The role of lymph node revealing solution on the improvement of lymph node harvest in colorectal cancer specimens.

AIM: The correct analysis of lymph node status is one of the most important parameters for the accurate pathological diagnosis of colorectal cancer. Our aim was to evaluate the number of lymph nodes among the specimens obtained from colorectal resections due to colorectal cancer, before and after the routine use of a lymph node revealing solution (LNRS). METHOD: Data from 780 surgical specimens from patients of both genders with colorectal cancer were studied. The cases were divided chronologically into two groups: the conventional group included 497 specimens treated with conventional methods, i.e. without the use of the LNRS (January 2000 to July 2007), and the LNRS group included 283 specimens examined through the routine use of this solution (August 2007 to July 2012). RESULTS: Most patients were female (57.4%) with a median age of 62 years. The median lymph node number was 18, and 75.9% of the cases (592) had 12 or more nodes dissected. Lymph node metastases were noted in 334 cases (42.8%). A median of 24 lymph nodes was dissected in the LNRS group compared to 15 in the conventional group (P < 0.001). The LNRS group had 9.2% of cases with fewer than 12 lymph nodes dissected compared with 32.6% in the conventional group (P < 0.001). CONCLUSIONS: The use of the LNRS increases the number of lymph nodes obtained from colorectal cancer surgical specimens and can help to reduce the number of cases with < 12 lymph nodes.

Immunological diagnosis of tuberculosis based on recombinant antigens ESAT-6 and CFP-10 in children from an endemic area in northeast Brazil.

Diagnostic tests for tuberculosis (TB) using interferon gamma (IFN-γ) responses produced by T lymphocytes after stimulation by early secretory antigen target 6 (ESAT-6), culture filtrate protein 10 (CFP-10) or purified protein derivate (PPD) were carried out using ELISA (enzyme-linked immunosorbent assay) in whole blood culture supernatants from children with suspected TB disease (n=21), latent TB infection (LTBI; n=17) and negative controls (NC; n=21) from Recife, Pernambuco, Brazil. The results were analysed using the ROC (receiver operating characteristic) curves and the areas under the curve (AUC) generated varied from 0.5 to 1.0 with higher values indicating increased discriminatory ability. Comparisons of AUCs were made using non-parametric assumptions, and the differences were considered significant if P<0.05. The ROC curve showed a statistical difference (P = 0.015) between the LTBI and NC groups with an AUC of 0.731, TB disease and NC (AUC=0.780; P=0.002) and a group with TB (latent infection+disease, n=38) and NC (AUC=0.758; P = 0.001) when the antigen used was ESAT-6. No statistical difference was found between the groups when CFP-10 or PPD was used. In conclusion, the ESAT-6 test may be the most appropriate for diagnosis of childhood TB, both LTBI and TB disease, when associated with epidemiological and clinical data, especially in endemic areas such as Brazil.

Análise da distribuição da região dos ferimentos provocados por morcegos hematófagos Desmodus rotundus (Geoffroy, 1810) em bovinos sob condições de campo / Topographic analysis of wounds caused by the vampire bat Desmodus rotundus (Geoffroy, 1810) in cattle under field conditions

Existem diversas espécies de morcegos no Brasil, sendo somente três hematófagas. O morcego Desmodus rotundus (Geoffroy, 1810), conhecido como "morcego vampiro comum", apresenta-se amplamente distribuído por todo território brasileiro e é encontrado em maior número do que as outras espécies de morcegos hematófagos. É responsável pela transmissão de diferentes doenças a outros mamíferos, especialmente a raiva nos herbívoros. Com o objetivo de avaliar regionalmente os ferimentos causados pelo D. rotundus em bovinos sob condições de campo, foram estudadas 12 propriedades rurais no período de março de 2005 a março de 2006 no Município de Valença, RJ. De um total de 580 bovinos examinados, foram observados 101 com ferimentos provocados por morcegos hematófagos (D. rotundus). Após análise dos resultados, pôde-se concluir que D. rotundus utiliza diferentes locais para se alimentar nos bovinos e as regiões corporais que apresentaram maior número de ferimentos foram a ântero e a póstero-superior. O elevado número de ferimentos encontrados, na metade superior dos bovinos (85,87%), justifica a adoção de metodologias de controle populacional de morcegos hematófagos que utilizem o contato corporal desses morcegos com o dorso do animal.

