Substrate modulation of osteoblast adhesion strength, focal adhesion kinase activation, and responsiveness to mechanical stimuli.

Osteoblast interactions with extracellular matrix (ECM) proteins are known to influence many cell functions, which may ultimately affect osseointegration of implants with the host bone tissue. Some adhesion-mediated events include activation of focal adhesion kinase, and subsequent changes in the cytoskeleton and cell morphology, which may lead to changes in adhesion strength and cell responsiveness to mechanical stimuli. In this study we examined focal adhesion kinase activation (FAK), F-actin cytoskeleton reorganization, adhesion strength, and osteoblast responsiveness to fluid shear when adhered to type I collagen (ColI), glass, poly-L-lysine (PLL), fibronectin (FN), vitronectin (VN), and serum (FBS). In general, surfaces that bind cells through integrins (FN, VN, FBS) elicited the highest adhesion strength, FAK activation, and F-actin stress fiber formation after both 15 and 60 minutes of adhesion. In contrast, cells attached through non-integrin mediated means (PLL, glass) showed the lowest FAK activation, adhesion strength, and little F-actin stress fiber formation. When subjected to steady fluid shear using a parallel plate flow chamber, osteoblasts plated on FN released significantly more PGE2 compared to those on glass. In contrast, PGE2 release of osteoblasts attached to FN or glass was not different in the absence of fluid shear, suggesting that differences in binding alone are insufficient to alter PGE2 secretion. The increased adhesion strength as well as PGE2 secretion of osteoblasts adhered via integrins may be due to increased F-actin fiber formation, which leads to increased cell stiffness.

Paracrine activation of extracellular signal-regulated kinase in a simple in vitro model of wounded osteoblasts.

The immediate signal-transduction response of osteoblasts to acute trauma is poorly characterized. We have developed a simple in vitro model for osteoblast trauma to investigate aspects of the molecular mechanisms of wound healing in bone. Herein we report the specific, rapid, and transient phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2 in osteoblasts as a response to disruption ("wounding") of a confluent monolayer. The mitogen-activated protein kinase (MAPK) cascades of p38 and stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) were not activated by this perturbation. The response to wounding was equivalent to the activation of ERK by the addition of exogenous growth factors, and the perturbation-dependent phosphorylation of ERK can be suppressed by an inhibitor of heparin-binding growth factors. Conditioned media from wounded monolayers can induce the phosphorylation of ERK in unperturbed monolayers. Using immunohistochemistry, it was demonstrated that the cells with increased levels of phosphorylated ERK were not localized to the wound edges. These results indicate that ERK activation is the result of an autocrine/paracrine response by osteoblasts to trauma. We speculate that osteoblasts respond to trauma with the release of soluble factors as part of an autocrine/paracrine modulation of the wound-healing process in bone.

Successful treatment of adults with severe Hantavirus pulmonary syndrome with extracorporeal membrane oxygenation.

OBJECTIVE: To describe our experience with the use of extracorporeal membrane oxygenation (ECMO) as a rescue therapy in adult patients with severe cardiopulmonary failure from Hantavirus pulmonary syndrome. DESIGN: Case series. SETTING: Tertiary referral center. PATIENTS: Patients with confirmed Hantavirus infection, who developed severe cardiopulmonary failure in which conventional therapy was assessed as being unsuccessful. INTERVENTIONS: Records of previous patients treated for Hantavirus pulmonary syndrome were reviewed and findings consistent with 100% mortality were found. MEASUREMENTS AND MAIN RESULTS: Findings associated with a 100% mortality rate were a) cardiac index of <2.5 L/min/m2; b) serum lactate concentration of >4.0 mmol/L (normal range 0.0 to 2.2); c) pulseless electrical activity or ventricular fibrillation or ventricular tachycardia; and d) refractory shock despite fluid resuscitation, and vasoactive medications. From 1994 to 1996, seven patients were admitted with confirmed Hantavirus pulmonary syndrome and severe cardiopulmonary failure. Three of the seven patients had at least two of the four criteria for a 100% mortality rate listed above, and appeared to be failing optimal conventional therapy. These three patients received support with venoarterial ECMO. The first patient was placed on ECMO during cardiac arrest and died. The next two patients who received ECMO for Hantavirus pulmonary syndrome survived after relatively short, uncomplicated ECMO runs, and were discharged without complications. CONCLUSIONS: ECMO successfully provided cardiopulmonary support in two patients with severe Hantavirus pulmonary syndrome who survived with a good outcome. Our experience suggests that ECMO is a beneficial therapy for patients critically ill with Hantavirus pulmonary syndrome.

