Cell cycle arrest induced by Pisosterol in HL60 cells with gene amplification

The leukemia cell line HL60 is widely used in studies of the cell cycle, apoptosis, and adhesion mechanisms in cancer cells. We conducted a focused cytogenetic study in an HL60 cell line, by analyzing GTG-banded chromosomes before and after treatment with pisosterol (at 0.5, 1.0, and 1.8 μg/ml), a triterpene isolated from Pisolithus tinctorius, a fungus collected in the Northeast of Brazil. Before treatment, 99% of the cells showed the homogeneously staining region (HSR) 8q24 aberration. After treatment with 1.8 μg/ml pisosterol, 90% of the analyzed cells lacked this aberration. We further performed a pulse test, in which the cells treated with pisosterol (0.5, 1.0, and 1.8 μg/ml) were washed and re-incubated in the absence of pisosterol. Only 30% of the analyzed cells lacked the HSR 8q24 aberration, suggesting that pisosterol probably blocks the cells with HSRs at interphase. No effects were detected at lower concentrations. At the highest concentration examined (1.8 μg/ml), pisosterol also inhibited cell growth, but this effect was not observed in the pulse test, reinforcing our hypothesis that, at the concentrations tested, pisosterol probably does not induce cell death in the HL60 line. The results found for pisosterol were compared with those for doxorubicin. Cells that do not show a high degree of gene amplification (HSRs and double-minute chromosomes) have a less aggressive and invasive behavior and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti-cancer therapy.     

Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence

Background

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models.

Methodology/Principal Findings

We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites.

Conclusions/Significance

Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.     

Trypanosoma cruzi: Genetic diversity influences the profile of immunoglobulins during experimental infection

The clonal evolution model postulated for Trypanosoma cruzi predicts a correlation between the phylogenetic divergence of T. cruzi clonal genotypes and their biological properties. In the present study, the linkage between phylogenetic divergence of the parasite and IgG, IgG1, IgG2a and IgG2b response has been evaluated during the acute and chronic phases of the experimental infection. Eight laboratory-cloned stocks representative of this phylogenetic diversity and including the lineages T. cruzi I (genotypes 19 and 20), T. cruzi II (genotype 32) and T. cruzi (genotype 39) have been studied. The results showed that the pattern of humoral immune response was correlated with T. cruzi genotype, and that stocks included in genotype 20 were responsible for the high IgG response in the acute and chronic phases. Moreover, T. cruzi I lineage was more efficient in over-expressing all subclasses of specific anti-parasite IgG than either T. cruzi II or T. cruzi lineages. Curiously, the alteration in the pattern of antibodies induced by Benznidazole treatment was related to the phase of the infection but not to the genotype of the parasite. The data suggest that genotypes of T. cruzi are able to drive levels/subclasses of specific IgG, hence giving rise to further concerns about the sensitivity of serological assays in the diagnosis of human Chagas disease.     

Design of a fungal cellulose-binding domain for PCR amplification and expression in E. coli

A new fungal cellulose binding domain (CBD) from Stachybotris sp. has been cloned. Multiple sequence alignment of the CBD from 34 fungi shows highest sequence identity at the ends of the domains. The two primers from these regions were amplified by PCR giving a 120-bp product. Two of these, from Trichoderma sp. and Stachybotris sp. were subsequently cloned, sequenced and confirmed to be of the CBD family. The CBD from Stachybotris sp. was expressed in E. coli fused to g3p of the M13 phage and with a c-myc tag. The secreted fusion protein adsorbed on acid-swollen cellulose thereby confirming its functionality.     

