The role of transforming growth factor-beta1 and oxidative stress in podoconiosis pathogenesis.

BACKGROUND: Podoconiosis (endemic nonfilarial elephantiasis) occurs in susceptible individuals who go barefoot in regions of irritant volcanic soil. Silicate particles absorbed via the skin are thought to induce an inflammatory process and a consequent endolymphangitis of the lower leg lymphatics. OBJECTIVES: To establish which oxidative stress biomarkers play a part in the inflammatory process, and to test whether transforming growth factor (TGF)-beta1 also has a pathogenetic role. PATIENTS AND METHODS: We enrolled 50 patients with early clinical stage disease, 43 patients with advanced stage disease and 35 local healthy controls. Oxidative stress biomarkers included serum total peroxides (TP), total antioxidant capacity (TAC), total nitrate plus nitrite (TN), malondialdehyde (MDA) and total superoxide dismutase (SOD) activity. The oxidative stress index (OSI) was also determined. Serum total TGF-beta1 was assayed using sandwich enzyme-linked immunosorbent assay. RESULTS: Compared with healthy controls, patients with early stage disease showed significantly higher mean levels of TP (P < 0.001), MDA (P < 0.05) and OSI (P < 0.01); and significantly lower mean concentrations of SOD (P < 0.001) and TGF-beta1 (P < 0.001). Mean levels of TGF-beta1 were even lower among patients with advanced stage disease (P < 0.001). Mean TAC levels were significantly lower among patients with advanced disease than either other group (P < 0.001). CONCLUSIONS: This is the first study, to our knowledge, to attempt to elucidate the molecular pathogenetic events in podoconiosis. We conclude that TGF-beta1 may have a pathogenetic role, with oxidative stress playing a minor role in the early stages of disease.

A study on biomarkers, cytokines, and growth factors in children with burn injuries.

Background. Burns are a unique injury which not only is devastating for the patients but also puts a great burden on society by consuming enormous health care resources. Despite improvements in burn wound care and treatment, understanding the role of pro-inflammatory and anti-inflammatory cytokines as well as the mechanisms responsible for the healing process remains to be clarified. Although leptin is regarded as a circulating hormone, it can exert a direct effect on T cells and monocytes, causing the release of cytokines. It may induce angiogenesis or influence angiogenic factors. The aim of the present work is to determine serum levels of leptin, tumour necrosis factor a (TNFa), interleukin-6 (IL-6), basic fibroblast growth factor (bFGF), procalcitonin (PCT), and C-reactive protein (CRP) in a group of children with thermal burns and to determine the changes in these parameters in relation to the duration of hospital stay, the presence of infection, and the total body surface area (TBSA) burned. Patients and methods. The study included 42 children with burns (22 males and 20 females; age range, 2 months to 7 years). The study also included 26 age-matched controls. Besides full clinical assessment, including assessment of TBSA burned and the presence or absence of sepsis, all the patients and controls had the following investigations performed: complete blood count, CRP, IL-6, TNFa, PCT, serum leptin, bFGF, and transforming growth factor a (TGFa). Results. The fatality rate in this study was 28.6%. Burn cases as a whole showed significantly higher values of white blood cells (WBC), CRP, PCT, TNFa, IL-6, leptin, bFGF, and TGFa than controls. Cases with sepsis showed significantly higher values of WBC, CRP, PCT, TNFa, and IL-6 than cases without sepsis. They showed significantly lower values of TGFa than cases without sepsis. Patients with larger TBSA (>30%) showed significantly higher levels of WBC, CRP, PCT, TNFa, IL-6, and leptin than cases with smaller TBSA. They showed significantly lower levels of bFGF and TGFa than patients with smaller TBSA. Non-survivors showed significantly higher levels of WBC, CRP, PCT, TNFa, and IL-6 than survivors. They showed significantly lower levels of leptin, bFGF, and TGFa than survivors. Correlation studies showed a significant positive correlation between TBSA and each of IL-6, TNFa, and leptin. Conclusions. Cytokines and leptin increased in severely burned patients, cases associated with sepsis, and in fatal cases, while bFGF and TGFa levels were lower in severe cases. This may point to impaired healing in such cases and to their poorer prognosis. Recommendations. It is highly recommended to monitor immunological parameters such as PCT and/or IL-6 for early detection of infectious complications following thermal injury. Leptin can be regarded as a novel treatment modality to diminish burn-induced inflammation, reduce post-burn immune dysfunction, and enhance burn healing.

