Formation of osteoblast-like cells from human mononuclear bone marrow cultures

Osteoblast-like cells are commonly found in the vicinity of osteoclasts formed in long-term human bone marrow cultures, and they are believed to be derived from osteogenic cell precursors belonging to the stromal cell system. This paper describes a new culture method for human osteoblasts from the adherent cell population of long-term human mononuclear bone marrow cultures. The cells obtained exhibited all the classic characteristics of osteoblasts. They contained high intracellular concentrations of alkaline phosphatase and they secreted the osteoblast-specific marker bone Gla protein. Collagen production was mainly (95-98%) procollagen type I propeptide and only minute quantities of procollagen type III propeptide were detectable by radioimmunoassay in the conditioned medium. After eight weeks the cells formed a mineralized matrix on exposure to beta-glycerophosphate and ascorbic acid. This system provides a model for the study of osteoblast differentiation in vitro and may form the basis for the use of defined media in bone cell cultures due to the presence of high concentrations of osteoblast precursors.     

Carboxyterminal telopeptide of type I collagen (ICTP) in predicting prognosis in epithelial ovarian cancer.

OBJECTIVE: The aim of this study was to assess the prognostic value of serum carboxyterminal telopeptide of type I collagen (ICTP) in ovarian cancer. Serum CA125 was used as a reference marker. METHODS: Forty-five patients with epithelial ovarian cancer were monitored with serial measurements of serum concentrations of ICTP, a degradation product of type I collagen likely to come about via the matrix metalloproteinase pathway. RESULTS: The patients with a good prognosis had significantly lower serum ICTP concentrations than the patients with a poor prognosis both before the operation and at all the postoperative time points studied (3, 6, 9, 12, 18, and 24 months), whereas a corresponding difference in CA125 was first seen after a 12-month follow-up. In multivariate regression analysis, the 9-month serum ICTP concentration remained the only independent prognostic indicator of all biochemical, clinical, and histological variables. The postoperative serum ICTP concentration did not correlate with the clinical stage, the grade of differentiation, or the presence of residual tumor. In contrast to ICTP, postoperative serum CA125 correlated with the clinical stage and the presence of residual tumor. CONCLUSIONS: Because our ICTP test does not detect defectively cross-linked carboxyterminal telopeptides of type I collagen, which is the predominant form in malignant ovarian tissue, the excess ICTP of ovarian cancer patients must originate from the tissue around the tumor, where the malignancy is causing tissue damage. As an indicator of invasion, the serum ICTP test opens up new possibilities to assess the clinical behavior of ovarian cancer and, in the future, also the effect of possible antiproteinase treatment in ovarian cancer.     

Sensitive immunoassays for the autoantibodies reacting against citrullinated carboxy-terminal telopeptides of type I and type II collagens in patients with rheumatoid arthritis.

We developed sensitive assay methods for autoantibodies recognizing the citrullinated synthetic peptides derived from type I and type II collagens in patients with rheumatoid arthritis (RA). These peptides were tested with the chemiluminescence method (Nichols Advantage System). In 44 RA patients out of 120, the sera showed increased binding of citrullinated synthetic C-telopeptide derived from the alpha1 chain of type I collagen (p=0.003 compared to controls). For a corresponding C-telopeptide pair from the alpha1 chain of type II collagen, 35 patient sera bound the citrullinated peptide more strongly than the arginine peptide, but the difference compared to the controls was not significant (p=0.074). Correlation between the two carboxy-telopeptides was r=0.473 (p<0.001). The anti-CCP assay (antibodies against citrullinated filaggrin sequence-derived peptides) was positive in 59% of our RA patients. There was no relationship between the anti-CCP results and the antibodies against collagen C-telopeptides, but both are increased in RA patients. We demonstrated autoantibodies in RA patients that bound citrullinated C-telopeptides derived from type I and type II collagen antigens. The peptide sequences detected (-YYXA and -YMXA) were different from that based on the cyclic filaggrin antigen (-STXG-, where X represents citrulline).     

Synthesis and degradation of type IV collagen in rat skeletal muscle during immobilization in shortened and lengthened positions

AIM: Type IV collagen is a major protein in basement membranes surrounding and supporting skeletal muscle cells. In the present study, we tested the hypotheses that immobilization down-regulates synthesis and up-regulates degradation of type IV collagen in skeletal muscle. METHODS: mRNA level and concentration of type IV collagen as well as mRNA levels and activities of proteins involved in its degradation were analysed from soleus (SOL), gastrocnemius (GAS) and extensor digitorum longus muscles after immobilization in shortened and lengthened positions for 1, 3 and 7 days. RESULTS: Following immobilization, type IV collagen mRNA level was decreased in SOL and GAS suggesting down-regulated synthesis of this protein. The mRNA level and activity of matrix metalloproteinase-2 (proMMP-2) were increased in all muscles, while the activity of tissue inhibitor of metalloproteinase-2 was decreased in SOL and GAS. These findings reflect an increased capacity for degradation of type IV collagen. CONCLUSIONS: As a consequence of decreased synthesis/degradation ratio immobilization reduced the concentration of type IV collagen in all muscles. The regulation of type IV collagen through synthesis and/or degradation seems, however, to be muscle specific. Immobilization in lengthened position seems to delay and partly decrease the net degradation of type IV collagen.     

