The bone disease associated with factor VIII deficiency in mice is secondary to increased bone resorption.

Osteopenia and osteoporosis have increasingly become a recognized morbidity of factor VIII (FVIII) deficiency. Recently, we demonstrated that FVIII knockout (KO) mice had significantly decreased bone mass and bone strength despite the fact that they did not have haemarthroses. The aim of this study was to explore the mechanism of bone disease associated with FVIII deficiency. We compared biochemical markers of bone formation and osteoclastogenesis, inflammatory cytokines, as well as static and dynamic histomorphometry of genetically engineered FVIII KO male mice to those of wild-type (WT) controls. At 20 weeks of age, FVIII KO mice, as well as WT controls, were sacrificed. Serum and bones were obtained at the time of sacrifice to study biochemical markers of bone formation (osteocalcin) and osteoclastogenesis (receptor activator of nuclear factor kappa-ß and osteoprotegerin), levels of inflammatory cytokines (interleukin-1α and interferon-ß) and to perform static and dynamic histomorphometry of tibia cancellous bone. There was no difference in the biochemical markers of bone formation or osteoclastogenesis. However, there were differences in the two bone-associated cytokines studied. In addition, histomorphometric examination revealed cancellous osteopenia in FVIII KO mice as evidenced by decreased bone area and trabecular number and increased trabecular separation. Bone formation parameters were normal in FVIII KO mice. In contrast, osteoclast-lined bone perimeter was increased. These data demonstrate that bone disease in FVIII KO mice is due to an increased rate of bone resorption.

Genetic regulation of bone mineral density in mice.

Peak bone mass is a major determinant of risk of osteoporotic fracture. Family and twin studies have found a strong genetic component to the determination of bone mineral density (BMD). However, BMD is a complex trait whose expression is confounded by environmental influences and polygenic inheritance. The number, locations and effects of the individual genes contributing to natural variation in this trait are all unknown. The extreme difficulty of dissecting out environmental factors from genetic ones in humans has motivated the investigation of animal models. Genetically distinct animal strains raised under strict environmental control are critical tools for defining genetic regulation. The availability of inbred strains, combined with its relative fecundity, has established the mouse as the best model system for the study of mammalian genetics and physiology. Importantly, genes identified in murine analyses can usually be readily mapped to particular human chromosomal regions because of the high degree of synteny that exists between the mouse and human genomes. We employed quantitative trait locus (QTL) analysis to examine peak BMD in 24 recombinant inbred (RI) mouse strains, derived from a cross between C57BL/6 (B6) and DBA/2 (D2) progenitors (BXD RI). The distribution of BMD values among these strains clearly indicated the presence of strong genetic influences, with an estimated narrow sense heritability of 35%. The differences in peak whole body BMD in the BXD strains were integrated with a large database of genetic markers previously defined in the RI BXD strains to generate chromosome map sites for QTL locations. This QTL analysis provisionally identified a number of chromosomal sites linked to BMD. In the second phase of our BMD QTL mapping efforts, we used three independent mouse populations (all derived from B6 and D2 progenitor strains) to confirm and narrow the genetic locations of 4 QTLs (on chromosomes 1, 2, 4, and 11) that strongly influence the acquisition of peak BMD in mice. Using a novel, fine-mapping approach (recombinant inbred segregation testing), we have succeeded in narrowing two of the BMD-related chromosomal regions and in the process eliminated a number of candidate genes. The homologous regions in the human genome for each of these murine QTLs have been identified in recent human genetic studies. In light of this, we believe that findings in mice should aid in the identification of specific candidate genes for study in humans.

Gender specificity in the genetic determinants of peak bone mass.