Brazil possesses a large amount of bat species, only 3 of which are haematophagous. The most abundant species, Desmodus rotundus (Geoffroy, 1810), is widely distributed throughout the Brazilian territory and it is involved in the transmission of several diseases to other animals, notably to herbivorous species, in the case of rabies. This study presents the topographical analysis of wounds caused by D. rotundus in bovines from 12 different rural properties in the county of Valença, state of Rio de Janeiro, where 101 bovines out of 580 examined in a year showed wounds caused by haematophagous bats. Analysis of the results leads to the conclusion that D. rotundus uses different sites to feed from the bovine's body and that the superior body region was the most frequently used. The increased number of wounds found in the bovine's superior half region (85.87%) will justify the use of bat population control methodologies which take into consideration the bat's corporal contact with the dorsal region of bovines.

Neolignans from plants in northeastern Brazil (Lauraceae) with activity against Trypanosoma cruzi.

Trypanosoma cruzi is the ethiological agent for Chagas disease in Latin America. This study aimed to test the trypanocidal effect of licarin A and burchellin isolated from plants in northeastern Brazil. These neolignans were tested on T. cruzi and on peritoneal macrophages, to evaluate drug toxicity. Epimastigote growth was inhibited in 45% with licarin A and 20% with burchellin with an IC(50)/96 h of 462.7 microM and 756 microM, respectively. Epimastigotes treated with licarin A presented swollen mitochondria and disorganized mitochondrial cristae, kDNA and Golgi complex. When treated with burchellin, they presented enormous autophagosomes and chromatin disorganization. Licarin A and burchellin were able to induce trypomastigote death with IC(50)/24 h of 960 microM and 520 microM, respectively. Although licarin A presented an IC(50) for trypomastigotes higher than for epimastigotes, both substances acted as therapeutic trypanocidal agents, because they were able to kill parasites without affecting macrophages. Due to our results, burchellin and licarin A need to be further analysed to observe if they may be used as alternative blood additive prophylaxis against Chagas disease, since it has been established that blood transfusion is an important mechanism in the transmission process.

Differences in peripheral blood lymphocyte phenotypes between Helicobacter pylori-positive children and adults with duodenal ulcer.

The immunological mechanisms involved in the development of duodenal ulcer, especially in childhood, are unclear. Helicobacter pylori-positive children and adults, with and without duodenal ulcer, were therefore compared with respect to CD4(+) T-cells, and CD8(+) T-cells, B-cells and B1a-cells, as well as cell activation (CD4(+)/HLA-DR(+) and CD8(+)/HLA-DR(+)) and co-stimulatory (CD4(+)/CD28(+) and CD8(+)/CD28(+)) markers, in peripheral blood. Children with and without duodenal ulcer differed significantly. In particular, there was a phenotypic change in CD8(+) T-cells from children with ulcer that involved a 200% increase in the number of CD8(+)/HLA-DR(+) cells/mm(3) and a decrease of 34.2% in the number of CD8(+)/CD28(+) cells/mm(3). This phenotype of chronically activated memory CD8(+) T-cells, which has also been observed in patients with AIDS and tuberculosis, is associated with disease severity and progression. A lower frequency of B1a-cells was also observed in the group of children with ulcer. Conversely, no difference between infected adults with and without ulcer was observed, but the percentage of CD4(+)/HLA-DR(+) cells was lower in adults with ulcer, suggesting that a down-regulated immune response may play a role in the development of duodenal ulcer in adults. Gastric inflammation correlated positively with CD4(+) and chronically activated CD4(+) T-cells in children and adults without duodenal ulcer, respectively. These results suggest that there are differences in the immunophenotyping profile between H. pylori-positive children and adults with duodenal ulcer, indicating the possibility of distinct immune mechanisms in the development of the disease according to age.

Biological activity of neolignans on the post-embryonic development of Chrysomya megacephala.

Phytochemicals endowed with hormonal, antihormonal, or toxic activity are potential agents for insect control. The effect of some neolignans on one of the most prevalent flies in urban areas, which constitutes a menace to public health, was investigated.

