Advantages of adipose tissue stem cells over CD34+ mobilization to decrease hepatic fibrosis in Wistar rats.

INTRODUCTION AND OBJECTIVES: Chronic liver inflammation may lead to hepatic cirrhosis, limiting its regenerative capacity. The clinical standard of care is transplantation, although stem cell therapy may be an alternative option. The study aim was to induce endogenous hematopoietic stem cells (HSCs) with granulocyte colony stimulating factor (G-CSF) and/or intravenous administration of adipose tissue-derived mesenchymal stem cells (MSCs) to decrease hepatic fibrosis in an experimental model. MATERIAL AND METHODS: A liver fibrosis model was developed with female Wistar rats via multiple intraperitoneal doses of carbon tetrachloride. Three rats were selected to confirm cirrhosis, and the rest were set into experimental groups to evaluate single and combined therapies of G-CSF-stimulated HSC mobilization and intravenous MSC administration. RESULTS: Treatment with MSCs and G-CSF significantly improved alanine amino transferase levels, while treatment with G-CSF, MSCs, and G-CSF+MSCs decreased aspartate amino transferase levels. Hepatocyte growth factor (HGF) and interleukin 10 levels increased with MSC treatment. Transforming growth factor ß levels were lower with MSC treatment. Interleukin 1ß and tumor necrosis factor alpha levels decreased in all treated groups. Histopathology showed that MSCs and G-CSF reduced liver fibrosis from F4 to F2. CONCLUSIONS: MSC treatment improves liver function, decreases hepatic fibrosis, and plays an anti-inflammatory role; it promotes HGF levels and increased proliferating cell nuclear antigen when followed by MSC treatment mobilization using G-CSF. When these therapies were combined, however, fibrosis improvement was less evident.

Cytokines profile in immunocompetent mice during Trichosporon asahii infection.

Trichosporon asahii is an opportunistic yeastlike fungus commonly associated with systemic infections in immunocompromised patients. Neutropenia is recognized as the main risk factor in infections by T. asahii; however, little is known about the cytokine response during trichosporonosis. Here, we evaluated systemic and local cytokine production and histological damage in immunocompetent mice during systemic infection with T. asahii. We found a significant increased presence of G-CSF, TNF-α, IFN-γ, and IL-6 in sera samples. High levels of G-CSF were found in organs (kidney, liver and spleen); meanwhile IL-10, IL-17A, IL-2, IL-4 and TNF-α were found in low levels. Neutrophils and fungal structures were found in early stage in analyzed organs. Our results demonstrated that T. asahii induces a systemic inflammatory response and G-CSF environment in infected organs in immunocompetent mice and neutrophil recruitment in analyzed tissue suggests the importance of these cells for fungal control.

Chitosan-G-Glycidyl Methacrylate/Au Nanocomposites Promote Accelerated Skin Wound Healing.

Herein, we report the synthesis of Au nanoparticles (AuNPs) in chitosan (CTS) solution by chemically reducing HAuCl4. CTS was further functionalized with glycidyl methacrylate (chitosan-g-glycidyl methacrylate/AuNP, CTS-g-GMA/AuNP) to improve the mechanical properties for cellular regeneration requirements of CTS-g-GMA/AuNP. Our nanocomposites promote excellent cellular viability and have a positive effect on cytokine regulation in the inflammatory and anti-inflammatory response of skin cells. After 40 days of nanocomposite exposure to a skin wound, we showed that our films have a greater skin wound healing capacity than a commercial film (TheraForm®), and the presence of the collagen allows better cosmetic ave aspects in skin regeneration in comparison with a nanocomposite with an absence of this protein. Electrical percolation phenomena in such nanocomposites were used as guiding tools for the best nanocomposite performance. Our results suggest that chitosan-based Au nanocomposites show great potential for skin wound repair.

Effects of Transportation Stress on Complete Blood Count, Blood Chemistry, and Cytokine Gene Expression in Heifers.

