Ivana Nascimento

Experimental Corynebacterium pseudotuberculosis primary infection in goats: kinetics of IgG and interferon-γ production, IgG avidity and antigen recognition by Western blotting

Corynebacterium pseudotuberculosis is the cause of caseous lymphadenitis (CLA) in small ruminants, a chronic granulomatous disease that provokes significant zootechnics losses to ovine and goat breeders in northern Brazil. The present work was conducted to analyse aspects of humoral and cellular immune responses after experimental infection. Eight goats were infected intradermally with a single dose of virulent C. pseudotuberculosis strain and specific IgG, interferon-gamma (IFN-gamma) production as well as IgG avidity and antigens pattern recognition dynamics against an excreted-secreted antigen were recorded during 20 weeks. At the end of the follow-up, animals were slaughtered and necropsied. Although no animals showed apparent clinical signs of infection at the end of the trial, IFN-gamma response, even more so than the humoral response, differentiated animals into two groups of high or medium/low response. The time-course of IFN-gamma production presented a short-lived primary response on day 5 after infection of animals of both groups, and a strong and long lasting secondary response starting on day 16 after infection in the high response group. The indirect ELISA used was able to detect a positive antibody titre between 6 and 11 days after infection in the two groups. IgG avidity index oscillated initially between 15 and 45%, and showed approximately 5% units increment during the 20 follow-up weeks. With only one individual exception, the qualitative antigens pattern recognition showed on day 11 after infection remained constant through the experiment. IgG avidity is highly correlated with IgG production, but could not be related with specific immunodominant bands. Both humoral and cellular responses kinetics presented a similar pattern of activation/deactivation but necropsy results suggested that the IFN-gamma test would be a very specific marker of CLA status.

Immunological characteristics of mesenchymal stem cells.

Although bone marrow is the main source, mesenchymal stem cells have already been isolated from various other tissues, such as the liver, pancreas, adipose tissue, peripheral blood and dental pulp. These plastic adherent cells are morphologically similar to fibroblasts and have a high proliferative potential. This special group of cells possesses two essential characteristics: self-renewal and differentiation, with appropriate stimuli, into various cell types. Mesenchymal stem cells are considered immunologically privileged, since they do not express costimulatory molecules, required for complete T cell activation, on their surface. Several studies have shown that these cells exert an immunosuppressive effect on cells from both innate and acquired immunity systems. Mesenchymal stem cells can regulate the immune response in vitro by inhibiting the maturation of dendritic cells, as well as by suppressing the proliferation and function of T and B lymphocytes and natural killer cells. These special properties of mesenchymal stem cells make them a promising strategy in the treatment of immune mediated disorders, such as graft-versus-host disease and autoimmune diseases, as well as in regenerative medicine. The understanding of immune regulation mechanisms of mesenchymal stem cells, and also those involved in the differentiation of these cells in various lineages is primordial for their successful and safe application in different areas of medicine.

Evaluation of the humoral and cellular immune response to different antigens of Corynebacterium pseudotuberculosis in Caninde´ goats and their potential protection against caseous lymphadenitis

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis, a disease that affects goats and sheep, and can cause severe economic losses. In this study, four different antigenic extracts were obtained from the attenuated strain T1, which was isolated in the state of Bahia (Brazil). Forty-four Canindé breed goats were divided in five groups, each receiving a different antigen solution and saline buffer as a control. The humoral response was monitored through the identification of specific IgG by indirect ELISA and Western Blotting, and the production of IFN-gamma was followed in order to observe the activation of cellular response. After twelve weeks of antigen inoculation, the animals were challenged with 2 x 10(5)CFU of a wild strain, also isolated in Bahia, and necropsy was performed on all animals twelve weeks afterwards. It was observed that the attenuated bacteria gave a protection of 33.3%, in addition to the weak humoral response elicited. Animals inoculated with secreted antigen associated with Freunds incomplete adjuvant and oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) showed a strong humoral response, but this inoculation could not prevent the spread of challenge bacteria in the majority of animals. These results demonstrate the immunogenic potential of the attenuated T1 strain in the development of a vaccine against caseous lymphadenitis in goats.

