Lethality rate, hematological alterations and viral load upon DENV-2 infection.

<p>WT or CCR1^–/–, CCR2^–/– and CCR4^–/– (KO) mice were infected i.p. with 10 LD₅₀ of DENV-2 and then monitored for lethality until day 14. In panel A, percentages of survival (n = 10–12). Hematological analysis were done at day 6 p.i. for changes in platelets count (B) hematocrit (C) and lymphocytes (D) in the blood of non-infected and infected-WT and KO mice. In panel E, viral loads recovered from the liver of WT and KO mice at day 6 p.i. shown as the log of PFU/100 mg of tissue. Results are expressed as mean ± SEM and are representative of at least two experiments (n = 5–12 mice). *** P<0.001 when compared to non-infected mice. # P<0.05, ## P<0.01 when compared to WT infected mice. NI: non-infected mice. NS: Not significant.</p

Histological changes in liver upon DENV-2 infection in mice.

<p>WT or KO mice were infected i.p. with 10 LD₅₀ of DENV-2 and then sacrificed at day 6 for tissue samples. Hematoxylin & Eosin stained liver sections from non-infected and DENV-2 infected WT, CCR1^–/–, CCR2^–/– and CCR4^–/– mice, showing different degrees of congestion, hemorrhage, hepatocyte degeneration and necrosis. Each slide presented in the panel is representative of at least 10 different fields (n = 5–6 mice). Magnification: 400X.</p

Liver inflammation and injury upon DENV-2 infection.

<p>WT or KO mice were infected i.p. with 10 LD₅₀ of DENV-2 and then sacrificed at day 6 for blood and tissue samples. AST (A) and ALT (B) were dosed in serum of WT and KO mice as markers of hepatic injury. MPO activity, as an index of neutrophil accumulation, was evaluated in liver (C). Concentrations of cytokines IL-6 (D) and IFN-γ (E) were evaluated in liver homogenates by ELISA and are expressed as pg per 100 mg of tissue. Results are expressed as mean ± SEM and are representative of at least two experiments (n = 5–6). * P<0.05, *** P<0.001 when compared to non-infected mice. # P<0.05, ## P<0.01, ### P<0.001 when compared to WT infected mice. NI: non-infected mice.</p

Cytokine and chemokine production in spleen upon DENV-2 infection in mice.

<p>WT or KO mice were infected i.p. with 10 LD₅₀ of DENV-2 and then sacrificed at day 6 for tissue samples. TNF-α (A), CCL2/JE (B), CCL3/MIP-1α (C), CCL5/RANTES (D) and CCL17/TARC (E) were evaluated in spleen homogenates by ELISA and are expressed as pg per 100 mg of tissue. Results are expressed as mean ± SEM and are representative of at least two experiments (n = 5–6 mice). * P<0.05, *** P<0.001 when compared to non-infected mice. # P<0.05, ## P<0.01, ### P<0.001 when compared to WT infected mice. NI: non-infected mice.</p

Chemokine production in liver upon DENV-2 infection in mice.

<p>WT or KO mice were infected i.p. with 10LD₅₀ of DENV-2 and then sacrificed at day 6 for tissue samples. CCL2/JE (A), CCL3/MIP-1α (B) and CCL5/RANTES (C) were evaluated in liver homogenates by ELISA and are expressed as pg per 100 mg of tissue. Results are expressed as mean ± SEM and are representative of at least two experiments (n = 5–6 mice). *P<0.05, *** P<0.001 when compared to non-infected mice. # P<0.05, ## P<0.01, ### P<0.001 when compared to WT infected mice. NI: non-infected mice.</p

Lymphocyte number and activation in CC chemokine receptors deficient mice upon DENV-2 infection.

<p>WT or KO mice were infected i.p. with 10 LD₅₀ of DENV-2 and then sacrificed at day 6. Splenic leukocytes were counted and then stained with specific antibodies. Flow cytometry, according to size and granularity, were performed as analysis. The numbers of specific cell populations are shown compared to total number of leukocytes in the spleen. Number of T lymphocytes CD3⁺CD4⁺ (A) and CD3⁺CD8⁺ (C) were evaluated in WT and KO mice. Activated T lymphocytes expressing CD69 were also evaluated for CD4⁺ (B) and CD8⁺ (D) populations. The number of CD3⁺DX5⁺ NKT cells (E) and their activation by CD69 expression as MFI, were also evaluated (F). Results are expressed as mean ± SEM and are representative of at least two experiments (n = 5–6 mice). *P<0.05, ** P<0.01, *** P<0.001 when compared to non-infected mice. # P<0.05, ## P<0.01 when compared to WT infected mice. NI: non-infected mice. MFI: Mean fluorescence intensity.</p

Cytokine production in serum upon DENV-2 infection in mice.

