Interleukin-1beta upregulates MMP-9 expression in stromal cells of human giant cell tumor of bone.

Giant cell tumor (GCT) of bone is a progressive, potentially malignant process that destroys skeletal tissue. It consists of multinucleated giant cells, which are hypothesized to be derived from a monocyte/macrophage lineage and mononuclear stromal cells, and the precise relationship of these cells is not fully understood. Recently, we demonstrated that the production of matrix metalloproteinase-9 (MMP-9) in GCT stromal cells is regulated by certain factor(s) secreted by the multinucleated giant cells. In the present study, we evaluated for the presence of interleukin-1beta (IL-1beta) and attempted to establish its possible role for the induction of MMP-9 in GCT stromal cells. Using enzyme-linked immunosorbent assay (ELISA), we have demonstrated that the primary GCT cultures secrete both IL-1beta and MMP-9. The addition of monoclonal antibody (mAb) against IL-1beta partially abrogated, but did not abolish, MMP-9 expression. Our results on gelatin zymography, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescence showed that GCT stromal cells did not express MMP-9, although treatment with IL-1beta induced MMP-9 expression in a dose-dependent manner, and the secretion peaked 24 h after stimulation and then plateaued. Studies with cycloheximide, a protein synthesis inhibitor, demonstrated that de novo protein synthesis is required for IL-1beta induced MMP-9 expression. Moreover, nuclear run-on analysis has revealed that IL-1beta significantly increased MMP-9 gene transcription in GCT stromal cells. The data suggest that IL-1beta secreted by the multinucleated giant cells in GCT may be one of the factors responsible for the induction of MMP-9 at the transcriptional level in GCT stromal cells in vivo. We conclude that GCT has a self-stimulatory system for the production of MMP-9, and the ability of stromal cells to produce MMP-9 with appropriate stimuli, such as IL-1beta, and possibly in concert with other cytokines may contribute to the aggressive and potentially malignant behavior of GCT.

Transcriptional regulation of MMP-9 expression in stromal cells of human giant cell tumor of bone by tumor necrosis factor-alpha.

We determined whether certain factor(s) secreted by multinucleated giant cells, which is of monocyte/macrophage lineage in giant cell tumor of bone (GCT), regulate the induction of matrix metalloproteinase (MMP)-9 expression in mononucleated stromal cells. Our data derived using enzyme linked immunosorbant assays (ELISAs) suggest that the GCT cells in primary culture produce both MMP-9 and tumor necrosis factor-alpha (TNF-alpha). Further, the MMP-9 expression in GCT primary cultures was partially abrogated by neutralizing antibody to TNF-alpha, suggesting that TNF-alpha secretion by the multinucleated giant cells may be one of the factors responsible for the production of MMP-9 by the stromal cells in vivo. In order to confirm this we examined the role of TNF-alpha on the induction of MMP-9 expression in bone GCT stromal cells. These cells express MMP-2, but not MMP-9. However, treatment of these cells with TNF-alpha induced the expression of MMP-9 in a concentration-dependent manner. Kinetic experiments revealed that the secretion of MMP-9 peaked 12 h post TNF-alpha stimulation. Immunofluorescence studies confirmed the expression of MMP-9 after stimulation of GCT stromal cells with TNF-alpha. Further, TNF-alpha-induced MMP-9 expression was completely blocked with neutralizing antibody to TNF-alpha, thereby demonstrating the specificity. In addition, the induction of MMP-9 expression by TNF-alpha was completely abrogated in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that de novo protein synthesis may be required. Nuclear run-on analysis demonstrated that treatment of GCT stromal cells significantly enhanced the MMP-9 gene transcription. Together, our data suggest that TNF-alpha secreted by the multinucleated giant cells up-regulates MMP-9 expression in GCT stromal cells by the induction of certain transcription factors, which in turn enhanced the rate of transcription of MMP-9 gene. These studies also suggest the existence of an essential cell-cell interaction in the regulation of MMP-9 expression in GCT.