The role of IFN-gamma and IL-4 in gastric mucosa inflammation associated with Helicobacter heilmannii type 1 infection.

Although Helicobacter heilmannii infection is less common than H. pylori infection in humans, it is considered to be of medical importance because of its association with gastritis, gastric ulcer, carcinoma, and mucosa-associated lymphoid tissue lymphoma of the stomach. However, there have been no studies evaluating the role of the Th cell response in H. heilmannii gastric infection. We evaluated the participation of pro-inflammatory and anti-inflammatory cytokines, IFN-gamma and IL-4, in H. heilmannii gastric infection in genetically IFN-gamma- or IL-4-deficient mice. The serum IFN-gamma and IL-4 concentrations were determined by ELISA. The gastric polymorphonuclear infiltrate was higher (P = 0.007) in H. heilmannii-positive than in H. heilmannii-negative wild-type (WT) C57BL/6 mice, whereas no significant inflammation was demonstrable in the stomach of H. heilmannii-positive IFN-gamma(-/-) C57BL/6 mice. The degree of gastric inflammatory cells, especially in oxyntic mucosa, was also higher (P = 0.007) in infected IL-4(-/-) than in WT BALB/c mice. Serum IFN-gamma levels were significantly higher in IL-4(-/-) than in WT BALB/c mice, independently of H. heilmannii-positive or -negative status. Although no difference in serum IFN-gamma levels was seen between H. heilmannii-positive (11.3 +/- 3.07 pg/mL, mean +/- SD) and -negative (11.07 +/- 3.5 pg/mL) WT BALB/c mice, in the group of IL-4(-/-) animals, the serum concentration of IFN-gamma was significantly higher in the infected ones (38.16 +/- 10.5 pg/mL, P = 0.04). In contrast, serum IL-4 levels were significantly decreased in H. heilmannii-positive (N = 10) WT BALB/c animals compared to the negative (N = 10) animals. In conclusion, H. heilmannii infection induces a predominantly Th1 immune response, with IFN-gamma playing a central role in gastric inflammation.

The role of IFN-gamma and IL-4 in gastric mucosa inflammation associated with Helicobacter heilmannii type 1 infection

Although Helicobacter heilmannii infection is less common than H. pylori infection in humans, it is considered to be of medical importance because of its association with gastritis, gastric ulcer, carcinoma, and mucosa-associated lymphoid tissue lymphoma of the stomach. However, there have been no studies evaluating the role of the Th cell response in H. heilmannii gastric infection. We evaluated the participation of pro-inflammatory and anti-inflammatory cytokines, IFN-gamma and IL-4, in H. heilmannii gastric infection in genetically IFN-gamma- or IL-4-deficient mice. The serum IFN-gamma and IL-4 concentrations were determined by ELISA. The gastric polymorphonuclear infiltrate was higher (P = 0.007) in H. heilmannii-positive than in H. heilmannii-negative wild-type (WT) C57BL/6 mice, whereas no significant inflammation was demonstrable in the stomach of H. heilmannii-positive IFN-gamma-/- C57BL/6 mice. The degree of gastric inflammatory cells, especially in oxyntic mucosa, was also higher (P = 0.007) in infected IL-4-/- than in WT BALB/c mice. Serum IFN-gamma levels were significantly higher in IL-4-/- than in WT BALB/c mice, independently of H. heilmannii-positive or -negative status. Although no difference in serum IFN-gamma levels was seen between H. heilmannii-positive (11.3 ± 3.07 pg/mL, mean ± SD) and -negative (11.07 ± 3.5 pg/mL) WT BALB/c mice, in the group of IL-4-/- animals, the serum concentration of IFN-g was significantly higher in the infected ones (38.16 ± 10.5 pg/mL, P = 0.04). In contrast, serum IL-4 levels were significantly decreased in H. heilmannii-positive (N = 10) WT BALB/c animals compared to the negative (N = 10) animals. In conclusion, H. heilmannii infection induces a predominantly Th1 immune response, with IFN-gamma playing a central role in gastric inflammation.

Dynamic development of Trypanosoma cruzi in Rhodnius prolixus: role of decapitation and ecdysone therapy.

