A novel marker for assessment of liver matrix remodeling: an enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M).

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.

Advances in osteoclast biology resulting from the study of osteopetrotic mutations.

Osteopetrosis is the result of mutations affecting osteoclast function. Careful analyses of osteopetrosis have provided instrumental information on bone remodeling, including the coupling of bone formation to bone resorption. Based on a range of novel genetic mutations and the resulting osteoclast phenotypes, we discuss how osteopetrosis models have clarified the function of the coupling of bone formation to bone resorption, and the pivotal role of the osteoclast and their function in this phenomenon. We highlight the distinct possibility that osteoclast activities can be divided into two separate avenues: bone resorption and control of bone formation.

Reversion of mtDNA depletion in a patient with TK2 deficiency.

Mutations in the thymidine kinase 2 (TK2) gene cause a myopathic form of the mitochondrial DNA depletion syndrome (MDS). Here, the authors report the unusual clinical, biochemical, and molecular findings in a 14-year-old patient in whom pathogenic mutations were identified in the TK2 gene. This report extends the phenotypic expression of primary TK2 deficiency and suggests that factors other than TK2 may modify expression of the clinical phenotype in patients with MDS syndrome.

Effects of taurine on polymorphonuclear phagocytosis activity in burned patients.

This study determines the effects of taurine (Tau) on phagocytosis of polymorphonuclear neutrophils (PMN) isolated from normal subjects (n = 41) and severely burned patients (n = 20). Phagocytosis was measured by nitroblue of tetrazolium (NBT) reduction in samples with and without latex bead stimulation. Taurine was added at doses of 0.2, 0.4, 0.8 and 1.6 mM to stimulated samples. In control cells there were statistically significant increases in phagocytosis after addition of Tau 0.8 mM and 1.6 mM to as compared to samples without Tau addition (295 +/- 23% and 330 +/- 35% vs. 248 +/- 18%; mean +/- S.E.; p < 0.05). A statistically significant increase in phagocytosis was observed in cells from the burned population after addition of Tau 1.6 mM (288 +/- 38% vs. 198 +/- 13%; mean +/- S.E.; p < 0.05). No changes in phagocytosis were found in cells from a subgroup of burn patients (n = 13) followed over 7, 15 and 21 days. These results indicate that taurine supplementation in vitro at doses of 0.8 to 1.6 mM improves the phagocytic capacity of neutrophils in healthy subjects and in patients with severe burn injury, mainly when neutrophil function is unaltered.

Role of putrescine in cell proliferation in a colon carcinoma cell line.

OBJECTIVES: Putrescine, the precursor for higher polyamine biosynthesis, is necessary for cell growth in mammals. Ornithine decarboxylase (ODC) activity and polyamine production are increased in neoplastic cells. Using colon cancer cell line derived from a tumor with high metastatic potential (CT-26), our objective was to study the effect of exogenous putrescine on ODC regulation, polyamine metabolism, and cell proliferation. METHODS: Cells cultured with fetal calf serum were exposed to 100, 550, and 1000 microM putrescine for 24 h. RESULTS: Intracellular free putrescine, determined by high-performance liquid chromatography, showed a statistically significant increase in exposed cells compared with controls and a significant correlation with levels of the metabolite present in the medium (r = 0.93; P < 0.001). Bromodeoxyuridine incorporation into newly synthesized DNA, a marker of cell proliferation, showed a statistically significant increase in the three putrescine groups as opposed to the control group. In samples with added aminoguanidine, significant increases in DNA synthesis were observed in the 550- and 1000-microM putrescine groups as opposed to the control group. Spermidine and spermine intracellular contents in all three putrescine-treated groups remained below control levels. No statistical differences in ODC enzymatic activity or ODC mRNA content were observed. Newly incorporated putrescine stimulated colon tumor cell growth. CONCLUSIONS: Because neither enhanced conversion into the higher polyamines nor aminoguanidine inhibition of proliferation was observed, we suggest that this effect can be attributed to the putrescine molecule itself.