Accelerated linear growth and advanced bone age in Sotos syndrome is not associated with abnormalities of collagen metabolism.

OBJECTIVES: To investigate whether the advanced bone age in Sotos syndrome is associated with alterations in type I collagen metabolism in bone. DESIGN AND METHODS: The metabolism of collagen was studied by analyzing the production, gene expression and degradation of type I collagen in dermal fibroblast strains from patients with Sotos syndrome and comparing them with fibroblasts from age-matched healthy subjects. Collagen production was determined as collagenase digestible radioactivity and collagen mRNA levels were measured by RT-PCR. Collagen degradation was assessed by specific collagenase assay and gelatin zymography. To determine the structural defects in type I collagen, the newly synthesized proteins were analyzed by SDS-PAGE before and after proteolytic digestion with pepsin. RESULTS: In the present study, we have demonstrated that the collagen production, secretion and degradation in Sotos syndrome is comparable to controls. In addition, no qualitative differences in mRNA transcripts for type I collagen were detected between the control and Sotos syndrome fibroblasts. The secretion and intracellular accumulation of procollagen is also comparable to controls. The analysis of both procollagen and collagen on SDS-PAGE did not exhibit any major structural changes as compared with controls. CONCLUSIONS: Our results on several aspects of collagen metabolism have demonstrated for the first time that collagen, the most abundant of mammalian proteins and the major constituent of bone, is normal in patients with Sotos syndrome. Therefore, it appears that the advanced bone age and accelerated linear growth seen in patients with Sotos syndrome may not be attributed to inherent abnormalities of collagen metabolism. The etiology and the pathogenesis of Sotos syndrome still remains unclear.

Regulation of MMP-9 (92 kDa type IV collagenase/gelatinase B) expression in stromal cells of human giant cell tumor of bone.

Matrix metalloproteinases (MMPs) play an important regulatory role in tissue morphogenesis, cell differentiation, tumor invasion and metastasis. Several authors have reported a direct correlation between the production of 72 kDa (MMP-2) and 92 kDa (MMP-9) type IV collagenases/gelatinases and the metastatic potential of cancer cells. Recently, we have identified the expression of both MMP-2 and MMP-9 in primary cultures of human giant cell tumor (GCT) of bone in vitro, and in tissue extracts in vivo. Interestingly, MMP-9 is not secreted by late-passaged GCT cells. It is possible that the production of MMP-9 is regulated by certain factor(s) secreted by the multinucleated giant cells in the primary culture. In order to test this hypothesis, the effect of primary-culture-conditioned medium on the expression of MMP-9 by late-passaged mononuclear stromal cells was examined. Adding conditioned medium from the primary GCT culture to the late-passaged stromal cells induced MMP-9, as evidenced by the presence of lytic bands at M(r) 92,000 and 72,000 on a gelatin zymogram. These enzyme activities were inhibited by EDTA, a well-known inhibitor of the MMPs. We confirmed these results by Western blotting using specific antibodies and RT-PCR for MMP-2 and MMP-9. Immunofluorescence studies with specific antibodies to MMP-9 further confirmed its expression by the passaged stromal cells cultured in the primary-culture-conditioned medium. The data indicate that MMP-2 and MMP-9 are produced by the mononuclear stromal cells when cultured in GCT primary-culture-conditioned medium. This suggests that multinucleated giant cells in primary cultures secrete a factor(s) that stimulates stromal cells to produce MMP-9, which, in turn, may contribute to the aggressive behavior of GCT.

Institutional change. Experiences in two departments.