Peak bone mass is a major determinant of osteoporotic fracture risk. Gender differences in peak bone mass acquisition are well recognized in humans and may account for a substantial share of the increased prevalence of fragility fractures in women compared with men. Skeletal development is regulated by both heritable and environmental factors. Experimental animal models provide a means to circumvent complicating environmental factors. In this study we examined the heritability of peak bone mineral density (BMD) in genetically distinct laboratory mouse strains raised under strict environmental control and sought to identify genetic loci that may contribute to gender differences in this skeletal phenotype. Peak whole body BMD of male and female mice from a panel of 18 recombinant inbred (RI) strains derived from a cross between C57BL/6 and DBA/2 progenitors (BXD) was measured by dual-energy X-ray absorptiometry (DXA). A highly significant relationship existed between body weight and BMD in the BXD RI mice (r2 = 0.25; p = 1 x 10(-43)). To allow for comparison between male and female RI strains, whole body BMD values were corrected for the influence of body weight. The distribution of weight-corrected BMD (WC-BMD) values among the strains indicated the presence of strong genetic influences in both genders, with an estimated narrow sense heritability of 45% and 22% in male and female mice, respectively. Comparison of RI strain results by two-way analysis of variance (ANOVA) revealed a significant strain-by-gender interaction (F1,17,479 = 6.13; p < 0.0001). Quantitative trait locus (QTL) analysis of the BXD RI strain series provisionally identified nine chromosomal sites linked to peak bone mass development in males and seven regions in females. In two cases, the provisional chromosomal loci were shared between genders, but in most cases they were distinct (five female-specific QTLs and six male-specific QTLs). QTL analysis of a genetically heterogeneous F2 population derived from the B6 and D2 progenitor strains provided additional support for the gender specificity of two loci. A significant phenotype-genotype correlation was only observed in male F2 mice at microsatellite marker D7Mit114 on chromosome 7, and a correlation at D2Mit94 on chromosome 2 was only observed in female F2 mice. The present data highlight the important role of gender in the genetic basis of peak bone mass in laboratory mice. Because the male phenotype is associated with considerable fracture risk reduction, an elucidation of the nature of that effect could provide the basis for novel diagnostic, preventative, or therapeutic approaches.

In silico mapping of complex disease-related traits in mice.

Experimental murine genetic models of complex human disease show great potential for understanding human disease pathogenesis. To reduce the time required for analysis of such models from many months down to milliseconds, a computational method for predicting chromosomal regions regulating phenotypic traits and a murine database of single nucleotide polymorphisms were developed. After entry of phenotypic information obtained from inbred mouse strains, the phenotypic and genotypic information is analyzed in silico to predict the chromosomal regions regulating the phenotypic trait.

Molecular epidemiologic features and antimicrobial susceptibility profiles of various ribotypes of Pseudomonas aeruginosa isolated from humans and ruminants.

OBJECTIVES: To assess automated ribotyping for characterization of Pseudomonas aeruginosa isolates and to identify their type prevalence and geographic distribution. SAMPLE POPULATION: 39 human and 56 ruminant P aeruginosa isolates. PROCEDURES: Isolates were identified by use of bacteriologic techniques and automated Pvull-based ribotyping. Susceptibility to antimicrobials was tested in vitro. Data were analyzed for index of discrimination; prevalence ratio; geographic distribution of ribotypes found only in humans, only in cows, or only in goats (single-host ribotypes); and geographic distribution of ribotypes found in humans and ruminants (multihost ribotypes). RESULTS: All isolates were typeable (45 ribotypes, 35 single-host ribotypes). Ribotyping index of discrimination was 0.976. More isolates (45.3%) than expected yielded multihost ribotypes (22% of all ribotypes). Although 8.6% of single-host ribotypes were found in 4 or more isolates, 60% of multihost ribotypes were found in 4 or more isolates. Ninety percent of multihost ribotypes were isolated from different geographic areas, whereas 3.0% of single-host ribotypes were isolated from different geographic areas. All ruminant isolates were susceptible to gentamicin and polymyxin B. In contrast, antibiogram profiles differed for human isolates from different geographic areas. Susceptibility to antimicrobials differentiated 6 isolates not distinguished by ribotyping. CONCLUSIONS AND CLINICAL RELEVANCE: Automated ribotyping with Pvull discriminated more isolates than in vitro antimicrobial susceptibility. In combination, both tests provided more information than either test alone. Given the greater prevalence and geographic distribution of multihost ribotypes, immunocompromised humans and lactating ruminants may have a greater risk for disease if exposed to multihost P aeruginosa ribotypes, compared with single-host ribotypes.