Flavonoids inhibit angiogenic cytokine production by human glioma cells

VEGF and TGF‐β1 are cytokines that stimulate tissue invasion and angiogenesis. These factors are considered as molecular targets for the therapy of glioblastoma. Bevacizumab, a recombinant humanized monoclonal antibody developed against VEGF, inhibits endothelial cell proliferation and vessel formation. Flavonoids obtained from Dimorphandra mollis and Croton betulaster have been described as proliferation inhibitors of a human glioblastoma derived cell line. VEGF and TGF‐β1 levels were dosed by ELISA in a GL‐15 cell line treated with bevacizumab and also with the flavonoids rutin, 5‐hydroxy‐7,4′‐dimethoxyflavone, casticin, apigenin and penduletin. Rutin reduced the VEGF and TGF‐β1 levels after 24 h but not after 72 h. The other flavonoids significantly reduced TGF‐β1 production. Bevacizumab reduced only the VEGF levels in the supernatant from GL‐15 cultures. These results suggest that the flavonoids studied, and commonly used in popular medicine, present an interesting subject of study due to their potential effect as angiogenic factor inhibitors. Copyright

Germline mutations in BRCA1, BRCA2, CHEK2 and TP53 in patients at high-risk for HBOC: characterizing a Northeast Brazilian Population.

Considering the importance of BRCA1, BRCA2, CHEK2 and TP53 in the development of hereditary early-onset breast and ovarian cancer and that the genetic susceptibility profile of the Northeast population from Brazil has never been analyzed, this study aimed to verify the frequency of mutations of clinical significance in these genes in high-risk hereditary breast and ovarian cancer (HBOC) syndrome patients from that region. DNA samples from 106 high-risk unrelated patients mostly from Bahia, the biggest state in the Northeast region, were analyzed. These patients underwent full BRCA1 gene sequencing, screening for common founder mutations in the BRCA2, CHEK2 and TP53 genes and genetic ancestry analysis with nine ancestry informative markers. The positive results were confirmed by two sequencing reactions. Three mutations of clinical significance were found: BRCA1 p.R71G (4.71%), 3450del4 (3.77%) and TP53 p.R337H (0.94%). The genetic ancestry analysis showed a high European ancestry contribution (62.2%) as well as considerable African (31.2%) and Amerindian (6.6%) ancestry contributions (r2=0.991); this degree of heterogeneity was also significant in the population structure analysis (r=0.604). This population is highly admixed with a different spectrum of genetic susceptibility, with the Galician founder mutation BRCA1 p.R71G accounting for 50% of all identified mutations in high-risk HBOC patients. TP53 p.R337H was also significantly frequent; thus, the combined screening of BRCA1/2 and TP53 should be offered to high-risk HBOC patients from Northeast Brazil.

BRCA1 and BRCA2 rearrangements in Brazilian individuals with Hereditary Breast and Ovarian Cancer Syndrome

Abstract Approximately 5-10% of breast cancers are caused by germline mutations in high penetrance predisposition genes. Among these, BRCA1 and BRCA2, which are associated with the Hereditary Breast and Ovarian Cancer (HBOC) syndrome, are the most frequently affected genes. Recent studies confirm that gene rearrangements, especially in BRCA1, are responsible for a significant proportion of mutations in certain populations. In this study we determined the prevalence of BRCA rearrangements in 145 unrelated Brazilian individuals at risk for HBOC syndrome who had not been previously tested for BRCA mutations. Using Multiplex Ligation-dependent Probe Amplification (MLPA) and a specific PCR-based protocol to identify a Portuguese founder BRCA2 mutation, we identified two (1,4%) individuals with germline BRCA1 rearrangements (c.547+240_5193+178del and c.4675+467_5075-990del) and three probands with the c.156_157insAlu founder BRCA2 rearrangement. Furthermore, two families with false positive MLPA results were shown to carry a deleterious point mutation at the probe binding site. This study comprises the largest Brazilian series of HBOC families tested for BRCA1 and BRCA2 rearrangements to date and includes patients from three regions of the country. The overall observed rearrangement frequency of 3.44% indicates that rearrangements are relatively uncommon in the admixed population of Brazil.