The interplay of half-site sequence and spacing on the activity of direct repeat thyroid hormone response elements.

Direct repeats of the hexamer AGGTCA can serve as response elements for vitamin D, thyroid hormone, or retinoic acid. The specificity of the response appears to reside in the spacing between the hexamers, with response elements for vitamin D restricted to direct repeats separated by a 3-base pair (bp) spacer, thyroid hormone a 4-bp spacer, and retinoic acid a 5-bp spacer (3-4-5 rule). Recently we have shown that the optimum thyroid hormone receptor binding site consists of an 8-bp sequence (TAAGGTCA), not a hexamer. Therefore we tested whether the 3-4-5 rule is valid for octamer sequence direct repeats. In transfection experiments octamer direct repeats with 3-, 4-, or 5-bp spacers conferred equivalently strong thyroid hormone responses, although a repeat with a 9-bp spacer was substantially weaker. For the 4- and 5-bp spacer constructs, the 5' half-site octamer had as strong an influence on thyroid hormone induction as did the 3' half-site octamer, although for the 3-bp spacer construct the 5' octamer was marginally less potent than the 3' octamer. Transfection and gel shift experiments did not suggest a simple correlation between the binding of thyroid hormone receptor-retinoid X receptor heterodimers and thyroid hormone induction from these response elements. We conclude that half-site sequence can override the effect of spacing in determining the hormone responsiveness of a direct repeat response element. In addition, the thyroid hormone response may not be due simply to the binding of thyroid hormone receptor-retinoid X receptor heterodimers to the DNA.

DNA binding specificity and function of retinoid X receptor alpha.

Retinoid X receptors are members of the erbA superfamily of ligand-inducible transcription factors. Similar to several other members of this gene family, retinoid X receptors are known to bind to the hexameric DNA sequence AGGTCA. After binding to a direct repeat of this hexamer with a one-base pair spacer, retinoid X receptor homodimers are able to activate transcription in the presence of the ligand 9-cis-retinoic acid. However, it is not known if AGGTCA represents the highest affinity binding site for retinoid X receptors. A combination of the electrophoretic mobility shift assay and polymerase chain reaction was used to isolate from a pool of random DNA those sequences that bind retinoid X receptors with highest affinity. This approach, combined with mutational analysis and DNA footprinting, led to the identification of the seven-base pair sequence GGGGTCA as the highest affinity retinoid X receptor binding site. A direct repeat of this sequence is substantially more active than a direct repeat of AGGTCA as a retinoid X response element.

Specificity and mechanism of thyroid hormone induction from an octamer response element.

Thyroid hormone response elements are specific DNA sequences that allow thyroid hormone receptors to confer ligand-dependent regulation of gene expression. The response elements characterized to date have been composed of varying arrangements of multiple copies of a conserved hexameric sequence. The traditional consensus half-site of these response elements is the sequence 5'-AGGTCA, although we have demonstrated recently that the optimal thyroid hormone receptor monomer binding site is 2 base pairs larger, 5'-TAAGGTCA. Since other members of this family of nuclear receptors also have been shown to use varying arrangements of the traditional hexamer sequence as response elements, we examined whether the octamer sequence was specific as a thyroid hormone response element. The studies reported here demonstrate that only thyroid hormone receptors confer ligand responsiveness to a reporter gene containing a single copy of the octamer sequence as a response element and that qualitative and quantitative differences in the binding of related nuclear receptors to this sequence can account for this functional specificity. We also have shown that thyroid hormone induction from the octamer response element occurs independently of retinoid X receptors, in contrast to the induction from traditional complex thyroid hormone response elements.

Nucleotide substitutions differentially affect direct repeat and palindromic thyroid hormone response elements.