Blood samples were obtained from 16 high-risk heifers; eight were newly arrived from a 40 h road trip (0 days post-arrival (DPA)), whereas the other eight heifers had been in the feedlot at 25 DPA. Both groups were transported from the southeast tropical region of Mexico to a feedlot in the northeast and were sampled on the same day. The complete blood count, blood chemistry, and cytokine gene expression were analyzed. Gene expression was analyzed using specific primers to amplify and quantify the cDNA reverse transcribed from the mRNA transcripts for tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-2. Higher values for hematocrit (p = 0.029), hemoglobin (p = 0.002), eosinophils (0.029), albumin (p = 0.014), alanine aminotransferase (p = 0.004), bilirubin (p = 0.003), cholesterol (p = 0.014), and cortisol (p = 0.051) were observed in the 0 DPA group than the 25 DPA group. In the electrophoresis of TNF-α amplification products, two non-specific bands were observed in the 0 DPA group. These bands were sequenced, and BLAST analysis suggested that they corresponded to bovine lymphotoxin and have not been reported previously related to stress. The TNF-α expression level was higher (p = 0.001) in the 25 DPA group than the 0 DPA group according to the semi-quantitative expression analysis. This may indicate a persistent inflammatory process that could be related to trauma and disease, which can negatively impact their subsequent health and growth performance. In conclusion, homeostatic disruption was apparent in the 0 DPA heifers, which showed higher cortisol and reductions in TNF-α levels and stress-induced bovine lymphotoxin (SIBL) co-expression.

Antiangiogenic and antitumor effects of IMMUNEPOTENT CRP in murine melanoma.

BACKGROUND: Skin cancers are common, and there has recently been a dramatic increase in their incidence, particularly in the occurrence of melanoma. Furthermore, relapse after curative surgical treatment of melanoma remains a significant clinical challenge and accounts for most of the mortality of this disease. OBJECTIVE: The aim of this study was to determine whether IMMUNEPOTENT CRP affects B16F10 melanoma cells and tumors growth and vascular endothelial growth factor (VEGF) production in vivo and in vitro. METHODS: B16F10 cells and B16F10-inoculated mice were treated with different concentrations of IMMUNEPOTENT CRP. Outcomes were then evaluated using MTT, TUNEL, Caspase-3, senescence, ELISA and colorimetric assays. Parameters related to survival and tumor weight were also assessed. RESULTS: IMMUNEPOTENT CRP decreased the viability of B16F10 cells by increasing apoptosis of the treated cells, and VEGF production was decreased both in vitro and in vivo. Furthermore, treatment prevented metastasis, delayed the appearance of tumors, decreased tumor weight and improved the survival of tumor-bearing mice. DISCUSSION: These observations suggest that IMMUNEPOTENT CRP can be used to suppress growth and metastasis by using targeting proteins such as VEGF.

Anti-inflammatory and antimicrobial activity of bioactive hydroxyapatite/silver nanocomposites.

Biomaterials are often used in orthopedic surgery like cavity fillings. However, related complications often require long-term systemic antibiotics, device removal, and extended rehabilitation. Hydroxyapatite/silver (HA/Ag) composites have been proposed as implantation biomaterials owing to the osteogenic properties of hydroxyapatite and to the antimicrobial efficiency of silver. Nevertheless, higher silver concentrations induce cytotoxic effects. The aim of this study was to synthesize and characterize HA/Ag nanocomposites that will allow us to use lower concentrations of silver nanoparticles with better antimicrobial efficiency and anti-inflammatory properties. The characterization of HA/Ag was performed by scanning electron microscopy, energy dispersive spectroscopy, X-ray diffraction, Fourier-transform infrared spectra, X-ray photoelectron spectroscopy, and laser diffraction. Bioactivity was evaluated under a simulated body fluid. The viability of osteoblast like-cells (MG-63) was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) and the antimicrobial activity was evaluated by the standard McFarland method. The detection of nitric oxide was measured by a colorimetric assay and the inflammatory cytokines by flow cytometry. We obtained particulate composites of calcium phosphates identified as hydroxyapatite and silver nanoparticles. The bioactivity of the HA/Ag nanocomposites on SFB was confirmed by apatite formations. The viability of MG-63 cells was not affected. We also found antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans owing to the presence of silver nanoparticles at non-cytotoxic concentrations. HA/Ag reduced the release of nitric oxide and decreased the secretion of IL-1 and TNF-α in cells stimulated with Lipopolysaccharide (LPS). In conclusion, the inflammatory and antimicrobial capacity of the HA/Ag nanocomposites, as well as its bioactivity and low cytotoxicity make it a candidate as an implantation biomaterial for bone tissues engineering and clinical practices in orthopedic, oral and maxillofacial surgery.

RNAi silencing of the WT1 gene inhibits cell proliferation and induces apoptosis in the B16F10 murine melanoma cell line.