Immunolocalization of FGF-2 and VEGF in rat periodontal ligament during experimental tooth movement

Objective This article aimed at identifying the expression of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) in the tension and pressure areas of rat periodontal ligament, in different periods of experimental orthodontic tooth movement. Methods An orthodontic force of 0.5 N was applied to the upper right first molar of 18 male Wistar rats for periods of 3 (group I), 7 (group II) and 14 days (group III). The counter-side first molar was used as a control. The animals were euthanized at the aforementioned time periods, and their maxillary bone was removed and fixed. After demineralization, the specimens were histologically processed and embedded in paraffin. FGF-2 and VEGF expressions were studied through immunohistochemistry and morphological analysis. Results The experimental side showed a higher expression of both FGF-2 and VEGF in all groups, when compared with the control side (P < 0.05). Statistically significant differences were also found between the tension and pressure areas in the experimental side. Conclusion Both FGF-2 and VEGF are expressed in rat periodontal tissue. Additionally, these growth factors are upregulated when orthodontic forces are applied, thereby suggesting that they play an important role in changes that occur in periodontal tissue during orthodontic movement.

The Flavonoid Rutin but not the Alkaloid Arborinine Induces Apoptosis in a B-Cell Hybridoma Cell Line

The effects of arborinine, an alkaloid extracted from Erthela bahiensis and of rutin, a flavonoid obtained from Dimorphandra mollis (Benth.), Brazilian medicinal plants, on the viability and function of a murine B-cell hybridoma as a tumor model were investigated. The flavonoid rutin at 50 microM induced an increase in the number of apoptotic cells of one- to fivefold and reductions in cellular proliferation and monoclonal antibody production. Less but still significant necrosis was also induced by rutin under the same experimental conditions. On the other hand, the alkaloid arborinine exerted no significant effects on the studied parameters.

Capillaria hepatica: alguns aspectos imunopatológicos da infecção espúria e da infecção verdadeira

In order to evaluate a potential protective role of spurious infection on the development of capillaria, mice were infected with non-embrionated eggs (spurious infection) followed by inoculation with embrionated eggs (true infection). The humoral immune response was evaluated through enzyme- linked immunosorbent assay (ELISA) that revealed a raise in anti-Capillaria hepatica IgG antibody two weeks after the first infection. Delayed type hypersensivity test was consistently negative. Histopathologic examination and the C. hepatica egg burden from different groups didnt show any significant variation. The humoral immune response induced by spurious infection do not protect mice against a true infection.

Different Effects of Arborinine Alkaloid Obtained from Brazilian Erthela baihensis on Spleen and Thymus Cells Stimulated in Vitro with Different Mitogens

The present study has examined the effects of arborinine, an alkaloid obtained from Erthela bahiensis, a Brazilian plant popularly used as diuretic, antidiabetic, antithermic and expectorant, on the viability and function of immune system cells in vitro using a murine model. Rat spleen and thymus cells were cultured with 10nM, 1µM e 10µM of the drug in the presence or absence of pokeweed (PWM), lipopolysaccharide (LPS), or concanavallin (ConA) mitogens. Cellular proliferation was analyzed by H3‐thymidin uptake after 48 and 72 hr. Our results showed an inhibitory effect of arborinine on splenocytes proliferation under ConA or PWM stimulation and increased apoptosis on splenocytes and thymocytes stimulated with PWM in 24 hr. A decrease was observed on Interferon gamma (IFN-γ) production by ConA- or LPS-stimulated splenocytes in 48 hr and 72 hr and ConA- or PWM-stimulated thymocytes in 72 hr. In contrast, an increase on lymphoproliferation was observed on LPS-stimulated splenocytes and ConA- or PWM-stimulated thymocytes in 48 hr. On this period, apoptosis decreased on LPS- or PWM-stimulated splenocytes and IFN-γ production increased in PWM stimulated thymocytes. Arborinine also induced a decrease on Interleukin-10 production by splenocytes and thymocytes stimulated with ConA or PWM. There was no significant variation on the necrosis rate of the cells treated with arborinine or any change on their viability or function values in the absence of mitogenic stimulus.