Induction of an adaptive response to quercetin, mitomycin C and hydrogen peroxide by low doses of quercetin in V79 Chinese hamster cells

The adaptive response is a phenomenon by which cells exposedto low, non-cytotoxic doses of a genotoxicant become significantlyresistant to a subsequent higher dose of the same or anothergenotoxic agent. Induction of the adaptive response has beenmainly studied using ionizing radiation and alkylating agentsas genotoxic agents. However, other mutagenic agents may warrantfurther study, since the adaptive response as a whole may bean important general biological mechanism to maintain geneticintegrity and thus could prevent carcinogenic initiation ofcells. The exposure to mutagenic agents present, or formed,in the diet is considered an important factor in the etiologyof human tumors and a considerable number of these agents havenot yet been identified or characterized. Flavonoids are a largegroup of polyphenolic quinoids found in a wide variety of ediblefruits and vegetables and a few, such as quercetin, presentgenotoxic activity in vitro. The mechanisms of mutagenicityof quercetin involve the production of oxygen radicals throughan autoxidation process dependent on pH value and the presenceof oxygen. Although there are few doubts regarding the mutagenicityof quercetin invitro, carcinogenicity of this flavonoid is stillcontroversial. In view of these conflicting results and theradiomimetic nature of the mutagenicity of flavonoids, we addressedthe question of cell exposure to quercetin at the low levelspresent in the diet leading to adaptation to further exposureto mutagens or carcinogens. The work reported here concernsinduction of an adaptive response by low doses of quercetinto challenging doses of quercetin and other compounds, namelyhydrogen peroxide and mitomycin C, using induction of chromosomalaberrations in V79 cells as the end point. ⁴To whom correspondence should be addressed: Tel: +351 1 3610290; Fax: +351 1 3622018; Email: jose.rueff{at}gene.unl.mailpac.pt     

Prevention of acute immunological lung lesion in rats by decomplementing treatment

The intravenous administration of nephrotoxic antibody serum to rats produced a rapid and pronounced reduction in the serum complement level; this was observed before lung lesions became apparent.

A total suppression of the acute immune lung change was observed in animals depleted of complement by treatment with heat-aggregated human γ-globulin or zymosan.

Albeit the experimental evidence presented is of indirect nature, it suggests that the complement system is involved in the mediation of the acute pulmonary injury following injection of nephrotoxic antibody serum.

     

<Emphasis Type="Italic">Trypanosoma evansi</Emphasis> in inbred and Swiss-Webster mice: distinct aspects of pathogenesis

Trypanosoma evansi (Trypanosomatidae, Kinetoplastida) is a salivarian trypanosomatid that infects eight mammal orders spread over America, Europe and Asia. In Brazil, T. evansi is the etiological agent of Mal de Cadeiras, a horse disease very often described in the region known as Pantanal do Mato Grosso. Few data concerning the genetic diversity and biology of subpopulations of T. evansi that circulate in Brazil are available. The factors that modulate the interaction of this parasite with its hosts also remain to be elucidated. Here we evaluated the course of experimental infection of six T. evansi isolates derived from domestic and wild animals in Swiss-Webster mice and three Mus musculus lineages. The follow-up included biological, immunological as well as biochemical and hematological parameters. The same isolates as well as three others were characterized by pulsed-field electrophoresis. Our results showed that T. evansi isolates displayed significant differences regarding behavior and morbidity patterns in the distinct mouse lineages. Nevertheless, these differences could not be correlated with pulsed-field electrophoresis profiles. Indeed, concerning this molecular marker, only microheterogeneity was observed. Moreover, we observed that the outcome of the infection is defined by both host genetic background and peculiarities (virulence factors) of the distinct T. evansi isolates. Anemia and hypoglycemia were the only features that could be observed in all mouse lineages, independently of the inoculated T. evansi subpopulation. In addition, our data also show that Mus musculus is a suitable model host for the study of the different pathogenetic features of T. evansi infection.     

Phase I testing of a malaria vaccine composed of hepatitis B virus core particles expressing Plasmodium falciparum circumsporozoite epitopes

We report the first phase I trial to assess the safety and immunogenicity of a malaria vaccine candidate, ICC-1132 (Malarivax), composed of a modified hepatitis B virus core protein (HBc) containing minimal epitopes of the Plasmodium falciparum circumsporozoite (CS) protein. When expressed in Escherichia coli, the recombinant ICC-1132 protein forms virus-like particles that were found to be highly immunogenic in preclinical studies of mice and monkeys. Twenty healthy adult volunteers received a 20- or a 50-microg dose of alum-adsorbed ICC-1132 administered intramuscularly at 0, 2, and 6 months. The majority of volunteers in the group receiving the 50-microg dose developed antibodies to CS repeats as well as to HBc. Malaria-specific T cells that secreted gamma interferon were also detected after a single immunization with ICC-1132-alum. These studies support ICC-1132 as a promising malaria vaccine candidate for further clinical testing using more-potent adjuvant formulations and confirm the potential of modified HBc virus-like particles as a delivery platform for vaccines against other human pathogens.     