Though the principle may seem simple or fundamental it has been our experience that the best way to develop clinician-educators in an academic setting is to value their contributions. This means that those contributions must lead to promotion, they should be valued by colleagues, they must be valued by the administration and the chairman, and they must be considered when determining faculty salary. As faculty members perceived that they were valued for teaching and clinical service. and would not be punished for the amount of time they were spending in these endeavors, there was a clear group of faculty who came forward to take on a primary teaching role. This group was not limited to general pediatricians or ambulatory pediatricians, but included some specialists who felt that their pediatric background was sufficient for them to teach in a primary care setting. Two of our leading teachers in the generalist curriculum are specialists in nephrology and hematology/oncology. Although this requires them to go back and increase their knowledge in general pediatrics, it is far less difficult according to these faculty members than they expected. Our specialists continue to maintain their specialty practices, but have oriented their didactic lectures and clinical teaching to specialty and general aspects of pediatrics. It is not difficult to teach about parenting and psychosocial skills when describing a complicated specialty patient and to orient the students and residents to the general care of such a patient. Although the majority of strategies described in this article deal with departmental and college initiatives, the reason that these strategies have become an integral part of the Department of Pediatrics is the changing health care environment in Nebraska. Managed care has mandated that physicians be more flexible and be willing to take on a primary care role within their specialty. This has made the transition for many faculty much easier and has been reinforced by financial reimbursement for their services. The transition would not have been as easy had there been no movement of the community toward primary care, or no shift in the university's interest in primary care as a mechanism for providing sufficient patient numbers to fulfill our teaching missions. Clinical research has become the area of focus for many of the primary care physicians and some specialists in the past few years, and the university is in the process of developing a clinical research center to allow for outpatient studies. Although the strategies summarized are specific to the University of Nebraska Medical Center, many of the principles could be adapted to other teaching programs. The most basic element is to tie reward and recognition to efforts in primary care.

The neuroimaging findings in Sotos syndrome.

We reviewed the neuroimaging studies of 40 patients with classic Sotos syndrome. The studies consisted of CT scans only in 4 patients and one or more MRI scans in 36 patients. The diagnosis of Sotos syndrome was made using well-established clinical criteria. The neuroimaging studies of each patient were evaluated subjectively by visual inspection and the chief findings were tabulated and grouped into five categories: 1) ventricular abnormalities, 2) extracerebral fluid spaces, 3) midline abnormalities, 4) migrational abnormalities, and 5) others. The most common abnormality of the cerebral ventricles was prominence of the trigone (90%), followed by prominence of the occipital horns (75%) and ventriculomegaly (63%). The supratentorial extracerebral fluid spaces were increased for age in 70% of the patients and the fluid spaces in the posterior fossa were increased in 70% also. A variety of midline abnormalities were noted but anomalies of the corpus callosum were almost universal. Gray matter heterotopias occurred in only 3 (8%) of 36 patients. Periventricular leukomalacia, presumably the result of prenatal or perinatal difficulties and unrelated to the basic condition, was the most common of the miscellaneous other abnormalities noted. The neuroimaging findings of Sotos syndrome are distinct enough to allow differentiation of this syndrome from other mental retardation syndromes with macrocephaly.

Teratogenic potential of cocaine.

Cocaine has been implicated as a potential cause of congenital abnormalities since the mid 1980s. Clinical studies have reported an increased risk of cardiovascular and central nervous system abnormalities as well as an increased incidence of limb reduction defects and intestinal atresias. The published data have not established an unequivocal link between cocaine and these abnormalities. The most compelling evidence for the role of cocaine as a teratogen is the increased risk of genitourinary tract defects. Although animal models have also yielded contradictory conclusions, it is intriguing to note that the abnormalities observed in these models are similar to those seen clinically. This review summarizes the clinical and basic research relating to the teratogenic potential of cocaine.

Expression of 72 kDa and 92 kDa type IV collagenases from human giant-cell tumor of bone.