The Wilms' tumor protein 1 (WT1) is essential for tumor cell proliferation and is highly expressed in various hematological and solid malignancies including human malignant melanoma. We investigated whether WT1 expression is essential for growth in the B16F10 murine melanoma cell line. Toward this end, we examined WT1 protein expression and WT1 isoforms (17AA+/17AA-, KTS+/KTS-) in this cell line. WT1 was silenced by two RNA interference constructs, designated WT1-1 and WT1-2. RNA interference-mediated reduction of WT1 protein expression significantly inhibited B16F10 cell viability. Loss of WT1 also increased caspase-3 and poly-ADP-ribose polymerase activation, as well as apoptotic body formation, chromatin condensation, and DNA fragmentation. Together, these findings implicate decreased WT1 protein levels in the induction of apoptosis. These results imply that WT1 plays a distinct role in B16F10 melanoma growth.

Effect of bovine dialyzable leukocyte extract on induction of cell differentiation and death in K562 human chronic myelogenous leukemia cells.

Differentiation induction therapy is an attractive approach in leukemia treatment due to the fact that in blast crisis stage, leukemic cells lose their differentiation capacity. Therefore, it has been proposed as a therapeutic strategy to induce terminal differentiation of leukemic blast cells into a specific lineage, leading to prevention of high proliferation rates. The aim of the present study was to demonstrate the potential of cell differentiation and death induced by bovine dialyzable leukocyte extract (bDLE) in the K562 cell line. For this purpose K562 and MOLT-3 human leukemic cell lines and primary human monocytes and murine peritoneal macrophages were exposed to bDLE, phorbol myristate acetate (PMA) and dimethyl sulfoxide for 96 h, and the viability, proliferation and cell cycle were evaluated. To determine the lineage that led to cell differentiation, Romanowsky staining was performed to observe the morphological changes following the treatments, and the expression of the surface markers cluster of differentiation (CD)14+, CD68+, CD163+ and CD42a+, as well as the phagocytic activity, and the production of nitric oxide (NO) (assessed by colorimetric assay), cytokines [interleukin (IL)-1ß, IL-6, IL-8 and tumor necrosis factor-α] and chemokines [chemokine (C-C motif) ligand (CCL)2, CCL5 and chemokine (C-X-C motif) ligand 8] in cell supernatants was assessed by flow cytometry. The results of the present study reveal that high doses of bDLE increase the cell death in K562 and MOLT-3 lines, without affecting the viability of human monocytes and murine peritoneal macrophages. Furthermore, low doses of bDLE induce differentiation in K562 cells towards a monocyte/macrophage lineage with an M2 phenotype, and induced moderately upregulated expression of CD42+, a megakaryocytic marker. Cell cycle arrest in the S and G2/M phases was observed in bDLE-treated K562 cells, which demonstrated similar phagocytic activity, NO levels and cytokine and chemokine production to that of PMA-treated cells. The present study demonstrates that bDLE exhibits an antileukemia effect, suggesting that it may be an effective candidate for leukemia treatment.

Silencing of Foxp3 delays the growth of murine melanomas and modifies the tumor immunosuppressive environment.

Forkhead box p3 (Foxp3) expression was believed to be specific for T-regulatory cells but has recently been described in non-hematopoietic cells from different tissue origins and in tumor cells from both epithelial and non-epithelial tissues. The aim of this study was to elucidate the role of Foxp3 in murine melanoma. The B16F10 cell line Foxp3 silenced with small interference Foxp3 plasmid transfection was established and named B16F10.1. These cells had lower levels of Foxp3 mRNA (quantitative real-time reverse transcription-polymerase chain reaction [0.235-fold]), protein (flow cytometry [0.02%]), CD25(+) expression (0.06%), cellular proliferation (trypan blue staining), and interleukin (IL)-2 production (enzyme-linked immunosorbent assay [72.35 pg/mL]) than those in B16F10 wild-type (WT) cells (P<0.05). Subcutaneous inoculation of the B16F10.1 cell line into C57BL/6 mice delayed the time of visible tumor appearance, increased the time of survival, and affected the weight of tumors, and also decreased the production of IL-10, IL-2, and transforming growth factor beta compared with mice inoculated with the B16F10 WT cell line. The B16F10.1 cells derived from tumors and free of T-cells (isolated by Dynabeads and plastic attachment) expressed relatively lower levels of Foxp3 and CD25(+) than B16F10 WT cells (P<0.05) in a time-dependent manner. The population of tumor-infiltrating lymphocytes of T CD4(+) cells (CD4(+), CD4(+)CD25(+), and CD4(+)CD25(+)Foxp3(+)) increased in a time-dependent manner (P<0.05) in tumors derived from B16F10 WT cells and decreased in tumors derived from B16F10.1 cells. Similar data were obtained from spleen cells. These results suggest that, in melanomas, Foxp3 partly induces tumor growth by modifying the immune system at the local and peripheral level, shifting the environment toward an immunosuppressive profile. Therapies incorporating this transcription factor could be strategies for cancer treatment.