Thyroid hormone receptors are transcription factors that bind to specific DNA sequences and regulate gene expression in a ligand-dependent manner. Thyroid hormone response elements can be composed of direct repeat or palindromic arrangements of half-sites. The traditional half-site has been considered to be the sequence 5'-AGGTCA, although we recently demonstrated that the optimal monomer-binding site is 2 base pairs larger, 5'-TAAGGTCA. It has generally been presumed that alterations in half-site sequence have commensurate effects on gene expression from direct repeat and palindromic response elements. However, in the present studies we demonstrate that palindromic elements are highly dependent on the presence of the two 5'-nucleotides (TA) for function, whereas this portion of the response element has minimal influence on hormone induction from direct repeat elements. Hormone induction correlates poorly with binding affinity for thyroid hormone receptor monomers, homodimers, or heterodimers with retinoid X receptors, either in the absence or presence of ligands. We speculate that the magnitude of hormone induction may reflect protein conformation differences induced by a combination of the presence of the appropriate 5'-nucleotides plus half-site orientation.

Safety of continuous nebulized albuterol for bronchospasm in infants and children.

OBJECTIVE: To determine the incidence of cardiotoxicity in infants and children who receive continuous nebulized albuterol (CNA) for bronchospasm. DESIGN: Prospective, case series. SETTING: A university pediatric intensive care and pediatric subacute units. PATIENTS: Nineteen infants and children who received CNA for at least 24 hours. INTERVENTIONS: None. MEASUREMENTS: Creatinine phosphokinase (CK) was measured at the time of admission and then at 12, 24, 48, and 72 hours while the patient received CNA. Isoenzyme CK-MB fractions were measured if CK concentration was > or = 250 IU/L. One electrocardiogram was obtained for each patient during CNA treatment. All patients had continuous cardiac monitoring during continuous nebulization therapy. MAIN RESULTS: Creatinine phosphokinase levels remained within normal limits for 16 patients during CNA treatment. Three patients had elevated CK and in two CK-MB fractions were elevated at one measurement. None of the electrocardiograms showed evidence of ischemia and no arrhythmias were noted during CNA therapy, even in the patients with elevated CK-MB fractions. CONCLUSIONS: Continuous albuterol therapy appears to be safe in our patient population as there was no significant evidence of cardiotoxicity. The significance of the transient elevation of CK-MB without other evidence of cardiotoxicity remains to be determined.

Nonbiased identification of DNA sequences that bind thyroid hormone receptor alpha 1 with high affinity.

Thyroid hormone receptors are transcription factors that bind to specific DNA sequences and regulate gene expression in a ligand-dependent manner. Although thyroid hormone receptors are known to bind to the hexamer 5'-AGGTCA, it is not known if this represents the optimal binding site. Therefore, a nonbiased strategy was used to identify DNA sequences which bind thyroid hormone receptor alpha 1 with high affinity. Such DNA sequences were isolated from a pool of random sequences using a strategy combining an electrophoretic mobility shift assay with the polymerase chain reaction. It was found that thyroid hormone receptor alpha 1 binds with highest affinity to the octamer 5'-TAAGGTCA. Mutation of the two 5'-nucleotides decreased the affinity of thyroid hormone receptor alpha 1 for this DNA sequence approximately 5-fold, and the importance of those nucleotides in receptor binding was confirmed by DNA footprinting. A single copy of the octamer sequence (but not the hexamer AGGTCA) could impart T3 responsiveness to a heterologous promoter in a transient transfection assay. The results indicate that the optimal binding site for thyroid hormone receptor alpha 1 is 2 base pairs larger than previously thought, and that a single binding site can function as a response element. In addition, we speculate that the optimal binding sites for thyroid hormone, vitamin D, and retinoic acid receptors may not be identical, as had previously been thought.

The functional equivalence of demineralized bone and tooth matrices in ectopic bone induction.

The objective of this study was to determine whether demineralized rat incisor matrices were a more potent inducer of ectopic endochondral bone formation than demineralized diaphyseal bone matrices derived from the same donors. Twenty-five-milligram disks of demineralized bone or tooth matrix obtained from adolescent Long-Evans rats were implanted in a standardized ectopic site. Biochemical and histometric measurements of bone formation revealed that the two matrices were functionally equivalent inducers of endochondral bone formation. The induced pellicle of bone reached a maturation point 18 days after implantation. Dentin matrix implants generated a significantly greater amount of mineralized tissue than did bone matrix implants. This difference could be explained on the basis of remineralization of the dentin particles to a greater degree than the bone matrix particles. Initial observations suggesting a more robust osteoinductive activity in demineralized incisor matrix can be attributed to the decreasing activity of bone matrix from older donors when compared to younger donors. The extent of osteoinduction by the two substrata was equivalent when the matrices were matched for age.