Usefulness of the polymerase chain reaction for monitoring cure of mice infected with different Trypanosoma cruzi clonal genotypes following treatment with benznidazole

The capacity of the polymerase chain reaction (PCR) to detect the DNA of Trypanosoma cruzi was evaluated in 90 blood samples from BALB/c mice infected with T. cruzi cloned stocks of genotypes 19 and 20 (T. cruzi I) and 39 and 32 (T. cruzi II), and treated with benznidazole. The results from the fresh blood examination, hemoculture, and ELISA allowed to group the treated animals into: cured (TC), dissociated (DIS) and non-cured (NC). The PCR detected T. cruzi DNA in 50.9%, 58.3% and 100.0% of the samples from TC, DIS and NC mice, respectively. These DNA possibly derives from live T. cruzi or from recently lysed parasites, suggests that these animals are in fact not cured. The difference between the PCR results and results obtained using other techniques was statistically significant and independent of the parasite genotype. The PCR described has therefore potential to be used in cure control of treated patients.     

Apolipoprotein E genotype is related to nitric oxide production in platelets

The presence of the epsilon4 allele of apolipoprotein E (APOE) is considered a risk factor for sporadic Alzheimer's disease (AD). Our recent data demonstrated that the systemic modulation of oxidative stress in platelets and erythrocytes is disrupted in aging and AD. In this study, the relationship between APOE genotype and oxidative stress markers, both in AD patients and controls, was evaluated. The AD group showed an increase in the content of thiobarbituric acid-reactive substances (TBARS) and in the activities of nitric oxide synthase (NOS) and Na, K-ATPase, when compared to controls. Both groups had a similar cGMP content and superoxide dismutase activity. APOE epsilon4 allele carriers showed higher NOS activity than non-carriers. These results suggest a possible influence of APOE genotype on nitric oxide (NO) production that might enhance the effects of age-related specific factor(s) associated with neurodegenerative disorders.     

Trypanosoma cruzi: blood parasitism kinetics and their correlation with heart parasitism intensity during long-term infection of Beagle dogs

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.     

Life Cycle of the Tick Haemaphysalis Leporis-palustris (Acari: Ixodidae) Under Laboratory Conditions

The life cycles of two separate populations (colonies A and B) of the rabbit tick, Haemaphysalis leporis-palustris, were studied under laboratory conditions. Domestic New Zealand rabbits, Oryctolagus cuniculus, and wild rabbits, Sylvilagus brasiliensis, were used as hosts for ticks from colony B and only O. cuniculus rabbits were used as hosts for ticks from colony A. Developmental periods were observed in an incubator at 27±1°C and RH 90±5%. Larvae from colonies A and B fed for 8.0±3.7 days and 8.5±1.3 days, respectively, on O. cuniculus. On S. brasiliensis larvae from colony B fed for 7.2±1.3 days. Nymphs from colony A fed for 8.1±1.4 days on O. cuniculus and nymphs from colony B fed for 8.1±1.0 days on S. brasiliensis. Only one engorged nymph from colony B was recovered from O. cuniculus. Females from colony A fed for 20.9±5.9 days on O. cuniculus and females from colony B fed for 18.6±2.4 days on O. cuniculus and 18.7±3.7 days on S. brasiliensis. Engorged larvae from colony A required 13.7±3.7 days to molt while engorged larvae from colony B required 11.8±3.0 and 11.5±1.8 days to molt, after having fed on O. cuniculus and S. brasiliensis, respectively. Engorged nymphs from colonies A and B required 16.3±1.9 days and 14.7±1.4 days to molt, respectively. Engorged females from colonies A and B required 4–7 and 3–5 days, respectively, to start oviposition. Mean egg incubation periods lasted for 33–34 days. For ticks from colony B, host species accounted for significant differences (p<0.05) in larval and nymphal feeding periods, oviposition weights and CEIs. Significant differences (p<0.05) between the two colonies when ticks fed on O. cuniculus were observed for larval and nymphal feeding and premolt periods, engorged female and oviposition weights and conversion efficiency indexes (CEI). S. brasiliensis were always a more suitable host for H. leporis-palustris than O. cuniculus. Significantly more larvae and nymphs engorged and molted when fed on S. brasiliensis (p<0.001). Females fed S. brasiliensis were more successful to lay fertile eggs and showed the highest engorged and egg mass weights, and the highest CEIs. Data of H. leporis-palustris fed on wild rabbits (one of its natural host species) are reported for the first time.