The expression of T regulatory cells (Foxp3), regulatory (interleukin [IL]-10 and transforming growth factor beta [TGF-β]) and proinflammatory (tumor necrosis factor alpha [TNF-α] and interleukin [IL]-1β) cytokines was quantified using real time polymerase chain reaction (qRT-PCR) in the liver of sheep during early stages of infection with Fasciola hepatica (1, 3, 9, and 18 days post-infection [dpi]). Portal fibrosis was also evaluated by Masson’s trichrome stain as well as the number of Foxp3+ cells by immunohistochemistry. Animals were divided into three groups: (a) group 1 was immunized with recombinant cathepsin L1 from F. hepatica (FhCL1) in Montanide adjuvant and infected; (b) group 2 was uniquely infected with F. hepatica; and (c) group 3 was the control group, unimmunized and uninfected. An overexpression of regulatory cytokines of groups 1 and 2 was found in all time points tested in comparison with group 3, particularly at 18 dpi. A significant increase of the number of Foxp3+ lymphocytes in groups 1 and 2 was found at 9 and 18 dpi relative to group 3. A progressive increase in portal fibrosis was found in groups 1 and 2 in comparison with group 3. In this regard, group 1 showed smaller areas of fibrosis than group 2. There was a significant positive correlation between Foxp3 and IL-10 expression (by immunohistochemistry and qRT-PCR) just as between portal fibrosis and TGF-β gene expression. The expression of proinflammatory cytokines increased gradually during the experience. These findings suggest the induction of a regulatory phenotype by the parasite that would allow its survival at early stages of the disease when it is more vulnerable.
Background The use of intraoperative fluorescence images with indocyanine green (ICG) has recently been described as an aid in decision-making during surgical procedures in adults.We present our first experiences with different laparoscopic procedures performed in children using ICG fluorescence images.
Material and Method We have used ICG fluorescence imaging technique in varicocele ligation, two nephrectomies, cholecystectomy, and one case of aortocoronary fistula closure. All procedures were performed through a minimally invasive approach. A high definition camera equipped with a visible infrared light source and gray-scale vision technology was used.After injection of ICG before or during the laparoscopic procedure, precise identification of vascular anatomy and bile duct architecture were easily identified. Fluorescence helped to assess blood flow from the spermatic vessels, define the variability of renal vascularization, and determine the precise location of the aortocoronary fistula. Biliary excretion of the ICG allowed the definition of the biliary tract.
Conclusion Fluorescein-assisted images allowed a clear definition of the anatomy and safe surgical maneuvers during surgical procedures. The ICG imaging system seems to be simple and safe. Larger and more specific studies are needed to confirm its applicability, expand its indications, and address its advantages and disadvantages.
Several immunomodulatory properties have been described in Fasciola hepatica infections. Apoptosis has been shown to be an effective mechanism to avoid the immune response in helminth infections. The aim of the present work was to study apoptosis in peritoneal leucocytes of sheep experimentally infected with F. hepatica during the early stages of infection. Five groups (n=5) of sheep were used. Groups 2-5 were orally infected with 200 metacercariae (mc) and sacrificed at 1, 3, 9 and 18days post-infection (dpi), respectively. Group 1 was used as the uninfected control (UC). Apoptosis was detected using three different methods 1) immunocytochemistry (ICC) with a polyclonal antibody anti-active caspase-3; 2) an annexin V flow cytometry assay using the Annexin V-FITC/propidium iodide (PI); and 3) transmission electron microscopy (TEM). The differential leucocyte count revealed that the majority of peritoneal granulocytes were eosinophils, which increased significantly at 9 and 18 dpi with respect to the uninfected controls. The ICC study revealed that the percentage of caspase-3 apoptotic peritoneal leucocytes increased significantly from 3 dpi onwards with respect to the uninfected controls. The flow cytometry annexin V assay detected a very significant (P<0.001) increase of apoptotic peritoneal macrophages, lymphocytes and granulocytes, which remained higher than in the UC until 18 dpi. Transmission electron microscopy studies also confirmed the presence of apoptosis in peritoneal eosinophils at 18 dpi. This is the first report of apoptosis induced by F. hepatica in the peritoneal leucocytes of sheep in vivo. The results of this work suggest the importance of apoptosis induction for the survival of the juvenile parasites in the peritoneal migratory stages of infection.
In this work we report the protection found in a vaccination trial performed in sheep with two different vaccines composed each one by a cocktail of antigens (rCL1, rPrx, rHDM and rLAP) formulated in two different adjuvants (Montanide ISA 61 VG (G1) and Alhydrogel®(G2)). The parameters of protection tested were fluke burden, faecal egg count and evaluation of hepatic lesions. In vaccinated group 1 we found a significant decrease in fluke burden in comparison to both unimmunised and infected control group (37.2%; p = 0.002) and to vaccinated group 2 (Alhydrogel®) (27.08%; p = 0.016). The lower fluke burden found in G1 was accompanied by a decrease in egg output of 28.71% in comparison with the infected control group. Additionally, gross hepatic lesions found in vaccine 1 group showed a significant decrease (p = 0.03) in comparison with unimmunised-infected group. The serological study showed the highest level for both IgG1 and IgG2 in animals from group 1. All these data support the hypothesis of protection found in vaccine 1 group.
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