Basement membrane forms widespread barriers to tumor invasion. It has been shown that tumor-secreted, basement membrane-degrading enzymes, namely metalloproteinases (MMPs) play an important role in tumor invasion and metastasis. In this study, we determined the enzymatic activity, content, and mRNA of both the 72 kDa (MMP-2) and 92 kDa (MMP-9) MMPs in primary cultures of human giant-cell tumor of bone (GCT) in vitro and in tissue extracts (in vivo). Gelatin zymography showed the presence of lytic bands at M(r) 121,000, 92,000, and 72,000, and these enzymatic activities were inhibited by EDTA, an inhibitor of MMPs. Western blots with antibodies specific for MMP-2 and MMP-9 confirmed the presence of MMP-2 and MMP-9 both in vitro and in vivo, but GCT cells at late passage showed only MMP-2. Northern blots using labeled cDNA probes specific for these molecules revealed the presence of 3.1 kb transcript for MMP-2 and a 2.9 kb transcript for MMP-9. Using specific antibodies to 72 kDa and 92 kDa type IV collagenases, we studied their cellular distribution by immunohistochemical means. Stronger immunoreactivity was found for 92 kDa type IV collagenase than 72 kDa type IV collagenase in the giant cells. It appears, therefore, that MMP-9 may play an important role in the malignant behavior of GCTs and suggests a potential therapeutic role for protease inhibitors in attempting to minimize the invasive behavior of GCTs.

Cocaine: how dangerous is it during pregnancy?

The risk of prematurity, stillbirth, small for gestational age, and central nervous system damage are clearly a consequence of maternal cocaine use during pregnancy. There is also the risk of concommitent use of alcohol which may cause long term developmental problems. Therefore cocaine should be avoided in pregnancy, but the good news is that if the child survives, the longterm outcome may be very favorable. The cocaine exposed child given proper parenting and educational support may well develop normally with a bright future. It is important not to be too pessimistic about the term infant exposed to cocaine since it may lead to a "self-fulfilling prophecy". Supporting the cocaine exposed child and their family is an important task for all health professionals who deal with children.

Biochemical and molecular teratology of fetal hydantoin syndrome.

Animal and human research has clearly shown that anticonvulsants are teratogens and pose a risk for fetal malformations. In the case of dilantin it appears that fetal susceptibility correlates with the fetal level of the microsomal detoxifying enzyme epoxide hydrolase. The genetics of seizures in the parents does not predict the risk for fetal teratogenesis. The clinician must work with a mother who has seizures prior to conception to achieve the best control of seizures with a single anticonvulsant at the lowest effective dose to minimize the teratogenic potential, but even if this is done there is still a risk of fetal malformations and developmental delays. Each pregnancy in a woman on anticonvulsants is at risk, and appropriate counseling should be accomplished before conception so the family can make an informed decision. The exact risk of teratogenesis is lower than previously recorded. Dilantin poses approximately a 10% risk, tegretol less than 10%, and valproic acid causes a threefold increase in the risk of neural tube defects as well as an increased risk of other malformations. The positive aspect is that with good medical management and good prenatal care approximately 90% of infants exposed to anticonvulsants in utero will not show evidence of teratogenesis. Finally, it is important to stress that all pregnancies carry a 3% risk for a major birth defect independent of any exposures or genetic history.

Preaxial ray reduction defects as part of valproic acid embryofetopathy.

It is well known that prenatal exposure of valproic acid (VPA) may be associated with the occurrence of neural tube defects (Robert and Gibaud, 1982). Additional adverse effects related to VPA exposure include craniofacial abnormalities, skeletal defects, brain defects, cardiovascular defects, and urogenital defects (DiLiberti et al., 1984; Winter et al., 1987; Huot et al., 1987; Jager-Roman et al., 1986; Martinez-Frias, 1990). Recently, radial ray reduction has been reported as a severe type of skeletal defect associated with VPA exposure (Jager-Roman et al., 1986; Huot et al., 1987; Verloes et al., 1990). We report two additional cases of this severe limb reduction defect as part of a broader pattern of altered morphogenesis in fetuses exposed to VPA and to confirm an association between such severe specific skeletal defects and VPA exposure. We briefly review teratologic studies in experimental animals which document the biologic plausibility of this association, and suggest that fetuses exposed to VPA undergo detailed prenatal ultrasonographic evaluation of the limbs.

Clinical and experimental studies linking oxidative metabolism to phenytoin-induced teratogenesis.