Retinoic acid can induce markers of endocrine transdifferentiation in pancreatic ductal adenocarcinoma: preliminary observations from an in vitro cell line model.

BACKGROUND AND HYPOTHESIS: The pancreatic ductal adenocarcinoma (HPAF) cells have a multipotent stem cell potential. It was hypothesised that all-trans-retinoic acid (atRA) can induce transdifferentiation of these cells into cells with an endocrine phenotype. MATERIAL AND METHODS: To explore this hypothesis, an in vitro system of cells was established. Some cells were treated with atRA at concentrations of 100 nmol/l (non-apoptosis-inducing) and 5 micromol/l (apoptosis-inducing) and harvested. Cells were examined for cell cycle kinetics, apoptosis (terminal deoxynucleotidyl transferase assay and p53 protein expression) and immunomorphological features of redifferentiation (MUC1 and DUPAN-2) and endocrine transdifferentiation (insulin, somatostatin, glucagon, neurone-specific enolase) by using immunoperoxidase staining methods. Levels of insulin, transforming growth factor (TGF) beta2, TGFalpha and epidermal growth factor receptor (EGFR) were measured by enzyme-linked immunosorbent assay (ELISA). The vehicle-treated cells served as a control group. RESULTS: When compared with untreated cells, cells treated with 100 nmol/l and 5 micromol/l atRA were observed to show (1) decreased proliferative activity (cpm) as indicated by decreased incorporation of thymidine labelled with hydrogen-3; (2) cell cycle arrest; (3) increased apoptotic activity associated with p53 protein overexpression; (4) upregulated expression of the transdifferentiation and redifferentiation markers; (5) morphological changes indicative of transdifferentiation (increased cell size and appearance of dendrites); (6) decreased production of EGFR; (7) upregulation of TGFalpha and TGFbeta2; and (8) increase in basal and glucose-induced insulin secretion. CONCLUSIONS: Functional endocrine transdifferentiation can be induced in HPAF lines by atRA. Further investigations are mandated to explore the underlying mechanisms of this transdifferentiation and to explore its in vivo extrapolation.

Establishment of human pancreatic ductal cells in a long-term culture.

Cultivation and preservation of human pancreatic ductal cells have remained a challenge. With a defined culture medium and refinement of culturing techniques, we have been able to maintain human pancreatic ductal cells without any genetic manipulation in culture for more than 16 months. Freshly isolated ductal fragments were placed on a rocker in M3:5 medium free of collagen for 14 days to remove fibroblasts and endocrine cells before allowing them to attach. The cells produced an excessive amount of mucin and expressed the duct specific cytokeratins (CK) 7 and 19, DU-PAN2, CA19-9, carbonic anhydrase II (CA II), and secretin receptors. During the course of the culture, however, the cells gradually lost the expression of CA II, secretin receptors, DU-PAN2, and CA 19-9 and assumed an undifferentiated phenotype, which showed an upregulation of transforming growth factor alpha (TGFalpha) and epidermal growth factor receptor (EGFR), an increase in the expression of Ki-67, and an increased binding to Phaseolus vulgaris leucoagglutinin (PHA-L) and tomato lectin. These ductal cells present a useful source with which to study physiologic aspects of ductal cells including differentiation.

Retinoic acid-dependent transforming growth factor-beta 2-mediated induction of MUC4 mucin expression in human pancreatic tumor cells follows retinoic acid receptor-alpha signaling pathway.

The MUC4 mucin is considered as the homologue of rat sialomucin complex (SMC, rat Muc4) due to its similar structural organization. Like SMC, MUC4 may also exist as two subunits: a mucin type unit known as MUC4alpha and a growth factor-like transmembrane subunit, MUC4beta. The expression of MUC4 in normal human pancreas is not detectable, but it is highly expressed in pancreatic tumor cells. In the present study, we investigated the regulation of MUC4 expression in human pancreatic tumor cells CD18/HPAF, exhibiting a high level of MUC4 transcripts and protein. When these cells were adapted to grow in the serum-free medium (CD18/HPAF-SF), the MUC4 expression was undetectable. Among several serum constituents, all-trans-retinoic acid (RA) induced the expression of MUC4 transcripts in a concentration- and time-dependent manner. The RA-mediated increase in the level of the MUC4 transcript coincided with an increased expression of transforming growth factor-beta2 (TGF-beta2) transcript. The antagonist of the retinoic acid receptor (RAR)-alpha (Ro41-5253) abrogated the expression of MUC4 and TGF-beta2 induced by RA. The exogenous addition of TGF-beta2 also increased the MUC4 expression. The TGF-beta-neutralizing antibody blocked the RA-induced as well as TGF-beta2-mediated MUC4 expression. In conclusion, induction of MUC4 expression in pancreatic carcinoma by RA is mediated through the RAR-alpha signaling pathway, and TGF-beta2 may serve as an interim mediator of this regulated expression.