Phenotypic characterization of mice bred for high and low peak bone mass.

In humans, peak bone mineral density (BMD) is a highly heritable trait and a strong determinant of subsequent osteoporotic fracture risk. To identify the genetic factors responsible for variation in peak BMD, investigators have turned to animal models. In this study we examined the heritability of BMD acquisition and characterized differences in skeletal geometry, histomorphometry, and biomechanical competence between two lines of mice artificially selected for extremes of peak whole body BMD. F2 progeny from a cross between C57BL/6 and DBA/2 inbred strains was used as the foundation population to develop lines selected for either high or low BMD. Whole body BMD was measured by dual-energy X-ray absorptiometry (DXA). By the third generation of selection, highest-scoring BMD (HiBMD) mice exhibited 14% greater peak BMD than lowest-scoring BMD (LoBMD) mice. The mean realized heritability of peak BMD was 36%. Femoral shaft cortical area and thickness and vertebral cancellous bone volume (BV) were significantly greater (16-30%) in the HiBMD line compared with the LoBMD line. Mean cancellous bone formation rates (BFRs) were 35% lower in HiBMD mice compared with LoBMD mice. Failure load and stiffness in the femoral shaft, femoral neck, and L6 vertebrae were all substantially greater (by 25-190%) in HiBMD mice. Thus, these divergently selected murine lines serve to illustrate some of the means by which genetic mechanisms can affect skeletal structure and remodeling. Identification of the individual genes influencing peak BMD in this experimental system will likely reveal some of the genetic determinants of overall bone strength.

Quantitative trait loci affecting peak bone mineral density in mice.

Peak bone mass is a major determinant of risk of osteoporotic fracture. Family and twin studies have found a strong genetic component to the determination of bone mineral density (BMD). However, BMD is a complex trait whose expression is confounded by environmental influences and polygenic inheritance. The number, locations, and effects of the individual genes contributing to natural variation in this trait are all unknown. Experimental animal models provide a means to circumvent complicating environmental factors, and the development of dense genetic maps based on molecular markers now provides opportunities to resolve quantitative genetic variation into individual regions of the genome influencing a given trait (quantitative trait loci, QTL). To begin to identify the heritable determinants of BMD, we have examined genetically distinct laboratory mouse strains raised under strict environmental control. Mouse whole-body bone mineral content by dual-energy X-ray absorptiometry (DXA) correlated strongly with skeletal calcium content by ashing, and peak whole-body BMD by DXA in female mice occurred at approximately 80-90 days of age. We therefore determined mean body weight and peak whole body BMD values in 12-week-old female mice from a panel of 24 recombinant inbred (RI) BXD strains, derived from a cross between C57BL/6 and DBA/2 progenitors. The distribution of body weight and BMD values among the strains clearly indicated the presence of strong genetic influences on both of these traits, with an estimated narrow sense heritability of 60% and 35%, respectively. The patterns of differences in body weight and peak whole body BMD in the BXD strains were then integrated with a large database of genetic markers previously defined in the RI BXD strains to generate chromosome map sites for QTL. After correction for redundancy among the significant correlations, QTL analysis of the BXD RI strain series provisionally identified 10 chromosomal sites linked to peak bone mass development in the female. Several of the identified sites map near genes encoding hormones, structural proteins, and cell surface receptors that are intricately involved in skeletal homeostasis. Four QTL for body weight were also identified. One of these loci was also strongly linked to inherited variation in BMD. This finding suggests that body weight and peak BMD may be influenced by linked genes or perhaps by common genes with pleiotropic effects. Our phenotyping in the RI BXD strains has allowed us to map a number of specific genetic loci strongly related to the acquisition of peak BMD. Confirmation of these findings will likely result in the understanding of which genes control skeletal health.

