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Biomedical subjects

R E Brenner

Publications and source records attributed to R E Brenner.

At least 19 recordsLinked to original sources
PubMed2003

Identification, quantification and isolation of mesenchymal progenitor cells from osteoarthritic synovium by fluorescence automated cell sorting.

OBJECTIVE: Identification, quantification and isolation of subpopulations with characteristics of mesenchymal progenitor cells (MPC) from the synovial membrane (SM) from patients with osteoarthritis (OA). METHOD: Cells from the SM of patients with end stage OA who underwent total knee joint replacement were enzymatically isolated. One aliquot was directly analyzed by fluorescence automated cell sorting (FACS) using various combinations of surface markers of bone marrow MPC (CD9, CD44, CD54, CD90, and CD166). Remaining cells were cultivated on plastic, expanded over several passages, analyzed by FACS again and tested for their osteo- and chondrogenic potential. The differentiation was analyzed by immuno-/histochemistry and by RT-PCR for the expression of lineage related marker genes. RESULTS: Using FACS analysis we could show that the relative proportion of subpopulations expressing triplicate combinations of CD9, CD44, CD54, CD90 and CD166 in the SM from OA patients varies between 3 and 10%. Upon cultivation their relative amount markedly increased to values between 24 and 48%. Within the heterogeneous cell populations it was possible to induce osteogenic and chondrogenic differentiation. Initial sorting for CD9/CD90/CD166 triplicate positive cells proved that this subpopulation contains cells with multipotency for mesenchymal differentiation and thus characteristics of MPC. CONCLUSION: Our results show that SM from OA patients contains cells that express typical combinations of MPC surface markers and have the potency of osteogenic and chondrogenic differentiation. Their relative enrichment during in vitro cultivation and the possibility of cell sorting to get more homogenous populations offer interesting perspectives for possible future therapeutic applications.

Aged↗
PubMed2002

Clinical phenotype and molecular diagnosis of multiple epiphyseal dysplasia with relative hip sparing during childhood (EDM2).

We report on a family of 19 individuals over four generations in which 12 members are affected with a variant of multiple epiphyseal dysplasia. Beginning in childhood, the disease leads to pain and stiffness of knees, ankles, elbows and finger joints. Some adult patients repeatedly suffer from free articular bodies resulting in locking of the joint. Finally, affected individuals are prone to the development of early degenerative joint disease. Mutation screening of candidate regions revealed a novel point mutation at position -1 in the COL9A2 exon 3/intron 3 splicing region. This G --> C substitution most probably induces an alteration of the splicing process. Family screening was carried out by both automated sequencing and by digestion of amplicons with BsaWI. We confirmed the nucleotide substitution in eight clinically affected family members as well as in three presymptomatic young children. Electron microscopy showed that the diameter of collagen fibrils from arthroscopically removed free articular bodies of two patients was not obviously different from that of normal articular cartilage. Together with previous reports our results indicate that mutations leading to skipping of exon 3 within the COL3 domain of the alpha2-chain of collagen type IX may be relatively common in patients with a special subtype of multiple epiphyseal dysplasia (MED) in which the hips are not markedly affected at early age (EDM2). In these patients and their families, mutation screening of the candidate regions may help to confirm the diagnosis, lead to appropriate advice for lifestyle and well based genetic counseling.

Adult↗
PubMed2002

Vascular endothelial growth factor stimulates chemotactic migration of primary human osteoblasts.