Biologic instability of pancreatic cancer xenografts in the nude mouse.

Tumor transplants into nude mice (NM) may reveal abnormal biological behavior compared with the original tumor. Despite this, human tumor xenografts in NM have been widely used to study the biology of tumors and to establish diagnostic and therapeutic modalities. Clearly, precise differences in the biology of a given tumor in human and in NM cannot be assessed. We compared the growth kinetics, differentiation pattern and karyotype of an anaplastic Syrian hamster pancreatic cancer cell line in NM and in allogenic hamsters. As with the original tumor, transplants in hamsters grew fast, were anaplastic and expressed markers related to tumor malignancy like galectin 3, TGF-alpha and its receptor EGFR at high levels. However, tumors in the NM were well-differentiated adenocarcinomas, grew slower, had increased apoptotic rate and had a high expression of differentiation markers such as blood group A antigen, DU-PAN-2, carbonic anhydrase II, TGF-beta(2) and mucin. Karyotypically, the tumors in the NM acquired additional chromosomal damage. Our results demonstrate significant differences in the morphology and biology of tumors grown in NM and the allogenic host, and call for caution in extrapolating data obtained from xenografts to primary cancer.

Lipoxygenase inhibition induced apoptosis, morphological changes, and carbonic anhydrase expression in human pancreatic cancer cells.

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism. We previously reported that 5-LOX and 12-LOX are upregulated in human pancreatic cancer cells and that blockade of these enzymes abolishes pancreatic cancer cell growth. The present study was aimed at evaluating the effect of LOX inhibition on differentiation and apoptosis in pancreatic cancer cells in parallel with growth inhibition. Four human pancreatic cancer cell lines, PANC-1, MiaPaca2, Capan2, and HPAF, were used. Apoptosis was evaluated by three separate methods, including DNA propidium iodide staining, DNA fragmentation, and the TUNEL assay. Morphological changes and carbonic anhydrase activity were used to determine the effect of LOX inhibitors on differentiation. The general LOX inhibitor NDGA, the 5-LOX inhibitor Rev5901, and the 12-LOX inhibitor baicalein all induced apoptosis in all four pancreatic cancer cell lines, as confirmed by all three methods, suggesting that both the 5-LOX and 12-LOX pathways are important for survival of these cells. Furthermore, NDGA, Rev5901, or baicalein resulted in marked cellular morphological changes in parallel with increased intracellular carbonic anhydrase activity, indicating that LOX blockade induced a more differentiated phenotype in human pancreatic cancer cells. Together with our previous findings, this study suggests that perturbations of LOX activity affect pancreatic cancer cell proliferation and survival. Blockade of LOX enzymes may be valuable for the treatment of human pancreatic cancer.

Optimization of treatment conditions for studying the anticancer effects of retinoids using pancreatic adenocarcinoma as a model.

Retinoids are natural differentiation-inducing compounds that are promising as anticancer agents. Cancer cell lines are valuable in the investigation of the potential of retinoids for the treatment of specific cancers. However, using different treatment conditions but the same cell lines, investigators have produced markedly contradictory results for the effectiveness of retinoids. The present study examined different factors in the treatment conditions that may have masked or interfered with the effects of retinoids and, thereby, resulted in this conflict. Our studies revealed that the effects of retinoids on cancer cell proliferation were influenced by serum, the choice of vehicle (DMSO vs ethanol) and its concentration, phenol red, the degree of cellular confluence, and the method of assessing proliferation (cell number or [3H]thymidine uptake vs the MTT assay). Optimized conditions were the use of serum-free, ethanol-free, and phenol red-free media, investigating cells in the log phase of growth, using