Decapitation and ecdysone therapy on the population dynamics of the Trypanosoma cruzi Dm28c clone in the stomach, small intestine and rectum of fifth-instar larvae of Rhodnius prolixus were investigated. Parasites were not found in the small intestine and rectum of decapitated insects after 10 days post-infection (p.i.). Decapitated ecdysone-supplemented insects sustained the flagellate infection in both gut compartments. In the rectum, the population density of parasites increased 5-fold in ecdysone-treated decapitated larvae and 7-fold in control insects. Epimastigote forms dominated with 40-65%, intermediate stages and round forms varied over 10-35% in the stomach, small intestine and rectum in both insect groups. Low numbers of metacyclic trypomastigotes were observed in the stomach and small intestine of the control group and decapitated insects supplemented with ecdysone but, at 15 days p.i., this form of flagellate reached about 20% in the rectum of the control insects. In the entire gut, at 30 days p.i., 23% of parasites in the control group and 8% in the decapitated insects treated with ecdysone were found. These results indicate that a head factor, possibly the prothoracicotropic hormone from the brain which stimulates ecdysone production by the prothoracic glands, may act directly or indirectly to stimulate the development of epimastigotes and round forms of the parasite and that a single ecdysone treatment is not able to fully reverse metacyclogenesis in decapitated R. prolixus.

Burchellin: effects on Triatoma infestans and on Trypanosoma cruzi within this vector.

Supplementation of blood with the neolignan burchellin (50 microg/ml), a compound from the arboreous Lauraceae Aniba burchelli, affected the ingestion of blood and the course of excretion of fourth- and fifth-instar larvae of Triatoma infestans, the latter especially within the first 4 h after feeding. The total resultant weight loss of treated fourth instars within 24 and 48 h after feeding was only 24% and 28% vs 41% and 48%, respectively, in untreated bugs. In fifth instars, the total weight losses of untreated bugs within 24 and 48 h after feeding were 38% and 41% whereas the weight of treated bugs decreased by 28% and 34%, respectively. In a treatment of Trypanosoma cruzi-infected fourth instars, burchellin significantly reduced the population density of the established infection in the rectum at 5 and 10 days after feeding. This was especially due to a significant increase in the number of the main dividing stage, the epimastigote.

Effects of lignoids on a hematophagous bug, Rhodnius prolixus: feeding, ecdysis and diuresis.

The effects of lignoids on feeding, ecdysis and diuresis in fourth-instar larvae of Rhodnius prolixus (Hemiptera) were investigated. Up to 100 microg/ml burchellin, podophyllotoxin, pinoresinol, sesamin, licarin A, or nordihydroguaiaretic acid (NDGA) in the diet did not induce antifeedant effects. Pinoresinol and NDGA significantly inhibited ecdysis. In experiments in vivo, burchellin and podophyllotoxin reduced the production of urine after feeding. 5-Hydroxytryptamine (5-HT), a diuretic hormone, partially counteracted this effect of burchellin. In experiments in vitro, using isolated Malpighian tubules, (i) burchellin reduced diuretic hormone levels in the hemolymph but not the amount of diuretic hormone stored in the thoracic ganglionic masses (including axons), (ii) burchellin decreased the volume of urine secreted by isolated Malpighian tubules, and (iii) 5-HT could not overcome the effect of burchellin upon the Malpighian tubules. We conclude that burchellin interfered with the release, but not with the production of diuretic hormone by the thoracic ganglionic mass or induced an antidiuretic hormone and directly affected the Malpighian tubules.

Rhodnius prolixus: effects of the neolignan burchellin on in vivo and in vitro diuresis.

Supplementation of blood with the neolignan burchellin (100 microg/ml), a compound from the arboreous Lauraceae Aniba burchelli, affected the course of excretion of fourth-instar larvae of Rhodnius prolixus, especially directly after feeding, and reduced the volume of feces/urine excreted within 6 h of feeding to about 18% and, on the simultaneous addition of the diuretic hormone analogue 5-hydroxytryptamine (5-HT), about 71% of that observed in untreated bugs. In the latter, 5-HT induced a significant 60% increase in excretion. Regardless of whether Malpighian tubules originating from unfed, untreated or fed, burchellin-treated bugs were incubated in vitro in the hemolymph of these bugs or in physiological saline supplemented with 5-HT with or without burchellin or in homogenates of thoracic ganglionic masses of untreated and treated bugs, burchellin was consistently found to affect the secretion rates. Therefore, burchellin not only depresses the release of the diuretic hormone or induces the release of antidiuretic factors but also directly affects the Malpighian tubules.