Recent studies have indicated a critical role for vascular endothelial growth factor (VEGF) during the process of endochondral ossification, in particular in coupling cartilage resorption with bone formation. Therefore, we studied the chemoattractive and proliferative properties of human VEGF-A on primary human osteoblasts (PHO) and compared these data with the effects of human basic fibroblast growth factor (bFGF) and human bone morphogenetic protein-2 (BMP-2). Furthermore, initial experiments were carried out to characterize VEGF-binding proteins on osteoblastic cells possibly involved in the response. For the first time, to our knowledge, we could demonstrate a chemoattractive effect of VEGF-A, but not VEGF-E, on primary human osteoblasts. The effect of VEGF-A was dose-dependent and did not reach a maximum within the concentration range tested (up to 10 ng/mL). The maximal effect observed was a chemotactic index (CI) of 2 at a concentration of 10 ng/mL. bFGF and BMP-2 exhibited maxima at 1.0 ng/mL with CI values of 2.5 and 2, respectively. In addition to its effect on cell migration, VEGF-A stimulated cell proliferation by up to 70%. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the expression of VEGF receptors VEGFR-1 (Flt-1), VEGFR-2 (Kdr), and VEGFR-3 (Flt-4), as well as neuropilin-1 and -2. An in vitro kinase assay failed to demonstrate activation of VEGFR-2 upon stimulation with either VEGF-E or VEGF-A, consistent with the idea that the effect of VEGF-A on primary human osteoblasts is mediated via VEGFR-1. Taken together, our data establish that human osteoblasts respond to VEGF-A, suggesting a functional role for this growth factor in bone formation and remodeling.

Aged↗
PubMed2002

Expression of collagen type I, II, X and Ki-67 in osteochondroma compared to human growth plate cartilage.

In order to characterize the consequences for the process of endochondral ossification we performed an immunohistochemical study and compared the expression of collagen type I, II and X as markers of cartilage differentiation and Ki-67 as a marker of cell proliferation in solitary (7-26 years, n=9) and multiple (11-42 years, n=6) osteochondromas with their expression in human fetal and postnatal growth plates. In fetal and young postnatal controls, we found a thin superficial layer of articular cartilage that stained positive for collagen type I while collagen II was expressed in the rest of the cartilage and collagen type X was restricted to the hypertrophic zone. Osteochondromas from children showed lobular collagen type II-positive areas surrounded by collagen type I. In adults, the separation of collagen type I- and type II-positive areas was more blurred, or the cartilaginous cap was missing. Collagen type X was detected in a pericellular distribution pattern within hypertrophic zones but also deeper between bone trabecula. The proliferative activity of osteochondromas from children younger than 14 years of age was comparable to postnatal growth plates, whereas in cartilage from individuals older than 14 years of age, we could not detect significant proliferative activity.

Adolescent↗
PubMed2001

Effects of dexamethasone on proliferation, chemotaxis, collagen I, and fibronectin-metabolism of human fetal lung fibroblasts.

Premature infants at risk for bronchopulmonary dysplasia (BPD) are often treated with dexamethasone (Dex), which has been shown to suppress inflammatory processes in the lung. To elucidate a possible direct influence on the fibroproliferative component of the disease, we studied the effects of Dex in therapeutic and supratherapeutic dosages (5-50 nmol/L) on proliferation, chemotaxis, procollagen I, and fibronectin metabolism of human fetal lung fibroblasts in vitro. Proliferation was inhibited by Dex in a dose-dependent manner. Chemotactic activity in response to conditioned medium of human fetal fibroblasts also showed a dose-dependent inhibition after pretreatment with Dex. The amount of procollagen I C-terminal propeptide and fibronectin per cell in the cell culture supernatant was increased in the presence of Dex. Our results show that Dex does not uniformly suppress the fibroproliferative activity of human fetal lung fibroblasts, which may explain in part the unsatisfactory long-term effects of Dex treatment in BPD.

Anti-Inflammatory Agents↗
PubMed2001

Classic, atypically severe and neonatal Marfan syndrome: twelve mutations and genotype-phenotype correlations in FBN1 exons 24-40.

Mutations in the gene for fibrillin-1 (FBN1) cause Marfan syndrome, an autosomal dominant disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular system. There is a remarkable degree of clinical variability both within and between families with Marfan syndrome as well as in individuals with related disorders of connective tissue caused by FBN1 mutations and collectively termed type-1 fibrillinopathies. The so-called neonatal region in FBN1 exons 24-32 comprises one of the few generally accepted genotype-phenotype correlations described to date. In this work, we report 12 FBN1 mutations identified by temperature-gradient gel electrophoresis screening of exons 24-40 in 127 individuals with Marfan syndrome or related disorders. The data reported here, together with other published reports, document a significant clustering of mutations in exons 24-32. Although all reported mutations associated with neonatal Marfan syndrome and the majority of point mutations associated with atypically severe presentations have been found in exons 24-32, mutations associated with classic Marfan syndrome occur in this region as well. It is not possible to predict whether a given mutation in exons 24-32 will be associated with classic, atypically severe, or neonatal Marfan syndrome.