Pulmonary artery sling with severe tracheobronchial stenosis: repair in infancy by tracheal resection and pulmonary artery reimplantation.

Pulmonary artery sling is an unusual anomaly. The results of surgical therapy have been unsatisfactory in the past because of poor long-term left pulmonary artery patency and failure to address concomitant primary tracheobronchial stenoses. Refinement of suture materials and microsurgical techniques have allowed earlier surgical treatment of tracheal stenosis and have led to improved pulmonary artery patency rates. Intermediate results of primary one-stage repair with tracheal resection and left pulmonary artery reimplantation or translocation in early childhood have been promising. It remains to be seen whether late anastomotic tracheal stenosis will be a problem. This constitutes the first report of a case of one-stage repair with tracheal resection and left pulmonary artery reimplantation in an infant.

Preliminary studies on pulp capping with demineralized dentin.

The use of osteoinductive demineralized dentin matrix in the repair of vital pulp exposures was examined. The pulps of the maxillary first molars of adult male Long Evans rats were exposed and capped with either demineralized dentin particles or guanidinium extracted demineralized dentin particles; then covered with a glass ionomer cement restoration. The animals were sacrificed after two weeks and the maxillae were dissected free, fixed in formaldehyde and demineralized. Histologic examination revealed that in those cases where the protective glass ionomer restoration was intact, both groups showed some healing. There was some preliminary indication that the extent of reactive matrix formation was greater in the osteoinductive group. In both groups, odontoblast-like cells appeared to incorporate some of the dentin particles into the reparative dentin bridge. This preliminary work suggests that osteoinductive factors present in demineralized incisor dentin might be used to guide the formation of a dentin bridge in a rat model for vital pulp exposures.

Characterization of a novel tumor necrosis factor-alpha-induced endothelial primary response gene.

The response of endothelial cells to the cytokine tumor necrosis factor-alpha (TNF) is complex, involving the induction and suppression of multiple genes and gene products. Differential screening of a TNF-stimulated, cycloheximide-treated human umbilical vein endothelial cell library has resulted in the cloning of several novel cDNAs whose protein products are involved in the primary response of the endothelium to TNF. One of these cDNAs, designated B12, is further characterized here. B12 is encoded by a 3.5-kilobase transcript and is induced rapidly and transiently by TNF. Transcript expression is found to be developmentally regulated in a tissue-specific manner, with B12 message being differentially expressed in the heart and liver during mouse embryogenesis. The open reading frame of B12 predicts a 316-amino acid sequence rich in charged residues, particularly at the carboxyl terminus, and has neither significant homology to other known proteins nor to any extent sequence motifs. B12 is found to be a highly conserved single-copy gene which is located in the q22----q23 region of human chromosome 17. Polyclonal antibodies raised against a large portion of the B12 open reading frame immunoprecipitate a 36-kilodalton polypeptide from wheat germ lysates programmed to translate in vitro transcribed B12 mRNA. The B12 protein is further shown to be induced in human umbilical vein endothelial cells by TNF, and the protein is shown to be rapidly degraded.

Total financial management.

Total financial management is a program designed by author Robert Katz, M.S., C.P.A., which says that by managing from the top down, the physician/administrator team can regain control of the practice and its major financial centers. His article describes the two-phased approach to total financial management.

Localization of the panhypopituitary dwarf mutation (df) on mouse chromosome 11 in an intersubspecific backcross.

Ames dwarf (df) is an autosomal recessive mutation characterized by severe dwarfism and infertility. This mutation provides a mouse model for panhypopituitarism. The dwarf phenotype results from failure in the differentiation of the cells which produce growth hormone, prolactin, and thyroid stimulating hormone. Using the backcross (DF/B-df/df X CASA/Rk) X DF/B-df/df, we confirmed the assignment of df to mouse chromosome 11 and demonstrated recombination between df and the growth hormone gene. This backcross is an invaluable resource for screening candidate genes for the df mutation. The df locus maps to less than 1 cM distal to Pad-1 (0.85 +/- 0.85 cM). Two new genes localized on mouse chromosome 11, Rpo2-1, and Edp-1, map to a region of conserved synteny with human chromosome 17. The localization of the alpha 1 adrenergic receptor, Adra-1, extends a known region of synteny conservation between mouse chromosome 11 and human chromosome 5, and suggests that a human counterpart to df would map to human chromosome 5.