Studies of the continuing susceptibility of group A streptococcal strains to penicillin during eight decades.

BACKGROUND: In view of the widespread use of penicillin for >50 years for the treatment of group A streptococcal infections, we examined the question of whether there has been a change in susceptibility to penicillin in group A streptococcal strains collected during a span of 80 years (1917 to 1997). METHODS: One hundred thirty-three group A streptococcal strains collected during 80 years were tested for changes in penicillin susceptibility. Three tests were used: (1) the microtiter broth minimal inhibitory concentration (MIC); (2) the minimal bactericidal concentration (MBC); and (3) the penicillin E strip MIC. RESULTS: The results indicate there has been no change in the susceptibility to penicillin in these group A streptococci during the past 80 years. The microtiter broth MIC90 for the oldest strains (0.032 microg/ml) was not significantly different from those collected most recently (0.032 microg/ml); there is no statistical difference between the raw MIC data for the four collection periods (P=0.468, analysis of variance on ranks). CONCLUSIONS: There has been no change in the susceptibility of group A streptococci during this time in spite of well-documented cases of penicillin resistance in other Gram-positive organisms and despite recognized resistance of group A streptococci to other antibiotics.

Consumer reaction to healthcare advertising.

How do consumers view healthcare advertising? This question, along with many others, was addressed in a national survey conducted by Market Strategies for The Alliance For Healthcare Strategy And Marketing, and presented during The Alliance's annual advertising and promotion conference last June.

Alcohol-induced bone disease: impact of ethanol on osteoblast proliferation.

The habitual consumption of even moderate quantities of alcohol (1 to 2 drinks/day) is clearly linked with reduced bone mass (osteopenia). Biochemical and histological evaluation of patients with alcoholic bone disease reveal a marked impairment in bone formation in the face of relatively normal bone resorption. Experiments using well-defined osteoblastic model systems indicate that the observed reductions in bone formation result from a direct, antiproliferative effect of ethanol on the osteoblast itself. As bone remodeling and mineralization are dependent on osteoblasts, it follows that the deleterious effect of alcohol on these cells would result in slowed bone formation, aberrant remodeling of skeletal tissue and, ultimately, osteopenia and fractures. The skeletal consequences of alcohol intake during adolescence, when the rapid skeletal growth ultimately responsible for achieving peak bone mass is occurring, may be especially harmful. The specific subcellular mechanisms whereby ethanol inhibits cell proliferation are, as yet, unknown. During the last few years, attention has shifted from nonspecific membrane perturbation effects to actions on certain signaling proteins. Specifically, there is increasing evidence that ethanol may exert significant effects on transmembrane signal transduction processes that constitute major branches of cellular control mechanisms. At present, abstinence is the only effective therapy for alcohol-induced bone disease. An improved understanding of the pathogenesis of alcohol-induced bone disease may eventually result in alternative therapeutic avenues for those who are unable to abstain.

Ethanol inhibits human osteoblastic cell proliferation.

The habitual consumption of alcoholic beverages is clearly associated with low bone mass and an increased prevalence of skeletal fractures. Microscopic analysis of skeletal tissue from alcoholic patients reveals reduced osteoblast number and suppressed bone formation activity with a relative sparing of resorptive indices. The decreased number of osteoblasts observed in alcoholic subjects results from either impaired proliferation or accelerated senescence. Polyamines and ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis, are essential for cell proliferation in a variety of cell types. To determine if the adverse effect of ethanol on osteoblast number involves modulation of polyamine biosynthesis, we examined the effect of ethanol on parameters of cell growth and ODC activity in a human osteoblast-like osteosarcoma cell line (TE-85). Ethanol markedly impaired DNA synthesis and cell proliferation in a dose-dependent fashion, but alkaline phosphatase activity (a marker of differentiated osteoblast function) remained intact, and accelerated apoptosis was not evident. Thus, the reduced osteoblastic cell number was a result of a direct effect on proliferative processes rather than a nonspecific toxic effect of ethanol to accelerate cell death. Induction of ODC activity was impaired in ethanol-exposed cell cultures in a dose-dependent fashion that paralleled the antiproliferative effects. Finally, supplemental polyamine administration substantially improved DNA synthesis in ethanol-exposed UMR 106-01 cell cultures. These data confirm a direct inhibitory effect of ethanol on osteoblast proliferation without overt cellular toxicity that may, in part, explain the reduced bone mass observed in those who consume excessive amounts of alcohol.