Bovine dialyzable leukocyte extract modulates the nitric oxide and pro-inflammatory cytokine production in lipopolysaccharide-stimulated murine peritoneal macrophages in vitro.

Lipopolysaccharides (LPS) released from Gram-negative bacteria after infection initiate an exagerated response that leads to a cascade of pathophysiological events termed sepsis. Monocytes or macrophages produce many of the mediators found in septic patients. Targeting of these mediators, especially tumor necrosis factor (TNF)-alpha and nitric oxide (NO), has been pursued as a mean of reducing mortality in sepsis. Bovine dialyzable leukocyte extract (bDLE) is a dialysate of a heterogeneous mixture of low-molecular-weight substances released from disintegrated leukocytes of the blood or tissue lymphoid. In this study, to determine whether bDLE modulates NO and pro-inflammatory cytokine production, murine peritoneal macrophages were treated with bDLE (0.05 or 0.5 U/mL) before LPS (20 mg/mL) stimulation, and also LPS-stimulated murine peritoneal macrophages were treated with bDLE (0.05 or 0.5 U/mL) at 0, 4, 8, 12, and 24 hours. The bDLE significantly decreased NO production, and also decreased TNF-alpha and interleukin (IL)-6 but increased IL-10 production in LPS-stimulated murine peritoneal macrophages. Our results demonstrate that bDLE plays an important role in modulating TNF-alpha, IL-6, and NO production through IL-10, and this may offer therapeutic potential in clinical endotoxic shock.

Bovine dialyzable leukocyte extract protects against LPS-induced, murine endotoxic shock.

The pathophysiology of endotoxic shock is characterized by the activation of multiple pro-inflammatory genes and their products which initiate the inflammatory process. Endotoxic shock is a serious condition with high mortality. Bovine dialyzable leukocyte extract (bDLE) is a dialyzate of a heterogeneous mixture of low molecular weight substances released from disintegrated leukocytes of the blood or lymphoid tissue obtained from homogenized bovine spleen. bDLE is clinically effective for a broad spectrum of diseases. To determine whether bDLE improves survival and modulates the expression of pro-inflammatory cytokine genes in LPS-induced, murine endotoxic shock, Balb/C mice were treated with bDLE (1 U) after pretreatment with LPS (17 mg/kg). The bDLE improved survival (90%), suppressed IL-10 and IL-6, and decreased IL-1beta, TNF-alpha, and IL-12p40 mRNA expression; and decreased the production of IL-10 (P<0.01), TNF-alpha (P<0.01), and IL-6 (P<0.01) in LPS-induced, murine endotoxic shock. Our results demonstrate that bDLE leads to improved survival in LPS-induced endotoxic shock in mice, modulating the pro-inflammatory cytokine gene expression, suggesting that bDLE is an effective therapeutic agent for inflammatory illnesses associated with an unbalanced expression of pro-inflammatory cytokine genes such as in endotoxic shock, rheumatic arthritis and other diseases.

WT1 silencing by RNAi synergizes with chemotherapeutic agents and induces chemosensitization to doxorubicin and cisplatin in B16F10 murine melanoma cells.

The Wilm's tumor gene (WT1), encoding a transcription factor that modulates the expression of certain genes that are involved in proliferation and apoptosis, is overexpressed in numerous solid tumors. WT1 is important for cell proliferation and in the diagnosis of melanoma. The objectives of this study were to investigate whether WT1 silencing is capable of synergizing with chemotherapeutic agents and whether this silencing is capable of sensitizing cancer cells to doxorubicin and cisplatin in the B16F10 murine melanoma cell line. In the present study, B16F10 cells were simultaneously treated with median lethal doses (LD50s) of WT1-1 or WT1-2 small hairpin RNAs (shRNAs) and chemotherapeutic agents. A total of 24 h post-transfection, a [3-(4,5-dimethylthiazol-2yl)-2,5- diphenyl tetrazolium bromide assay] MTT assay was performed. To determine whether shRNA interference (shRNAi) is capable of sensitizing B16F10 cells to chemotherapeutic agents, cells were transfected with an LD50 of each of the recombinant plasmids, treated with varying concentrations of doxorubicin or cisplatin 24 h post-transfection, and analyzed 48 h later for inhibition of cell proliferation using the MTT assay. We observed that WT1-RNAi and the two chemotherapeutic agents acted synergistically to inhibit B16F10 cell proliferation. The greatest inhibition of cell proliferation was observed with the WT1-2/cisplatin (91%) and WT1-1/cisplatin combinations (85%). WT1 silencing using shRNAi induced the chemosensitization of cells to doxorubicin and cisplatin, with the greatest inhibition (85%) of cell proliferation being observed in the cells treated with the WT1-2/cisplatin 6 ng/µl combination. Our results provide direct evidence that WT1 gene silencing has a synergistic effect with chemotherapeutic drugs and sensitizes B16F10 melanoma cells to doxorubicin and cisplatin. This suggests that these combination strategies are potentially utilized in melanoma therapy.