Antimicrobial resistance of Salmonella serotypes isolated from slaughter-age pigs and environmental samples

The aim of this study was to determine the antimicrobial resistance patterns of Salmonella strains isolated from slaughter-age pigs and environmental samples collected at modern swine raising facilities in Brazil. Seventeen isolates of six serotypes of Salmonella enterica subsp. enterica were isolated out of 1,026 collected samples: Salmonella Typhimurium (1), Salmonella Agona (5), Salmonella Sandiego (5), Salmonella Rissen (1), Salmonella Senftenberg (4), and Salmonella Javiana (1). Resistance patterns were determined to extended-spectrum penicillin (ampicillin), broad-spectrum cephalosporins (cefotaxime and ceftriaxone), aminoglycosides (streptomycin, neomycin, gentamicin, amikacin, and tobramycin), narrow-spectrum quinolone (nalidixic acid), broad-spectrum quinolone (ciprofloxacin and norfloxacin), tetracycline, trimethoprim, and chloramphenicol. Antimicrobial resistance patterns varied among serotypes, but isolates from a single serotype consistently showed the same resistance profile. All isolates were resistant to tetracycline, streptomycin, and nalidixic acid. One isolate, Salmonella Rissen, was also resistant to cefotaxime and tobramycin. All serotypes were susceptible to ceftriaxone, norfloxacin, ciprofloxacin, ampicillin, gentamicin, and chloramphenicol. The high resistance to tetracycline and streptomycin may be linked to their common use as therapeutic drugs on the tested farms. No relation was seen between nalidixic acid and fluoroquinolone resistance.     

A dinucleotide mutation in the endothelin-B receptor gene is associated with lethal white foal syndrome (LWFS); a horse variant of Hirschsprung disease

Lethal white foal syndrome (LWFS) is a congenital anomaly of horses characterized by a white coat colour and aganglionosis of the bowel, which is similar to Hirschsprung disease (HSCR). We decided to investigate possible mutations of the endothelin-B receptor gene ( EDNRB ) in LWFS as recent studies in mutant rodents and some patients have demonstrated EDNRB defects. First, we identified a full-length cDNA for horse EDNRB . This cDNA fragment contained a 1329 bp open reading frame which encoded 443 amino acid residues. The predicted amino acid sequence was 89, 91 and 85% identical to human, bovine and mouse as well as rat EDNRB respectively, but only 55% identical to the human, bovine and rat endothelin A receptor (EDNRA). Secondly, sequence analysis, together with allele-specific PCR and the amplification- created restriction site (ACRS) technique, revealed a dinucleotide TC-- >AG mutation, which changed isoleucine to lysine in the predicted first transmembrane domain of the EDNRB protein. This was associated with LWFS when homozygous and with the overo phenotype when heterozygous.      

T and B cell responses to neutrophil cytoplasmic antigens in systemic vasculitis.

The systemic vasculitides (SV) are characterized by the presence of autoantibodies to neutrophil cytoplasmic antigens (ANCA). The role of T cells in SV is uncertain. We studied human and murine T cell responses to human neutrophil cytoplasmic antigens in vitro. T cells from mice immunized with the neutrophil extract showed dose-dependent antigen-specific proliferation, restricted by the MHC class II E molecule. Myeloperoxidase (MPO) was not an important target antigen for murine T cells. Peripheral blood lymphocytes (PBLs) were obtained from 36 patients with SV, 31 before the start of immunosuppressive therapy, and from 11 healthy controls. T cell responses to the neutrophil extract in vitro did not differ between patients and controls: there were only low levels of antigen-specific proliferation, and this could not be amplified by in vitro selection. In 3 patients and 2 normals, PBLs were also tested after the depletion of CD8+ cells; this did not unmask T cell reactivity to neutrophil extract. The lack of demonstrable T cell reactivity to this antigen preparation may indicate that T cells do not play an important effector role in these diseases. A solid-phase spot ELISA was adapted to demonstrate autoantibody-producing B cells in vitro. Low numbers of ANCA-producing B cells could be demonstrated in the majority of patients. B cells producing antibody to MPO could be demonstrated in most patients and in three laboratory staff, but not in normals from outside the laboratory. In 2 patients, sequential B cell spot ELISAs were performed during the introduction of therapy, and autoantibody-producing B cells rapidly decreased in number. This assay may therefore be useful in monitoring the effects of treatment at the cellular level.     

Aberrant cellular retinol binding protein 1 (CRBP1) gene expression and promoter methylation in prostate cancer

AIMS: Retinoids are involved in cell growth, differentiation, and carcinogenesis. Their effects depend on cytosolic transport and binding to nuclear receptors. CRBP1 encodes a protein involved in this process. Because altered CRBP1 expression and promoter hypermethylation occur in several tumours, these changes were investigated in prostate tumorigenesis. METHODS: The CRBP1 promoter was assessed by methylation specific polymerase chain reaction on tissue samples from 36 radical prostatectomy specimens (paired normal tissue, adenocarcinoma, and high grade prostatic intraepithelial neoplasia (HGPIN)), 32 benign prostatic hyperplasias (BPHs), and 13 normal prostate tissue samples from cystoprostatectomies. Methylation of DNA extracted from microdissected tissue was examined blindly. CRBP1 expression was assessed by immunohistochemistry on formalin fixed, paraffin wax embedded tissue. RESULTS: Loss of CRBP1 expression was seen in 15 of 36 adenocarcinomas and 18 of 36 HGPINs. Fifteen adenocarcinomas and nine HGPINs showed overexpression, whereas the remainder showed normal expression. BPH displayed normal expression. No significant associations were found between CRBP1 expression and Gleason score or stage. CRBP1 promoter hypermethylation was found in 17 of 36 adenocarcinomas, three of 35 HGPINs, one of 36 normal prostate tissues from the same patients, none of 32 BPHs, and none of 13 normal prostate tissues from cystoprostatectomies. Loss of expression and hypermethylation of CRBP1 were not significantly associated. CONCLUSIONS: Altered CRBP1 expression and hypermethylation are common in prostate carcinoma, although CRBP1 hypermethylation is not an early event in tumorigenesis. Moreover, both adenocarcinoma and HGPIN show frequent CRBP1 overexpression. The molecular mechanisms underlying altered CRBP1 expression in prostate cancer deserve further study.