Adolescent↗
PubMed2001

Characterization of a human preadipocyte cell strain with high capacity for adipose differentiation.

OBJECTIVE: To develop and to characterize a human preadipocyte cell strain with high capacity for adipose differentiation serving as a model for studying human adipocyte development and metabolism in vitro. METHODS: Cells were derived from the stromal cells fraction of subcutaneous adipose tissue of an infant with Simpson-Golabi-Behmel syndrome (SGBS). Adipose differentiation was induced under serum-free culture conditions by exposure to 10 nM insulin, 200 pM triiodothyronine, 1 microM cortisol and 2 microM BRL 49653, a PPAR gamma agonist. RESULTS: During the differentiation process SGBS cells developed a gene expression pattern similar to that found in differentiating human preadipocytes with a characteristic increase in fat cell-specific mRNAs encoding lipoprotein lipase (LPL), glycero-3-phosphate dehydrogenase (GPDH), GLUT4, leptin and others. Differentiated SGBS cells exhibited an increase in glucose uptake upon insulin stimulation and in glycerol release upon catecholamine exposure. SGBS adipocytes were morphologically, biochemically and functionally identical to in vitro differentiated adipocytes from healthy subjects. However, while preadipocytes from healthy control infants rapidly lost their capacity to differentiate after a few cell divisions in culture, SGBS cells maintained their differentiation capacity over many generations: upon appropriate stimulation 95% of SGBS cells of generation 30 developed into adipocytes. A mutation in the glypican 3 gene was not detected in the patient. Thus, it remains unclear whether the molecular alteration in SGBS cells is also responsible for the high differentiation capacity and further investigations are required. CONCLUSION: The human cell strain described here provides an almost unlimited source of human preadipocytes with high capacity for adipose differentiation and may, therefore, represent a unique tool for studying human fat cell development and metabolism. International Journal of Obesity (2001) 25, 8-15

Adipocytes↗
PubMed2001

Non-insulin dependent diabetes mellitus (NIDDM) and patterns of osteoarthritis. The Ulm osteoarthritis study.

OBJECTIVES: Despite recent advances regarding the role of diabetes with respect to cartilage metabolism, epidemiologic data to quantify the role of diabetes in the development of osteoarthritis (OA) are sparse. METHODS: OA patterns were studied in 809 patients with knee or hip joint replacement due to OA. NIDDM was defined by a history of physician diagnosed diabetes or use of antihyperglycemics. RESULTS: Patients with NIDDM had more often bilateral OA (adjusted odds ratio (OR)=2.2; 95% confidence interval (CI): 0.8-6.4). No association between NIDDM and generalized OA (adjusted OR= 1.0; 95%CI: 0.5-1.9) was observed. CONCLUSIONS: Our results are consistent with the hypothesis that NIDDM might be a potentially important systemic risk factor for knee and hip OA, but chance cannot be ruled out as an alternative explanation.

Aged↗
PubMed2000

Yunis-Varon syndrome: evidence for a lysosomal storage disease.

We present clinical and neuropathological findings in a female infant with Yunis-Varon syndrome (YVS) comprising absence of thumbs and halluces, aphalangia of fingers and toes, hypoplasia of clavicles, severely undermineralized skeleton (especially skull), microcephaly, and multiple nonskeletal anomalies. The patient also had a Dandy-Walker malformation, hydrocephalus, and hypertension, which were not reported previously in YVS. The infant excreted an abnormal unidentified oligosaccharide. The patient died at day 108 with severe neurological impairment. Autopsy showed prominent intraneuronal inclusions with vacuolar degeneration, mainly in the thalamic, dentate nuclei, cerebellar cortex, and inferior olivary nuclei. No storage phenomena were observed in other tissues. These findings strongly suggest that a lysosomal storage disorder is involved in the pathogenesis of Yunis-Varon syndrome.

