Experimental Biology and Medicine

Diabetic foot ulcers are an important complication of diabetes mellitus characterized by chronic, non-healing ulcers resulting from poor proliferation and migration of fibroblasts and keratinocytes, thus impairing a correct re-epithelialization of wounded tissues. This healing process can be modulated by neuropeptides released from peripheral nerves; however, little is known regarding the role of neurotensin (NT) as a modulator of human keratinocyte function under hyperglycemic conditions. Therefore, this work is focused on the effect of NT in human keratinocytes, under normal and hyperglycemic conditions at different functional levels, namely NT receptors, cytokine, and growth factor expression, as well as proliferation and migration. Human keratinocyte cells were maintained at either 10/30 mM glucose and treated with or without NT (10 nM). The results show that NT did not affect keratinocyte viability. In addition, NT and all NT receptor expression levels were significantly reduced by hyperglycemia; however, NT treatment stimulated expression of NT and neurotensin receptor 2 (NTR2) while neurotensin receptor 1 (NTR1) and neurotensin receptor 3 (NTR3) expression levels were unchanged. Keratinocyte proliferation was not affected by NT and hyperglycemia, while cell migration was reduced by NT treatment.

These results demonstrated that hyperglycemic conditions strongly impaired endogenous NT and NTR2 expression in keratinocytes. Despite the addition of exogenous NT to stimulate the endogenous NT and NTR2 expression, these changes do not translate into functional modifications on keratinocytes, particularly in terms of migration, proliferation, and production of cytokines or growth factors. These results suggest that NT production by keratinocytes may exert a paracrine effect on other skin cells, namely fibroblasts, macrophages, and dendritic cells for correct wound healing.

Dendritic cells (DCs) play an important role in atherosclerosis plaque formation, but the mechanism has not been elucidated clearly. This study is designed to establish a method for tracing DCs in vivo facilitating the investigation of the DCs’ specific roles in atherosclerosis. Rabbit DCs labeled by different concentrations of ultra small superparamagnetic iron oxide (USPIO) were injected into atherosclerosis rabbit model and traced with magnetic resonance imaging (MRI). Results showed that USPIO labeling nearly have no cytotoxicity to DCs in low concentrations (<500 µg/mL) but induced some decrease of cell viability at high concentrations (>500 µg/mL). Moreover, USPIO labeling, from 200 to 2000 µg/mL, caused a dose-dependent decrease of the mitochondrial membrane potential in DCs. The high labeling concentration (2000 µg/mL) triggered necrosis instead of apoptosis in DCs. By T2WI and fs T2WI sequence imaging comparison, DCs were found to exist in rabbit abdominal artery plaques after 24 h of transplantation and in spleen after one week detected by Prussian blue staining of tissue sections. We concluded that about 200 µg/mL USPIO is ideal to effectively label DCs for MRI tracing in vivo without a threat to cell viability. Combining USPIO labeling and MRI to track the movement of injected DCs in vivo is a feasible method.

Evidence indicates that high-grade serous ovarian carcinoma (HGSOC) may originate from lesions within the distal fallopian tube epithelium (FTE). Our previous studies indicate that fallopian tube epithelial cells from carriers of germline mutations in breast cancer susceptibility genes exhibit a pro-inflammatory gene expression signature during the luteal phase, suggesting that delayed resolution of postovulatory inflammatory signaling may contribute to predisposition to this ovarian cancer histotype. To determine whether exposure of tubal epithelial cells to periovulatory follicular fluid alters expression of inflammation-associated genes, we used an ex vivo culture system of bovine oviductal epithelial cells. Oviductal cells grown on collagen IV-coated transwell membranes assumed a cobblestone appearance and immunocytochemistry for FoxJ1 and Pax8 indicated that both ciliated and secretory epithelial cells were maintained in the cultures. Oviductal cells were exposed to human follicular fluid or culture medium for 24 h following which total cellular RNA was extracted at various time points. Expression of genes associated with inflammation was determined by quantitative real-time RT-PCR. Exposure to follicular fluid transiently increased the transcript levels of interleukin 8 (IL8) and cyclooxygenase 2 (PTGS2), and decreased the expression of mitochondrial superoxide dismutase (SOD2), glutathione peroxidase 3 (GPX3), disabled homolog 2 (DAB2), and glucocorticoid receptor (NR3C1). Tumor necrosis factor (TNF) and IL6 levels were also decreased while those of nicotinomide phosphoribosyltransferase (NAMPT) were unaffected. This study demonstrates that periovulatory follicular fluid can act directly upon oviductal epithelial cells to alter gene expression that might contribute to early carcinogenic events. Furthermore, these findings illustrate the potential use of bovine oviductal cells to study signaling events implicated in ovarian carcinogenesis.