Factors associated with Helicobacter pylori infection by a cagA-positive strain in children.

Although infection with a cagA-positive Helicobacter pylori strain is considered a risk factor for the development of duodenal peptic ulcer in adults, this association has not been demonstrated in children. The presence of cagA was investigated by polymerase chain reaction in H. pylori strains isolated from 27 children with duodenal ulcer and 53 without duodenal ulcer. All patients (100%) with duodenal ulcer and 33 (62.3%) without ulcer were colonized by a cagA-positive strain (P=.00007). A cagA-positive status was also associated with a more marked macroscopic gastritis, with a greater inflammatory infiltrate of both mononuclear and polymorphonuclear cells in the antral and oxyntic gastric mucosae and degenerative and regenerative changes of the gastric mucosa. Increased cagA positivity was also associated with increased age, but no association between cagA-positive status and sex was observed.

Effects of some lignans and neolignans on the development and excretion of Rhodnius prolixus.

The effects of six lignans and neolignans as inhibitors of ecdysis and on the water balance in fourth-instar larvae of Rhodnius prolixus were studied by oral, topical and continuous contact treatments. The main results may be summarised as follows: (i) burchellin, pinoresinol, sesamin, licarin A and nordihydroguaiaretic acid (NDGA) did not cause feeding inhibition at doses of 100 micrograms/ml blood; podophyllotoxin had no antifeedant effect but caused a high moulting inhibition and significant toxicity when applied either orally or topically; (ii) the highest ecdysis inhibitory effects were observed with pinoresinol and NDGA when applied orally at a dose of 100 micrograms/ml (58% and 50% of moulting inhibition, respectively); burchellin inhibited 30% of the moulting at this concentration; (iii) by topical treatment none of the compounds presented any influence on the moulting cycle; and (iv) podophyllotoxin and burchellin significantly reduced the excretion of the insect in 24 h; the other compounds had no effect on excretion. The implications of these findings in relation to the pertinent biological events in R. prolixus are discussed.

Omeprazole, clarithromycin and furazolidone for the eradication of Helicobacter pylori in patients with duodenal ulcer.

AIM: To evaluate the efficacy of omeprazole plus clarithromycin and furazolidone in Helicobacter pylori eradication and duodenal ulcer healing in Brazilian patients. METHODS: Forty H. pylori-positive patients with duodenal ulcer were randomized to receive 20 mg omeprazole o.m. or b.d. for 1 month plus 500 mg clarithromycin (b.d. ) and 200 mg furazolidone (b.d.) for 1 week. RESULTS: Three months after the end of the treatment the eradication rates were 90% by intention-to-treat analysis, and 97% by per protocol analysis. Mild side-effects were observed in 25 patients, none of whom abandoned the protocol. No difference was observed between the 20 mg and 40 mg omeprazole daily doses. Cure or significant improvement of the symptoms and of the histological alterations were observed after H. pylori eradication. CONCLUSION: Our results demonstrate that clarithromycin and furazolidone in combination with omeprazole are a good alternative for H. pylori eradication in Brazilian patients with duodenal ulcer.

Microbiological and histological study of the gastrointestinal tract of germ-free mice infected with Helicobacter trogontum.