The research presented in this article concerns the proposed mechanism of phenytoin-induced teratogenicity that focuses on oxidative metabolites as sources of reactive species in clinical studies and by testing paradigms in animal models. The clinical aspect involved determining whether at-risk fetuses could be detected prenatally on the basis of low or deficient epoxide hydrolase activity. In 19 pregnancies monitored by amniocentesis, we predicted an adverse outcome in four infants on the basis of low enzyme activity. When examined neonatally, all four infants had the dysmorphic features of the "fetal hydantoin syndrome." In an animal model of phenytoin-induced teratogenesis, the level of fetal exposure to oxidative metabolites was decreased by coadministration of the cytochrome P-450-inhibiting antiepileptic drug stiripentol, which significantly reduced the incidence of phenytoin-induced congenital malformations in two of the three inbred mouse strains, thus providing support for the hypothesis that oxidative metabolites are critical in mediating phenytoin teratogenesis.

Prenatal prediction of risk of the fetal hydantoin syndrome.

The well-known teratogenicity of several anticonvulsant medications is associated with an elevated level of oxidative metabolites that are normally eliminated by the enzyme epoxide hydrolase. In this study, we attempted to determine whether infants who are at risk for congenital malformations could be identified prenatally by the measurement of epoxide hydrolase activity. Before fetuses at risk could be identified, it was necessary to measure epoxide hydrolase activity in a randomly selected sample of amniocytes from 100 pregnant women. According to a thin-layer chromatographic assay, the randomly selected sample population had an apparently trimodal distribution, suggestive of an enzyme regulated by a single gene with two allelic forms. Fetuses homozygous for the recessive allele would have low epoxide hydrolase activity and would therefore be at risk if exposed to anticonvulsant drugs during gestation. In a prospective study of 19 pregnancies monitored by amniocentesis, an adverse outcome was predicted for four fetuses on the basis of low enzyme activity (less than 30 percent of the standard). In all four cases, the mother was receiving phenytoin monotherapy, and after birth the infants had clinical findings compatible with the fetal hydantoin syndrome. The 15 fetuses with enzyme activity above 30 percent of the standard were not considered to be at risk, and all 15 neonates lacked any characteristic features of the fetal hydantoin syndrome. These preliminary results suggest that this enzymatic biomarker may prove useful in determining which infants are at increased risk for congenital malformations induced by anticonvulsant drugs.

Wiedemann-Beckwith syndrome in apparently discordant monozygotic twins.

We report 3 pairs of monozygotic (MZ) twins, one twin showing typical Wiedemann-Beckwith syndrome (WBS) with minimal or no expression of the condition in the co-twin. These cases are documented, and three previously reported MZ twin pairs are reviewed. Phenotypic concordance for this syndrome in MZ twin pairs has not been reported. Many cases of familial occurrence have been published and different modes of inheritance have been postulated. Based on the twin-twin variability seen in our patients, it seems the most likely mechanism of inheritance is an autosomal dominant mutation with environmental modification of expressivity, or reduced phenotrance.

Familial aortic dissection in absence of ascending aortic aneurysms: a lethal syndrome associated with precocious systemic hypertension.

The interaction of elevated blood pressure and aortic metabolism in the genesis of aortic dissection is uncharacterized. A kindred with fatal familial aortic dissection in association with precocious systemic hypertension and in absence of a definable connective tissue syndrome has undergone genealogical, clinical, pathological, and biochemical evaluation. Six family members spanning three generations have died of acute dissection. Five men died at a mean age of 28 years (range 22-34), while the proband's paternal grandmother died at 62 years of age. All were hypertensive. A constellation of subtle clinical features points toward deficient integrity of connective tissues; however, major hallmarks of known connective tissue syndromes including aortic root ectasia or aneurysms are absent. Studies of cultured dermal and aortae fibroblasts of two of the proband's brothers mitigate against Ehlers-Danlos IV syndrome. This family's susceptibility to aortic dissection reflects the synergistic liability of coexistent elevated blood pressure and metabolic abnormalities in the genesis of aortic degeneration.