Abnormalities, Multiple↗
PubMed1999

Perinatal hypophosphatasia: diagnosis and detection of heterozygote carriers within the family.

We report on two families in which one or two children had a severe disorder of skeletal development detected by prenatal ultrasonography. The children died postnatally and showed typical radiological and biochemical findings of perinatal hypophosphatasia. Biochemical analysis revealed a low activity of alkaline phosphatase (AP) and a high value of pyridoxal-5-phosphate (PLP), one of its natural substrates. The screening for mutations of the tissue nonspecific alkaline phosphatase (TNSALP) gene showed homozygosity for a point mutation (G 317 --> D) in the two affected children of the first family. The affected child of the second family was homozygous for a nonsense mutation (R 411 --> X). Family screening revealed that the determination of AP and PLP is helpful for detection of heterozygotes. However, heterozygote children had values of AP in the lower normal range during phases of rapid growth. The determination of PLP proved to be more sensitive in these cases. It should be kept in mind that during the last trimester of gestation there is an increase in maternal AP activity and a normalization of PLP due to placental AP, which is not affected. Therefore, in the course of a prenatal diagnosis in an index case, paternal blood should be analyzed in parallel. For detailed genetic counseling and early prenatal diagnosis in following pregnancies, the possibility of mutation analysis should be used.

Alkaline Phosphatase↗
PubMed1996

In vitro studies on clonal growth of chondrocytes in thanatophoric dysplasia.

Thanatophoric dysplasia (TD) is characterized by a disorganized growth plate with markedly reduced proliferative and hypertrophic cartilage zones. Therefore, we studied in vitro the proliferation rates of articular chondrocytes from five TD patients and age-matched controls in response to bFGF, IGF-I, IGF-II, and TGF-beta 1. In human fetal controls bFGF was the most potent growth factor. Clonal growth the articular chondrocytes in response to bFGF was reduced in two of five TD patients and slightly below the range of controls in a third case. Stimulation of chondrocyte proliferation by IGF I and II was reduced in the patient whose response to bFGF was most markedly impaired. The effect of TGF-beta 1 ranged from normal to slightly elevated values in TD fetuses. These results indicate heterogeneity of the underlying defects in TD. Low proliferative responses of chondrocytes to bFGF and IGF-I/II are likely to play a key role in the pathogenesis of some cases. In two of five patients studied, the mechanisms of bFGF and IGF-signal transduction are candidates for the primary molecular defect.

Cartilage, Articular↗
PubMed1995

Expression of osteoblastic markers in cultured human bone and fracture callus cells.

We compared the expression of osteoblastic markers in cultured human cells isolated from fracture calluses of various histological states of development with that in cells from adult and fetal bone. Adult osteoblasts and all callus cells produced almost exclusively type I collagen, whereas fetal osteoblasts produced also considerable amounts of type III collagen in vitro. 1,25-Dihydroxyvitamin D3 induced the synthesis of osteocalcin in all bone and callus cells but to varying extents. Fetal bone cells and early-stage callus cells synthesized less than 10% the amount of osteocalcin produced by adult bone cells. Late-stage callus cells produced intermediate levels of osteocalcin. Fetal bone cells and early-stage callus cells responded to parathyroid hormone with a less pronounced increase in intracellular cAMP than did adult bone cells. Late-stage callus cells showed the best response to parathyroid hormone. The activity of alkaline phosphatase was highest in fetal bone cells. These observations show that cells isolated from fetal bone and from fracture callus tissues express a pattern of markers clearly relating them to the osteoblastic lineage. On the basis of the different patterns of osteoblastic markers expressed in vitro we conclude that functionally distinct subtypes of osteoblasts do exist in different mineralized tissues and at different developmental stages.

Adult↗
PubMed1995

Glibenclamide stimulates growth of human chondrocytes by IGF I dependent mechanisms.