The intrinsic antimicrobial activity of selected sclerosing agents in sclerotherapy.

BACKGROUND: Detergent sclerosing agents may have intrinsic antimicrobial properties. In addition, they may have synergistic effects with other antibiotics such as penicillin. They may induce suppression of intrinsic resistance to penicillin in Staphylococcus aureus. OBJECTIVE: It is in this setting that the present study was carried out in order to determine the degree of suppression of resistance to methicillin and oxacillin in S. aureus by two detergent sclerosing solutions. METHODS: Four strains of S. aureus including a quality control strain were isolated. The minimal inhibitory concentration (MIC) of Sotradecol 1.0% and Polidocanol 0.5% were determined in Mueller Hinton Broth. These dilutions were subsequently seeded with 10(5) organisms of the strain of S. aureus being tested. Serial dilutions of penicillin were made and then the sclerosing agents were added in the appropriate dilutions. RESULTS: Sotradecol 1.0% produced a MIC of 1/64 in two strains of S. aureus and 1/128 in two other variant strains. Polidocanol 0.5% produced a MIC of 1/64 against two strains of S. aureus and an MIC of 1/8 and 1/4 with two other variant strains. In addition, in three of the four S. aureus strains both sclerosing agents had synergistic activity with penicillin and augmented its activity approximately 16-fold. CONCLUSION: This study presents the first successful modification in which detergent sclerosing solutions influence methicillin resistance in a Staphylococcal species. This points out a new potential therapeutic indication for this class of agents.

Inhibition of osteoblastic cell proliferation and ornithine decarboxylase activity by ethanol.

Low bone mass and an increased prevalence of skeletal fractures are evident in the alcoholic population. Histomorphometric analysis of skeletal tissue from alcoholic patients reveals reduced osteoblast number and suppressed bone formation activity, with relative sparing of resorptive indexes. The decreased number of osteoblasts observed in alcoholic subjects results from either impaired proliferation or accelerated senescence. Polyamines and ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis, are essential for cell proliferation in a variety of cell types. To determine whether the consequences of ethanol on osteoblast number involve the modulation of polyamine biosynthesis, we examined the effect of ethanol on parameters of cell growth and ODC activity in a rat osteoblast-like osteosarcoma cell line (UMR 106-01). Ethanol markedly impaired DNA synthesis and cell proliferation in a dose-dependent fashion. Difluoromethylornithine, a specific inhibitor of ODC activity, induced a similar inhibition of UMR 106-01 cell proliferation, indicating the importance of the polyamine pathway in this osteoblastic cell line. Induction of ODC activity was impaired in ethanol-exposed cell cultures in a dose-dependent fashion that paralleled the antiproliferative effects. Finally, supplemental polyamine administration substantially improved DNA synthesis in ethanol-exposed UMR 106-01 cell cultures. These data confirm a direct inhibitory effect of ethanol on osteoblast proliferation that may in part explain the reduced bone mass observed in subjects who consume excessive amounts of alcohol. These findings also suggest that altered polyamine metabolism may be an important mechanism responsible for the antiproliferative effects of ethanol on the osteoblast.

Chiral resolution of cationic drugs of forensic interest by capillary electrophoresis with mixtures of neutral and anionic cyclodextrins.

