenic activity of the PHF was evaluated using in ovo chick chorio-allantoic membrane (CAM) assay and in vivo cautery induced corneal neovascularization assay in rats. Anticataract potential was evaluated using steroid induced cataract in developing chick embryos, sodium selenite induced cataract in rat pups and galactose induced cataract in rats. The antioxidant activity was evaluated using di-phenyl picryl hydrazyl (DPPH) radical scavenging assay. Anti-inflammatory activity was evaluated in vitro using inhibition of LTB₄ formation in human WBCs and in vivo using carrageenan induced paw edema assay in rats. The cytotoxicity was evaluated against HeLa cancer cell lines using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore evaluation of the intraocular penetration of the PHF was carried out in rabbit eyes via aqueous humor paracentesis and further analysis using LC-MS/MS.

Results

PHF significantly inhibited VEGF induced proliferation of new blood vessels in CAM assay and inhibited the cautery induced corneal neovascularization in rats. Additionally, PHF showed noticeable delay in the progression of cataract in the selenite and galactose induced cataract models whereby the PHF treated lenses were graded for stages II and III respectively. However, the PHF did not show any anticataract activity in the hydrocortisone induced cataract model. Moreover, PHF exhibited anti-inflammatory activity whereby it showed 39.34% inhibition of LTB₄ formation and significantly inhibited carrageenan induced paw edema in rats. Eight compounds of PHF viz. camphor, casticin, curcumin-II, quercetin, rosmarinic acid, γ-terpinene, β-pinene and dipentene exhibited transcorneal penetration in rabbit eyes.

Conclusion

The significant antiangiogenic and anti-inflammatory activities evinced by the PHF merits further investigation for ocular neovascular and inflammatory diseases in humans.     

Repetitive sequences in the ribosomal intergenic spacer of Trypanosoma cruzi

A fragment of Trypanosoma cruzi ribosomal intergenic spacer (IGS) located at 6.7 kb from the 3′ end of the 24S rRNA gene was analyzed. This IGS fragment is characterized by the presence of three types of repetitive elements (designated Spacer Repetitive Elements, SRE), short direct repeats (5-6 bp) and chi-like recombinational sequences. SRE elements are composed of relatively short repeats (43–145 bp) which show variabilities consisting of nucleotide changes, insertions and deletions. SRE-1 element (145 bp) has a short oligo(dA) tail at the end of the repeat and can be found flanked by other SRE elements. SRE elements are species-specific, suggesting that probes based on them may be diagnostic for Trypanosoma cruzi.