Antitumor activity of colloidal silver on MCF-7 human breast cancer cells.

BACKGROUND: Colloidal silver has been used as an antimicrobial and disinfectant agent. However, there is scarce information on its antitumor potential. The aim of this study was to determine if colloidal silver had cytotoxic effects on MCF-7 breast cancer cells and its mechanism of cell death. METHODS: MCF-7 breast cancer cells were treated with colloidal silver (ranged from 1.75 to 17.5 ng/mL) for 5 h at 37°C and 5% CO2 atmosphere. Cell Viability was evaluated by trypan blue exclusion method and the mechanism of cell death through detection of mono-oligonucleosomes using an ELISA kit and TUNEL assay. The production of NO, LDH, and Gpx, SOD, CAT, and Total antioxidant activities were evaluated by colorimetric assays. RESULTS: Colloidal silver had dose-dependent cytotoxic effect in MCF-7 breast cancer cells through induction of apoptosis, shown an LD50 (3.5 ng/mL) and LD100 (14 ng/mL) (*P < 0.05), significantly decreased LDH (*P < 0.05) and significantly increased SOD (*P < 0.05) activities. However, the NO production, and Gpx, CAT, and Total antioxidant activities were not affected in MCF-7 breast cancer cells. PBMC were not altered by colloidal silver. CONCLUSIONS: The present results showed that colloidal silver might be a potential alternative agent for human breast cancer therapy.

Expression of prostate apoptosis response (Par-4) is associated with progesterone receptor in breast cancer.

BACKGROUND: The prostate apoptosis response (Par-4) gene encodes a proapoptotic protein that selectively induces apoptosis in cancer cells after diverse apoptotic stimuli. Par-4 expression and its association with other biomarkers have not been reported in breast cancer. The purpose of this study was to determine Par-4 expression in breast cancer samples and its association with other biomarkers and clinical factors (T-stage, age, nodal status). METHODS: Paraffin-embedded section samples of breast cancer were evaluated by immunohistochemical analysis to determine Par-4, estrogen receptor (ER), progesterone receptor (PgR), c-erbB2, Ki67, p53 and bcl-2 expression. The correlation between Par-4 and the other biomarkers and clinical factors was determined by multivariate analysis. RESULTS: Thirty five percent (n=21) of samples were PAR-4 positive and 64.4% (n=38) were negative. The hormonal status was 64% ER positive (n=38), 35% ER-negative (n=21) and 40.7% PgR positive (n=24), 59.3% PgR negative (n=35). The majority (90%) of the samples presented clear cytoplasmic localization and a small portion (10%) was cytoplasmic and nuclear. Univariate analysis indicates that the Par-4 expression has a significant inverse association (p=0.04) only with expression of PgR and not with the other variables analyzed. Normal breast tissue analyzed was negative for Par-4 immunostaining. CONCLUSIONS: Our results suggest that, in breast cancer, Par-4 plays a similar tumor suppressor gene role as reported in endometrial carcinoma.

Adjuvant effects of crystal proteins from a Mexican strain of Bacillus thuringiensis on the mouse humoral response.

In this study, we determined the adjuvant effects of the crystal (Cry) proteins, p130, p98, and p64-62, on the immune response of mice to both sheep red blood cells (SRBC) and ovalbumin (OVA). The administration of p130, p98, and p64-62 Cry proteins to Balb/c mice induced a significant (p<0.01) increase in the production of anti-SRBC antibody-secreting cells (ASC). The p64-62 Cry proteins demonstrated the best ability to induce the production of IgA and IgG antibodies to SRBC (p<0.05), and IgM, IgA, and IgG antibodies to OVA (p<0.05). Additionally, Cry proteins did not produce any side effects associated with their administration to Balb/c mice. We suggest the potential use of the p64-62 Cry proteins as adjuvants for the administration of heterologous antigens.