Intestinal ornithine decarboxylase in short bowel syndrome patients with oral diet.

The major consequence of extensive intestinal resection is loss of absorptive surface area, which results in malabsorption of nutrients; this condition is known as short-bowel syndrome (SBS). Patients with extensive small intestinal resection and colectomy leading to jejunostomy have the most severe SBS. Ornithine decarboxylase (ODC) plays a central role in cell proliferation and in the process of gut adaptation. Polyamine synthesis in crypt cells mediates the action of extracellular growth factors on DNA synthesis and mitotic activity. The aim of this study was to examine ODC expression and activity, diamine oxidase (DAO) activity and polyamine levels in the jejunal mucosa and red blood cells of SBS patients with a jejunostomy. The study group consisted of 6 patients (4 men and 2 women, mean age 55.8+/-9.8 years), who had undergone extensive small bowel resection and colectomy. All patients were maintained on cyclic parenteral nutrition and non-restricted oral nutrition. Two groups of patients operated on for unrelated reasons were included as the jejunum control group (n=6) and the ileum control group (n=13). Non statistical differences were observed in polyamine levels of red blood cells versus the control group (spermidine: 21.0+/-3.6 vs. 17.7+/-1.1 and spermine: 17.1+/-8.6 vs. 13.2+/-1.6 nmol/ml RBC, respectively). No significant decreases in putrescine and spermidine levels were observed between the groups, but spermine levels in SBS jejunum were significantly lower than the controls (P<0.05). In SBS patients a significant decrease in ODC and DAO activity were observed vs jejunum. A significant decrease in ODC-mRNA abundance was found for the SBS patients as compared to the two control groups (P<0.05). These results suggest that in SBS patients with jejunostomy intestinal adaptation may be impaired.

[Polyamines in the gastrointestinal tract]. / Poliaminas en el tracto gastrointestinal.

Polyamines are extremely important for cell growth, a fact that is reflected in the strict control of their synthesis and breakdown. In the small intestine, the polyamines play a fundamental role in all processes involving tissue regeneration, such as healing of stress-related ulcers, post-hepatectomy hepatic regeneration, adaptation syndrome after fasting and all processes with enterocyte hyperplasia or hypertrophy. In the gastrointestinal tract, there is a polyamine gradient in the villi-crypt cell axis and along the digestive tube, itself; the segments with greatest luminal content are the jejunum and the colon. Endogenous polyamine synthesis is stimulated by the diet and normal bacterial flora, which, in turn, regulate the amount and concentration of polyamines. Other hormonal and active peptide components (e.g. gastrin, epidermal growth factor, growth hormone) also have an influence on the pathway of polyamine synthesis. The interaction of these factors as related to the intestinal adaptive response is reviewed.

In vitro effects of melatonin on cell proliferation in a colon adenocarcinoma line.

The effect of melatonin on inhibition of cell growth was studied in CT-26, a murine colon carcinoma-derived cell line. Cells growing in exponential phase were exposed to low (10(-7)-10(-10) M) and high doses (1, 2 and 3 x 10(-3) M) of melatonin during 24 h. Synthesis of DNA was measured by 5-bromo-2'-deoxyuridine incorporation. There was no effect at low doses, but a statistically significant correlation was found between the decrease in DNA synthesis and the dose of melatonin used (r = -0.52, P < 0.001). This implied the following percentages of inhibition: 1 mM, 22%; 2 mM, 25%; 3 mM, 47%. Potential cell membrane damage by high doses of melatonin was investigated by lactate dehydrogenase measurement and no significant levels were observed. Analysis with a single saturation technique showed no detectable oestradiol receptors in this cell type; therefore, we can assume that the effects occurring with the addition of melatonin were not mediated by modulation of this hormone on oestrogen receptors. The decreases in cell growth were attributed to a moderate, but significant antiproliferative action of melatonin on this non-hormone-dependent cell line.