Gelsolin amyloid angiopathy causes severe disruption of the arterial wall

Hereditary gelsolin amyloidosis (HGA) is a dominantly inherited systemic disease reported worldwide. HGA is characterized by ophthalmological, neurological, and dermatological manifestations. AGel amyloid accumulates at basal lamina of epithelial and muscle cells, thus amyloid angiopathy is encountered in nearly every organ. HGA patients have cardiovascular, hemorrhagic, and potentially vascularly induced neurological problems. To clarify pathomechanisms of AGel angiopathy, we performed histological, immunohistochemical, and electron microscopic analyses on facial temporal artery branches from 8 HGA patients and 13 control subjects. We demonstrate major pathological changes in arteries: disruption of the tunica media, disorganization of vascular smooth muscle cells, and accumulation of AGel fibrils in arterial walls, where they associate with the lamina elastica interna, which becomes fragmented and diminished. We also provide evidence of abnormal accumulation and localization of collagen types I and III and an increase of collagen type I degradation product in the tunica media. Vascular smooth muscle cells appear to be morphologically and semi‐quantitatively normal, only their basal lamina is often thickened. In conclusion, angiopathy in HGA results in severe disruption of arterial walls, characterized by prominent AGel deposition, collagen derangement and severe elastolysis, and it may be responsible for several, particularly hemorrhagic, disease manifestations in HGA.     

High dietary intake of retinol leads to bone marrow hypoxia and diaphyseal endosteal mineralization in rats

Vitamin A (retinol) is the only molecule known to induce spontaneous fractures in laboratory animals and we have identified retinol as a risk factor for fracture in humans. Since subsequent observational studies in humans and old animal data both show that high retinol intake appears to only have small effects on bone mineral density (BMD) we undertook a mechanistic study of how excess retinol reduces bone diameter while leaving BMD essentially unaffected. We fed growing rats high doses of retinol for only 1 week. Bone analysis involved antibody-based methods, histology, pQCT, biomechanics and bone compartment-specific PCR together with Fourier Transform Infrared Spectroscopy of bone mineral. Excess dietary retinol induced weakening of bones with little apparent effect on BMD. Periosteal osteoclasts increased but unexpectedly endosteal osteoclasts disappeared and there was a reduction of osteoclastic serum markers. There was also a lack of capillary erythrocytes, endothelial cells and serum retinol transport protein in the endosteal/marrow compartment. A further indication of reduced endosteal/marrow blood flow was the increased expression of hypoxia-associated genes. Also, in contrast to the inhibitory effects in vitro, the marrow of retinol-treated rats showed increased expression of osteogenic genes. Finally, we show that hypervitaminotic bones have a higher degree of mineralization, which is in line with biomechanical data of preserved stiffness in spite of thinner bones. Together these novel findings suggest that a rapid primary effect of excess retinol on bone tissue is the impairment of endosteal/marrow blood flow leading to hypoxia and pathological endosteal mineralization.     

Effect of the menopause and hormone replacement therapy on the carboxy-terminal pyridinoline cross-linked telopeptide of type I collagen

We investigated the effect of the menopause and postmenopausal hormone replacement therapy (HRT) on the serum concentration of carboxy-terminal pyridinoline cross-linked telopeptide of type I collagen (ICTP), a potential new biochemical marker of bone resorption. A group of 44 healthy postmenopausal women, aged 45–54 years, had about 19% higher serum ICTP than did a group of 42 healthy premenopausal women aged 35–50 years (3.6±0.8 µg/l v 3.0±0.7 µg/l (mean ±SD);p<0.01), although there was a large overlap in the values. The 44 postmenopausal women also participated in a longitudinal clinical study, in which 20 received HRT and 24 received a placebo. Compared with the placebo group, those who received HRT had a significant (p<0.05) decrease in ICTP of about 12% at the end of 1 year of treatment, but again there was considerable overlap in the values. The menopause-and HRT-induced changes in ICTP were less than those seen in serum osteocalcin, serum total alkaline phosphatase, and fasting urinary excretion of hydroxyproline, calcium, pyridinoline and deoxypyridinoline. We conclude that the menopause increases and HRT decreases ICTP, although these changes are less pronounced than those seen in other biochemical markers of bone turnover.     

Stromal cells can be cultured and characterized from diagnostic bronchoalveolar fluid samples obtained from patients with various types of interstitial lung diseases

Increased proliferation of stromal cells is a typical feature encountered in several lung diseases. The objective of this study was to evaluate the success of standardized process for culturing stromal cells from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples collected from various patients and to characterize the cultured cells. Small volumes (average 15 mL) of BAL fluid samples were collected from 98 patients who underwent bronchoscopy and BAL for diagnostic purposes. The cells were cultured in vitro and characterized by immunohistochemistry, electron microscopy, flow cytometry and differentiation tests. Cells could be cultured from 62% of samples with the success rate varying with the disease (p = 0.003). Cultures from samples of the patients with idiopathic pulmonary fibrosis, non‐specific interstitial pneumonia, connective tissue disorder associated interstitial lung disease and allergic alveolitis had a higher success rate than samples derived from control lung (p < 0.001, 0.03, 0.03 and 0.044, respectively). Smokers had a higher success rate compared with non‐smokers (p = 0.035). The cultured cells were fibroblasts or myofibroblasts, but shared also similarities with progenitor‐type cells. The study shows that mesenchymal cells can be cultured and studied from small volumes of diagnostic BAL fluid samples from patients with several different types of lung diseases.