Chiral resolution of a number of cationic drugs of forensic interest (amphetamine, methamphetamine, cathinone, methcathinone, cathine, cocaine, propoxyphene, and various alpha-hydroxyphenethylamines) is achieved via capillary electrophoresis (CE) with added cyclodextrins (CDs), including novel mixtures of neutral and anionic CDs. In the latter studies, resolution and migration speed are readily adjusted by varying the ratio of the two added CDs, as the anionic CD acts as a counter-migrating complexing reagent. The neutral CD, heptakis(2,6-di-O-methyl)-beta-CD, was found suitable for the analysis of illicit cocaine and khat leaves (Catha edulis Forsk), which contain (-)-alpha-aminopropiophenone ((-)-cathinone), (+)-norpseudoephedrine (cathine), (-)-norephedrine, and trace levels of the phenylpentenylamines (+)-merucathinone, (+)-merucathine, and possibly (-)-pseudomerucathine. The use of mixtures of the neutral and the anionic CD (beta-CD sulfobutyl ether IV) was found suitable for the analysis of illicit amphetamine, methamphetamine, methcathinone, and propoxyphene. A model is presented for the impact of mixtures of neutral and anionic CDs on migration behavior and chiral resolution in CE.

Antineurofilament and antiretinal antibodies in AIDS patients with cytomegalovirus retinitis.

Sera obtained from AIDS patients with cytomegalovirus (CMV) retinitis before and after treatment with foscarnet, AIDS patients with human immunodeficiency virus (HIV) retinopathy, AIDS patients without retinal disease, and normal healthy controls with and without positive CMV serologies were assayed for the presence of antibodies against the 200-kDa outer, 160-kDa middle, and 68-kDa core subunits of the neurofilament triplet. Additional studies were performed to determine the presence of antibodies reactive with proteins extracted from crude human retinal antigen preparations. Antibodies against the 200-, 260-, and 68-kDa proteins of the neurofilament triplet were detected in 15 of 15 AIDS patients with CMV retinitis. The expression of these antibodies was unaffected, qualitatively, by successful treatment with foscarnet. In contrast, only 30% of patients with HIV retinopathy unrelated to CMV, fewer than 35% of AIDS patients with positive CMV titers but without evident retinitis, and fewer than 25% of healthy controls with positive or negative CMV titers possessed antibodies against any of the triplet proteins (P < 0.001). Antibodies against several clusters of retinal antigens were also identified in the sera of patients with CMV retinitis. In summary, the data indicate that retinal elements damaged by CMV infection induce an antibody response against the 200-, 160-, and 68kDa components of the neurofilament triplet as well as other, as yet undefined retinal antigens.

Determination and in-depth chromatographic analyses of alkaloids in South American and greenhouse-cultivated coca leaves.

Methodology is described for the detection and/or determination of cocaine and minor alkaloids in South American coca as well as in greenhouse- and tropical-cultivated field coca of known taxonomy. Coca leaf from Bolivia, Peru, Ecuador and Colombia were subjected to the determination of cocaine, cis- and trans-cinnamoylcocaine, tropacocaine, hygrine, cuscohygrine and the isomeric truxillines. The greenhouse samples were cocaine-bearing leaves of the genus Erythroxylum and included E. coca var. coca, E. novogranatense var. novogranatense and E. novogranatense var. truxillense, and the alkaloids determined were cocaine, ecgonine methyl ester, cuscohygrine, tropacocaine and the cinnamoylcocaines. The tropical-cultivated coca were E. novogranatense var. novogranatense and E. coca var. coca. Cocaine and minor alkaloids were isolated from basified powdered leaf samples using a toluene extractant, followed by acid-Celite column chromatography. The isolated alkaloids were determined by capillary gas chromatography with flame ionization or electron-capture detection. Methodology is also presented for the isolation and mass spectral analysis of numerous trace-level coca alkaloids of unknown structure.