<p>WT or KO mice were infected i.p. with 10 LD₅₀ of DENV-2 and then sacrificed at day 6 for blood samples. IL-6 (A) and IFN-γ (B) were evaluated in serum by ELISA and are expressed as pg/ml. Results are expressed as mean ± SEM and are representative of at least two experiments (n = 5–6 mice). ** P<0.01, *** P<0.001 when compared to non-infected mice. # P<0.05, ## P<0.01, ### P<0.001 when compared to WT infected mice. NI: non-infected mice.</p

Nanocomposite Treatment Reduces Disease and Lethality in a Murine Model of Acute Graft-versus-Host Disease and Preserves Anti-Tumor Effects

<div><p>Graft versus host disease (GVHD) is an immunological disorder triggered by bone marrow transplantation that affects several organs, including the gastrointestinal tract and liver. Fullerenes and their soluble forms, fullerols, are nanocomposites with a closed symmetrical structure with anti-inflammatory and anti-oxidant properties. The present study evaluated the effects of treatment with the fullerol (C60(OH)18-20) in the development and pathogenesis of GVHD in a murine model. Mice with experimental GVHD that were treated with the fullerol showed reduced clinical signs of disease and mortality compared with untreated mice. Treatment with the fullerol decreased the hepatic damage associated with reduced hepatic levels of reactive oxygen species, pro-inflammatory cytokines and chemokines (IFN-γ TNF-α, CCL2, CCL3 and CCL5) and reduced leukocyte accumulation. The amelioration of GVHD after treatment with the fullerol was also associated with reduced intestinal lesions and consequent bacterial translocation to the blood, liver and peritoneal cavity. Moreover, the fullerol treatment alleviated the GVHD while preserving effects of the graft against a leukemia cell line (GFP+P815). In summary, the fullerol was effective in reducing the GVHD inflammatory response in mice and may suggest novel ways to treat this disease.</p></div

Fullerol treatment reduces hepatic injury in mice with experimental GVHD.

<p>GVHD was induced by the transfer of splenocytes from semi allogeneic C57BL/6J donors to B6D2F1 mice. Fullerol (10 mg/Kg in 100 ml of PBS, i.p.) was given to the B6D2F1 mice 30 min before transplantation and every 48 hours thereafter during the entire duration of the experiments. The mice that received splenocytes from the syngeneic (B6D2F1) mice did not develop the disease and were considered the control group. After the induction of GVHD, the mice were killed and the liver sampled for histopathological analysis and scoring at 20 days post-transplantation (A). Histological aspects of the H&E-stained liver sections in the control, GVHD, and fullerol treated mice, respectively (B-D). The scale bar = 50 μm for all the panels. The results are presented as the mean ± SEM (n = 6); * and #, P < 0.05 when compared with the control and GVHD groups, respectively.</p

Fullerol treatment decreased the accumulation of leukocytes in the liver at 20 days after transplant.

<p>GVHD was induced by the transfer of splenocytes from semi allogeneic C57BL/6J donors to B6D2F1 mice. Fullerol (10 mg/Kg in 100 ml of PBS, i.p.) was given to the B6D2F1 mice 30 min before transplantation and every 48 hours thereafter during the entire duration of the experiments. The mice that received splenocytes from the syngeneic (B6D2F1) mice did not develop the disease and were considered the control group. Liver samples were collected 10 and 20 days after transplantation, and the accumulation of macrophages and neutrophils was analyzed in this tissue through the activity of N-acetyl-β-D-glucosaminidase (NAG) and myeloperoxidase (MPO), respectively. The relative numbers of macrophages 10 days (A) and 20 days (B) after transplantation are shown. The relative numbers of neutrophils 10 days (C) and 20 days (D) after transplantation are shown. The results are presented as the mean ± SEM (n = 6), * and #P < 0.05 compared with the control and GVHD groups, respectively.</p