In rats and men the sulfonylurea glibenclamide augmented skeletal growth. However, with the design of the in vivo studies it was not possible to distinguish whether the growth promoting effect of glibenclamide was mediated by the augmented peripheral insulin or IGF-I levels or if the sulfonylurea had a direct effect on chondrocytes. We therefore measured clonal growth of isolated human chondrocytes in response to glibenclamide in vitro. Cells were isolated from human nose septal cartilage and incubated in a semi-solid medium. Colony formation in response to glibenclamide and IGF-I was determined. Glibenclamide stimulated clonal growth of chondrocytes in a bell-shaped fashion (p < 0.001). 50 ng/ml glibenclamide as the maximal dose augmented colony formation to 144 +/- 9% compared to clonal growth without glibenclamide in the incubation medium, which was designated as 100%. Basal values were obtained with 200 ng/ml glibenclamide. Insulin-like growth factor-I (IGF-I) at 3 ng/ml (118 +/- 4%) and 25 ng/ml (149 +/- 8%, p < 0.02) stimulated growth of chondrocytes. To elucidate the possible mechanism of glibenclamide on clonal growth, chondrocytes were incubated with the sulfonylurea and the IGF-I receptor antibody alpha IR-3. The antibody completely abolished the effect of glibenclamide on colony formation. The results suggest that the growth promoting effect of glibenclamide on isolated human chondrocytes is mediated by IGF-I dependent mechanisms.

Adolescent↗
PubMed1995

Hydroxylation of collagen type I: evidence that both lysyl and prolyl residues are overhydroxylated in osteogenesis imperfecta.

The composition of the collagens secreted into the media of fibroblast cultures of 39 patients with osteogenesis imperfecta (OI) was the same in controls and OI cultures. An abnormal migration pattern of collagens upon SDS-PAGE was evident in one third of the cultures investigated. Lysyl and prolyl hydroxylation of HPLC-purified alpha 1(I) chains was elevated in about 60% of cultures. The degree of hydroxylation was highest in the lethal forms. The extent of lysyl and prolyl hydroxylation showed a strong correlation (r = 0.74, P < 0.001). While high levels of hydroxylation are frequently observed in OI patients, a direct correlation between lysyl or prolyl hydroxylation and fracture rate or growth retardation could not be established.

Adolescent↗
PubMed1994

Bone resorption assessed by immunoassay of urinary cross-linked collagen peptides in patients with osteogenesis imperfecta.

Urinary excretion of type I collagen cross-linked N-telopeptides was studied in 52 children and adolescents with osteogenesis imperfecta (OI) and found to be above the 75th percentile of controls in 44 of the patients. OI patients suffering from fractures during the preceding 6 months had significantly higher values (p < 0.05). In contrast, patients with better motor performance tended to have lower values (p = 0.059). The concentration of urinary type I collagen cross-linked N-telopeptides was positively correlated with urinary calcium excretion (p < 0.05), which was found to be elevated in 20 of the patients. Our results show that during childhood and adolescence in OI not only the synthesis but also the turnover of mature cross-linked type I collagen is disturbed and provide evidence that bone resorption rates are elevated.

Adolescent↗
PubMed1994

Ehlers-Danlos syndrome type VII: phenotype and genotype.

A patient suffering from a severe form of Ehlers-Danlos syndrome is presented (EDS type VII). The presence of bilateral congenital hip dislocation, generalized joint hypermobility and a soft hyperelastic skin with abnormal scarring suggested a specific collagen type I defect. SDS-PAGE analysis of collagens secreted into the medium of fibroblast cultures showed a retarded migration of more than half of the alpha 2(I) chains. CNBr peptide mapping of the HPLC-purified altered chain localized the mutant locus to the N-terminal region of the protein. cDNA analysis of the corresponding gene COL1A2 revealed, in addition to the expected collagen sequence, a transcript missing the entire exon 6. This exon encodes a major crosslinking site within collagen fibres as well as the N-propeptidase cleavage site. The skipping of exon 6 is caused by a splice site mutation substituting an A for a G at the first nucleotide of intron 6.

Base Sequence↗