Use of pcr-rflp of thefla a gene for detection and subtyping of Campylobacter jejuni strains Potentially related to Guillain-barré syndrome, isolated from humans and animals

The objectives of the present study were the subtyping of Campylobacter jejuni subsp. jejuni strains obtained from humans and different animal species using PCR-RFLP, and the detection, by means of the same technique, of strains related to serotype PEN O19:LIO 7, the main C. jejuni serotype linked to Guillain-Barré Syndrome (GBS). Seventy C. jejuni strains isolated from human feces (n=33), primates (n=15), dogs (n=5), swine (n=2), bovines (n=1), abortion material from goats (n=2) and poultry carcasses (n=12), all collected in the state of São Paulo, were subtyped by means of PCR-RFLP of fla A gene, using restriction endonucleases Hae III, Afa I and Mbo I. Seven subtypes were observed when using the enzyme Hae III; eight when using Mbo I; and seven when using Afa I. The combination of the three endonucleases led to 16 fla-RFLP subtypes, from which ten subtypes shared strains of human and animal origin. From these, seven subtypes were observed in human and broiler strains. In eight subtypes, the other animal species shared patterns with human strains. It was inferred that, besides broilers, swine, goats, dogs and primates may be sources of infection for human in São Paulo. PCR-RFLP is a highly discriminatory technique that may be applied to molecular epidemiology studies of samples from different origins. Besides, the study also enabled the detection of two human strains and two primate strains related to serotype PEN O19: LIO 7.

In vitro evaluation of the cytotoxic and trypanocidal activities of Ampelozizyphus amazonicus (Rhamnaceae)

Ampelozizyphus amazonicus Ducke is a tree commonly found in the Amazon region and an extract of its stem bark is popularly used as an antimalarial and anti-inflammatory agent and as an antidote to snake venom. Ursolic acid; five lupane type triterpenes: betulin, betulinic acid, lupenone, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid, and 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid, and three phytosteroids: stigmasterol, sitosterol and campesterol, have been isolated from stem extracts of A. amazonicus Ducke. Their structures were characterized by spectral data including COSY and HMQC. In an in vitro biological screening of the isolated compounds, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid was cytotoxic against the SKBR-3 human adenocarcinoma cell line (1 to 10 mg/mL), while 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid exhibited cytotoxicity against both SKBR-3 human adenocarcinoma and C-8161 human melanoma tumor cell lines (>0.1 mg/mL). In the present study, different extracts and some fractions of this plant were also investigated for trypanocidal activity due to the presence of pentacyclic triterpenes. The triterpene classes are potent against Trypanosoma cruzi. The bioassays were carried out using blood collected from Swiss albino mice by cardiac puncture during the parasitemic peak (7th day) after infection with the Y strain of T. cruzi. The results obtained showed that A. amazonicus is a potential source of bioactive compounds since its extracts and fractions isolated from it exhibited in vitro parasite lysis against trypomastigote forms of T. cruzi at concentrations >100 æg/mL. Fractions containing mainly betulin, lupenone, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid, and 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid showed more activity than crude extracts.