γ-Tocotrienol, a member of the vitamin E family of compounds, displays potent antiproliferative and cytotoxic effects in a variety of cancer cell types at treatment doses that have little or no effect on normal cell viability or growth. Autophagy is a tightly regulated lysosomal self-digested process that can either promote cell survival or programmed cell death, but the role of autophagy in mediating γ-tocotrienol-induced cytotoxicity in breast cancer is not presently completely understood. Mouse (+SA) and human (MCF-7 and MDA-MD-231) mammary tumor cells lines were exposed to 0–40 µmol/L γ-tocotrienol for a 24 h treatment period. γ-Tocotrienol treatment caused a relatively large increase in the accumulation of monodansylcadaverine (MDC)-labeled vacuoles, a marker of autophagosome formation, in all tumor cell lines. Results also showed that γ-tocotrienol treatment induced an increased conversion of microtubule-associated protein, 1A/1B-light chain 3, from its cytosolic form (LC3B-I) to its lipidated form (LC3B-II), increased Beclin-1 levels, and increased acridine orange staining as determined by flow cytometry analysis, providing further evidence of γ-tocotrienol-induced autophagy in these mammary cancer cell lines. In contrast, similar treatment with γ-tocotrienol was not found to increase autophagy marker expression in immortalized mouse (CL-S1) and human (MCF-10 A) normal mammary epithelial cell lines. Treatment with γ-tocotrienol also caused a reduction in PI3K/Akt/mTOR signaling and a corresponding increase in the Bax/Bcl-2 ratio, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) levels in these cancer cell lines, suggesting that γ-tocotrienol-induced autophagy may be involved in the initiation of apoptosis. In summary, these findings demonstrate that the cytotoxic effects of γ-tocotrienol are associated with the induction of autophagy in a mouse and human mammary cancer cells.

Vitamin A supplementation improves status, which may protect against malarial infection. Provitamin A carotenoid biofortified staple crops may provide a more sustainable approach to alleviate vitamin A deficiency than supplementation, but the impact of febrile illness on food intake must be considered in malaria endemic regions. Morbidity data and food logs from a three-month efficacy trial on provitamin A biofortified (orange) maize in preschool Zambian children (n = 181, age 3–5 years) were systematically analyzed over time to determine the impact of malaria on food intake. Nutrients examined included macronutrients, iron, zinc, and vitamin A. Comparisons based on individual intakes in healthy and malarial states over three-day intervals were made including children from both the orange and white maize groups (n = 100). Malaria prevalence did not differ overall or between treatment groups over time (all P > 0.05). Lower nutrient intakes were observed for all variables during malaria outbreaks (food 289 ± 412 g; energy 248 ± 346 kcal; carbohydrate 42 ± 62 g; protein 8 ± 12 g; fat 5 ± 7 g; iron 1 ± 2 mg; zinc 1 ± 1 mg; vitamin A 58 ± 100 retinol activity equivalents; all P < 0.05). No differences were observed between nutrient decreases in orange and white maize groups (P > 0.05). Considering the impact of malaria on food and nutrient intakes and increased vitamin A utilization and excretion due to the acute phase response, biofortification targets for provitamin A carotenoids may need to be elevated in malaria endemic regions.