Helicobacter spp. have been the focus of considerable research because of the role of this genus in gastrointestinal diseases. We infected NIH germ-free mice with Helicobacter trogontum, a recently described intestinal bacterium of rats, in order to study the distribution of this bacterium in the gastrointestinal tract and the histopathological changes it can induce in this host. Sixteen mice were challenged with a single dose of H. trogontum (test group) and killed one and six weeks after inoculation (eight animals at each point). Eight animals were challenged with 0.85% saline alone (control group) and killed at the same time points (four at each point). Fragments from the gastric and intestinal mucosa were obtained for microbiological and histological examination. H. trogontum was isolated from the cecum and colon of all test mice and also from the gastric mucosa of several of them. All infected animals presented histological changes in at least one region of the bowel. Alterations in the gastric mucosa were also observed mainly in the six-week-infected group. The predominant histological change observed was a moderate diffuse inflammatory infiltrate of mononuclear cells in the lamina propria, often accompanied by a mild infiltration of polymorphonuclear cells. Two animals presented focal infiltration of inflammatory cells in the liver, although no bacteria were found in the liver of any animal. H. trogontum is an intestinal species that is able to elicit inflammatory responses in other regions of the gastrointestinal tract such as the gastric mucosa and the liver of gnotobiotic mice.

Neolignans inhibit Trypanosoma cruzi infection of its triatomine insect vector, Rhodnius prolixus.

Two neolignans, burchellin and nordihydroguaiaretic acid (NDGA), were toxic only to Trypanosoma cruzi clone Dm28c maintained in brain heart infusion (BHI) medium at a concentration of 100 microg/ml, not 10 microg/ml. When Rhodnius prolixus was fed with epimastigotes of T. cruzi and treated simultaneously with a single dose of burchellin or NDGA at 10 pg/ml of blood meal the number of parasites in the gut decreased. Whereas burchellin was only partially active, NDGA drastically reduced the number of epimastigotes and metacyclic trypomastigotes of T. cruzi in the excreta (urine plus feces). When the insect larvae were pretreated with burchellin or NDGA at 20 days before the infection with T. cruzi a significant reduction in the number of parasites in the gut occurred. However, when both compounds were applied at 20 days after the establishment of T. cruzi infection, although burchellin significantly reduced the gut infection, neither compound could abolish the infection entirely within the subsequent 15 days.

The interrelationship between Helicobacter pylori vacuolating cytotoxin and gastric carcinoma.

OBJECTIVE: We aimed to determine whether cytotoxin-positive Helicobacter pylori strains are associated with gastric carcinoma. METHODS: We studied 130 patients: 57 H. pylori-positive patients with gastric carcinoma, 53 H. pylori-positive patients without gastric carcinoma, and 20 H. pylori-negative subjects. The ability of H. pylori strains to produce vacuolating cytotoxin was tested in INT-407 and HeLa cells. The presence of antibodies to cytotoxin was investigated in blood serum from all subjects by immunoblotting. Fragments of the gastric mucosa from patients without gastric carcinoma and H. pylori-negative subjects were obtained for histopathological study. RESULTS: Considering the results as a whole, 40 (70.2%) patients with and 22 (41.5%) without gastric carcinoma were colonized by cytotoxin-positive strains. Antibodies against cytotoxin were not observed in the serum from 17 (29.8%) gastric carcinoma patients and from 31 (58.5%) patients without gastric carcinoma. H. pylori strains isolated from these patients did not produce cytotoxin in vitro. In regard to cytotoxin positivity, a significant difference was observed between patients with and without gastric carcinoma (p=0.004; odds ratio [OR]: 3.3; 95% confidence interval [CI]: 1.4-7.9). Higher scores of mononuclear (p=0.0001) and polymorphonuclear (p=0.000003) cells were observed in the antral mucosa from H. pylori-positive patients without gastric carcinoma infected by cytotoxin-positive strains than in those harboring cytotoxin-negative strains. CONCLUSION: Cytotoxin-producing H. pylori strains were more frequently observed in patients with gastric carcinoma and this aspect emphasizes the role of cytotoxin in the genesis of the tumor.

cagA-positive Helicobacter pylori and risk for developing gastric carcinoma in Brazil.

To evaluate a possible association between infection with cag A-positive strains and gastric carcinoma increased risk we studied 119 Helicobacter pylori-positive patients with gastric carcinoma and 119 matched controls. The presence of cag A gene was investigated by PCR in H. pylori isolates and in gastric biopsy specimens. A significant association was found between cag A-positive status and distal gastric carcinoma for both the intestinal and diffuse types of tumor for both males and females. On the other hand, no association was observed between cag A-positive status and proximal gastric carcinoma.