The Pit-1 transcription factor gene is a candidate for the murine Snell dwarf mutation.

Two nonallelic mouse mutations with severe dwarf phenotypes are characterized by a lack of growth hormone, prolactin, and thyroid stimulating hormone. The cells that normally synthesize these pituitary hormones express a common transcription factor called GHF-1 or Pit-1. Using an intersubspecific backcross, we have demonstrated tight linkage of the Pit-1 and Snell dwarf (dw) genes on mouse chromosome 16. No recombination was observed between Pit-1 and dw in 110 individuals examined. Southern blot analysis of genomic DNA reveals that the Pit-1 gene is rearranged in C3H/HeJ-dwJ/dw mice but not in coisogenic +/+ animals, providing molecular evidence that a lesion in the Pit-1 gene results in the Snell dwarf phenotype. Demonstration of low levels of Pit-1 expression in Ames dwarf (df) mice implies that both Pit-1 and df expression may be required for pituitary differentiation.

Radiation-sterilized insoluble collagenous bone matrix is a functional carrier of osteogenin for bone induction.

The influence of gamma radiation on the role of the collagenous substratum as a carrier for proteins which cause bone induction was examined. Osteoinductive demineralized bone matrix was extracted by 4 M guanidinium hydrochloride. The insoluble collagenous bone matrix (ICBM) obtained was not osteoinductive; however, when reconstituted with partially purified osteogenin, bone induction was restored. In order to apply the principle of bone induction to clinical use, methods of sterilization must be optimized to maintain the osteoinductive activity of bone allografts. The inactive substratum was irradiated and reconstituted with an active, partially purified bone extract and bioassayed. Irradiation of the ICBM by a Cobalt 60 source at a dose of 1 and 3 Mrads had no deleterious effect on the functional role of the substratum.

Premature alveolar bone loss in Erdheim-Chester disease.

Erdheim-Chester disease is a rare histiocytosis also known as lipoid granulomatosis. Oral findings have not been reported previously to our knowledge. This case report documents evidence of oral sequelae of Erdheim-Chester disease. A patient whose course was followed for 10 years at the National Institutes of Health had premature alveolar bone resorption. He underwent full-mouth extraction at age 29 years because of severe periodontitis. Histopathologic evidence of Erdheim-Chester disease was demonstrated in the periodontal soft tissues. In the ensuring years, accelerated resorption of the residual ridges precluded the use of conventional dentures. We recommend early preventive dental management for patients with Erdheim-Chester disease.

Oral squamous cell carcinoma arising in a patient with long-standing lichen planus. A case report.

The risk of malignant transformation of oral lichen planus remains a controversial point. Many previous reports have been discounted on the basis of inadequate information or lack of histologic confirmation of lichen planus. We report a well-documented case of long-standing cutaneous and oral lichen planus in which squamous cell carcinoma of the dorsal portion of the tongue occurred. There is an apparent difference in the sites of oral carcinomas in patients with lichen planus compared with the general population. This suggests that lichen planus increases the risk of oral cancer in affected sites.

An analysis of compound and complex odontomas.

Three hundred ninety-six odontomas were included in this study, with each case assigned to one of three categories using commonly accepted histologic criteria for compound and complex forms. Analysis revealed that compound odontomas were the most common type (70 percent of all cases). They were most common in the 11 to 15-year-old age-group and in the maxillary incisor area or the canine regions of either jaw. There was a nearly equal sex distribution. Complex odontomas showed similar age and sex distribution; they had a greater tendency to occur in the molar regions. Complex odontomas were also associated with unerupted teeth more often than compound odontomas. Tooth agenesis in the area of an odontoma or the impaction of a primary tooth by an odontoma occurred infrequently. This study showed that there is a correlation between the site of an odontoma and the age at which it is generally treated.