Peritoneal (PD) and hemodialysis (HD) represent the leading renal replacement therapies in advanced chronic kidney disease (CKD). Although absolutely necessary to ensure patient survival, these treatments are responsible for considerable biological alterations primarily due to the un-physiological contact of blood and tissues with bioincompatible devices or plastificants. Although extensively described, this complex dialysis-related deregulated bio-molecular machinery is still not completely known. Therefore, to select a set of genes deregulated in patients on dialysis treatment and to assess the possible differences between dialysis modalities, we measured the expression level of 132 genes involved in proteoglycans (PGs) biosynthesis/metabolism by microarray in peripheral blood mononuclear cells (PBMCs), biological elements involved in the inflammatory/immune response, from 5 healthy subjects (HS), 9 CKD, 10 PD, and 17 HD patients. We focused on PGs biosynthesis/metabolism pathways because of their involvement in the onset and development of several CKD-related clinical complications. Statistical analysis/bioinformatics identified 70 genes discriminating HD/PD patients from HS/CKD subjects (P < 0.009, FDR < 5%). Twenty-five genes were up-regulated (e.g. HPSE, VCAN, and VEGFA) and 45 down-regulated (e.g. IDS and HEXA) in PD/HD compared to HS/CKD. Gene expression and plasma activity of Heparanase (HPSE), one of the top selected up-regulated genes in PD/HD, validated microarray results. In addition, for the second part of the study, HPSE plasmatic activities were first assessed in an independent testing-group (7 HS, 10 CKD, 17 PD, and 11 HD), and then correlated with high-sensitive C reactive protein (HS-CRP) measurements. HPSE activity was higher in PD and HD versus CKD/HS and it correlated with HS-CRP levels (R²= 0.37, P = 0.007). Lipopolysaccharide (LPS)-stimulated PBMCs showed a significant up-regulation of HPSE mRNA level (P = 0.04). Our results revealed that dialysis treatments induce change in the transcriptomic pattern of biosynthetic proteoglycans in PBMCs with an up-regulation of HPSE. Our selected genes could be useful in the future as potential biomarkers and new therapeutic targets.

Increased frequency and risk of infection is one of the well described complications of sickle cell anemia (SCA). Dietary supplementation in children with SCA and growth retardation improved growth and decreased incidence of infection. We investigated the impact of a high protein diet on weight gain, hematological profile, and immune cytokine levels in the Berkeley model of SCA, 16 of which were randomized to either regular mouse diet with 20% of calories from protein (n = 8) or a test feed with 35% of calories from protein (n = 8). Control mice (C57BL/6, n = 16) were correspondingly randomized, and were all feed ad libitum for three months with actual intake estimated by subtracting the weight of gnaw waste from that of the feed given. Blood was collected at sacrifice by cardiac puncture and plasma levels of T helper cell 1 (TH1) and TH2 associated cytokines were measured using a multiplex antibody immobilized bead assay. SCA mice receiving the 35% protein diet had modest improvements in weight, red blood cell count, and hemoglobin level, with a slight decrease in reticulocyte count compared with SCA mice on the regular mouse diet. Furthermore, they also had significantly higher plasma levels of cytokines tumor necrosis factor (TNF)-α (P = 0.02), interferon (IFN)-γ (P = 0.01), interleukin 10 (IL-10; P = 0.02), and IL-4 (P = 0.02) compared with those that received the 20% protein diet. We conclude that providing additional protein calories to transgenic SCA mice increased the plasma levels of acute inflammatory cytokines associated with immune response to infection, which might partly explain decreased episodes of infection observed among supplemented children with SCA.

Local anesthetic drugs are extensively used in dentistry. However, the cytotoxic effects of these pharmaceutical compounds remain unclear. In this work, we have evaluated the cell viability and cell function of human oral mucosa fibroblasts exposed to different concentrations of lidocaine for increasing incubation times, using a global screening methods including structural, metabolic and microanalytical analyses. Our results demonstrate that lidocaine is able to alter cell viability and function even at low concentrations and times, although the effect of lidocaine concentration was more important than the incubation time. First, the structural analysis methods revealed that ≥5% concentrations of lidocaine are able to significantly reduce cell viability. Then, the metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and water-soluble tetrazolium salt (WST-1) assays suggest that concentrations starting from 1% were able to significantly hinder cell physiology. Finally, electron-probe X-ray microanalysis confirmed the deleterious effects of lidocaine and allowed us to demonstrate that these effects are associated to an apoptosis process of cell death. Therefore, care should be taken when lidocaine is clinically used, and the lowest efficient concentrations should always be used. Furthermore, these results suggest that the comprehensive evaluation method used in this work is accurate and efficient for screening of local anesthetics.

The antiallergic effects of traditional medicines have long been studied. Traditional Korean medicine, Citrus sunki and bamboo salt, has been used for the treatment of allergic diseases in Korea. K-ALL, composed of Citrus sunki and bamboo salt, is a newly prepared prescription for allergic patients. To develop the new antiallergic agent, we examined the effects of K-ALL through in vivo and in vitro models. K-ALL and naringin (an active compound of K-ALL) significantly inhibited histamine release from rat peritoneal mast cells. This inhibitory effect of K-ALL on histamine release was higher than effects from other known histamine inhibitors such as bamboo salt, Citrus sunki or disodium cromoglycate. K-ALL significantly inhibited systemic anaphylactic shock induced by the compound 48/80 and passive cutaneous anaphylaxis induced by the IgE. K-ALL also inhibited production and mRNA expression of inflammatory cytokines induced by phorbol 12-myristate 13-acetate and the calcium ionophore A23187 on HMC-1 cells (a human mast cell line). In the ovalbumin-induced allergic rhinitis animal model, rub scores, histamine, IgE, inflammatory cytokines and inflammatory cell counts were all reduced by the oral or nasal administration of K-ALL (pre and posttreatment). These results indicate the great potential of K-ALL as an active immune modulator for the treatment of mast cell-mediated allergic diseases.

Chemotherapy-induced myeloablation prior to allogeneic hematopoietic stem cell transplantation (HSCT) may be associated with severe toxicity. The current understanding of the pathophysiology of oral and gastrointestinal (GI) toxicity is largely derived from studies in rodents and very little is known from humans, especially children. We hypothesized that milk-fed piglets can be used as a clinically relevant model of GI-toxicity related to a standard conditioning chemotherapy (intravenous busulfan, Bu plus cyclophosphamide, Cy) used prior to HSCT. In study 1, dose–response relationships were investigated in three-day-old pigs (Landrace × Yorkshire × Duroc, n = 6). Pigs were given one of three different dose combinations of Bu and Cy (A: 4 days Bu, 2 × 1.6 mg/kg plus 2 days Cy, 60 mg/kg; B: 4 days Bu, 2 × 0.8 mg/kg plus 2 days Cy, 30 mg/kg; C: 2 days Bu at 2 × 1.6 mg/kg plus 1 day Cy, 60 mg/kg) and bone marrow was collected on day 11. Histology of bone marrow samples showed total aplasia after treatment A. Using this treatment in study 2, Bu–Cy pigs showed lowered spleen and intestinal weights and variable clinical signs of dehydration, sepsis, and pneumonia at tissue collection. Oral mucositis was evident as ulcers in the soft palate in 4/9 Bu–Cy pigs and villus height and brush-border enzyme activities were reduced, especially in the proximal intestine. There were no consistent effects on tissue cytokine levels (IL-8, IL-6, IL-1β, TNF-α) or blood chemistry values (electrolytes, liver transaminases, bilirubin, alkaline phosphatase), except that blood iron levels were higher in Bu–Cy pigs. We conclude that a myeloablative Bu–Cy regimen to piglets results in clinical signs comparable to those seen in pediatric patients subjected to myeloablative treatment prior to HSCT. Piglets may be used as a model for investigating chemotherapy-induced toxicity and dietary and medical interventions.

Human mesenchymal stem/stromal cells (MSCs) have been explored in a number of clinical trials as a possible method of treating various diseases. However, the effect of long-term cell expansion in vitro on physiological function and genetic stability is still poorly understood. In this study, MSC cultures derived from bone marrow and liver were evaluated for the presence of aberrant cells following long-term expansion. In 46 independent cultures, four batches of transformed MSCs (TMCs) were found, which were all beyond the culture period of five weeks. These aberrant cells were first identified based on the appearance of abnormal cytology and the acquirement of growth advantage. Despite common MSC markers being diminished or absent, TMCs remain highly susceptible to lysis by allogenic natural killer (NK) cells. When transplanted into immunodeficient mice, TMCs formed sarcoma-like tumors, whereas parental MSCs did not form tumors in mice. Using a combination of high-resolution genome-wide DNA array and short-tandem repeat profiling, we confirmed the origin of TMCs and excluded the possibility of human cell line contamination. Additional genomic duplication and deletions were observed in TMCs, which may be associated with the transformation event. Using gene and microRNA expression arrays, a number of genes were identified that were differentially expressed between TMCs and their normal parental counterparts, which may potentially serve as biomarkers to screen cultures for evidence of early transformation events. In conclusion, the spontaneous transformation of MSCs resulting in tumorigenesis is rare and occurs after relatively long-term (beyond five weeks) culture. However, as an added safety measure, cultures of MSCs can potentially be screened based on a novel gene expression signature.

Endothelial injury resulting from deleterious interaction of gas microbubbles occurs in many surgical procedures and other medical interventions. The symptoms of vascular air embolism (VAE), while serious, are often difficult to detect, and there are essentially no pharmaceutical preventative or post-event treatments currently available. Perfluorocarbons (PFCs), however, have shown particular promise as a therapeutic option in reducing endothelial injury both in- and ex-vivo. Recently, we demonstrated the effectiveness of Oxycyte, a third-generation PFC formulated in a phosphotidylcholine emulsion, using an in vitro model of VAE developed in our laboratory. This apparatus allows live cell imaging concurrent with precise manipulation of physiologically sized microbubbles so that they may be brought into individual contact with human umbilical vein endothelial cells dye-loaded with the Ca²⁺ sensitive Fluo-4. Herein, we expand use of this fluorescence microscopy-based cell culture model. Specifically, we examined the concentration dependence of Oxycyte in reducing both the amplitude and frequency of large intracellular Ca²⁺ currents that are both a hallmark of bubble contact and a quantifiable indication that abnormal intracellular signaling has been triggered. We measured dose dependence curves and fit the resultant data using a modified Black and Leff operational model of agonism. The half maximal inhibitory concentrations of Oxycyte for (i) inhibition of occurrence and (ii) amplitude reduction were 229 ± 49 µM and 226 ± 167 µM, respectively. This investigation shows the preferential gas/liquid interface occupancy of the PFC component of Oxycyte over that of mechanosensing glycocalyx components and validates Oxycyte’s specific surfactant mechanism of action. Further, no lethality was observed for any concentration of this bioinert PFC, as it acts as a competitive allosteric inhibitor of syndecan activation to ameliorate cell response to bubble contact.

Sutherlandia frutescens is widely used in indigenous medicine for the treatment of stress- and anxiety-related disorders, and although anecdotal evidence has been scientifically confirmed, relatively little data are available on its potential mechanisms of action. We manipulated a rodent model of acute psychological stress by acutely administering a low dose (4 mg/kg body mass) of S. frutescens extract 30 min prior to stress exposure (1 h restraint), to elucidate both its central and peripheral mechanisms of action in the context of acute stress. After 1 h of exposure to stress, acute restraint resulted in a significant increase in plasma corticosterone levels (56 ± 33 versus 499 ± 50 ng/ml; P < 0.0001) and anterior pituitary adrenocorticotropic hormone (ACTH) levels (0.066 ± 0.017 versus 0.202 ± 0.033% fluorescent area; P = 0.07), while decreasing hippocampal glucocorticoid receptor (GR) and gamma-aminobutyric acid (GABA)(A)α1 receptor levels (both P < 0.05). While the low dose of S. frutescens administered did not seem to have an effect on the down-stream stress response, it abolished the stress-induced down-regulation of GR, in a manner independent of GABA(A)α1 receptor. Results suggest a non-sedative effect of low-dose S. frutescens and points to central mechanisms of action that is in support of the anecdotal claims for its effectiveness as complimentary treatment in chronic stress-associated diseases.