Endophthalmitis caused by Enterobacter cloacae

Cataract extraction with placement of intraocular lenses is the most common intraocular procedure done today, with endophthalmitis as its most devastating complication. To our knowledge, this is only the third reported case of postoperative endophthalmitis caused by the gram-negative bacillus, Enterobacter cloacae. The course of infection, the treatment, and the laboratory identification of this organism are presented. It is important to add this proven case of gram-negative endophthalmitis to our growing knowledge of causes of this disease because, despite aggressive treatment with vitrectomy and intravitreal antibiotics, the infection was so virulent that it led to eventual phthisis bulbi.     

Characterization of arylamine N-acetyltransferase in Enterobacter aerogenes

N-acetyltransferase (NAT) activity was determined by incubation of purified Enterobacter aerogenes enzyme with 2-aminofluorene (2-AF) as the substrate, followed by high pressure liquid chromatography assays. The NAT activity from E. aerogenes was 0.58 +/- 0.08 nmol/min/mg protein for 2-AF. The values of apparent K(m) and Vmax were 0.72 +/- 0.14 mM and 2.45 +/- 0.29 nmol/min/mg protein, respectively, for 2-AF. The optimal pH value for the enzyme activity was 7.5 for the 2-AF tested. The optimal temperature for enzyme activity was 37 degrees C for the 2-AF substrate. The molecular weight of NAT from E. aerogenes was 44.9 kD. Among a series of divalent cations and salts, Zn2+, Ca2+, and Fe2+ were demonstrated to be the most potent protease inhibitors, and only ethylenediaminetetraacetic acid significantly protected the NAT. Iodoacetamide, in contrast to other agents, markedly inhibited NAT.     

Osteoblastic responses to TGF-beta during bone remodeling

Bone remodeling depends on the spatial and temporal coupling of bone formation by osteoblasts and bone resorption by osteoclasts; however, the molecular basis of these inductive interactions is unknown. We have previously shown that osteoblastic overexpression of TGF-beta2 in transgenic mice deregulates bone remodeling and leads to an age-dependent loss of bone mass that resembles high-turnover osteoporosis in humans. This phenotype implicates TGF-beta2 as a physiological regulator of bone remodeling and raises the question of how this single secreted factor regulates the functions of osteoblasts and osteoclasts and coordinates their opposing activities in vivo. To gain insight into the physiological role of TGF-beta in bone remodeling, we have now characterized the responses of osteoblasts to TGF-beta in these transgenic mice. We took advantage of the ability of alendronate to specifically inhibit bone resorption, the lack of osteoclast activity in c-fos-/- mice, and a new transgenic mouse line that expresses a dominant-negative form of the type II TGF-beta receptor in osteoblasts. Our results show that TGF-beta directly increases the steady-state rate of osteoblastic differentiation from osteoprogenitor cell to terminally differentiated osteocyte and thereby increases the final density of osteocytes embedded within bone matrix. Mice overexpressing TGF-beta2 also have increased rates of bone matrix formation; however, this activity does not result from a direct effect of TGF-beta on osteoblasts, but is more likely a homeostatic response to the increase in bone resorption caused by TGF-beta. Lastly, we find that osteoclastic activity contributes to the TGF-beta-induced increase in osteoblast differentiation at sites of bone resorption. These results suggest that TGF-beta is a physiological regulator of osteoblast differentiation and acts as a central component of the coupling of bone formation to resorption during bone remodeling.     

Bone response to surface-modified titanium implants: studies on the early tissue response to machined and electropolished implants with different oxide thicknesses

The bone formation around titanium implants with varied surface properties is investigated. Machined and electropolished samples with and without thick, anodically formed surface oxides were prepared, surface characterized and inserted in the cortical bone of rabbits (1, 3 and 6 weeks). Scanning electron microscopy, scanning Auger electron spectroscopy and atomic force microscopy revealed marked differences in oxide thickness, surface topography and roughness, but no significant differences in surface chemical composition, between the different groups of implants. Light microscopic morphology and morphometry showed that all implants were in contact with bone and had a large proportion of bone within the threads at 6 weeks. The smooth, electropolished implants, irrespective of anodic oxidation, were surrounded by less bone than the machined implants after 1 week. After 6 weeks the bone volume as well as the bone-implant contact were lower for the merely electropolished implants than for the other three groups. Our study shows that a high degree of bone contact and bone formation are achieved with titanium implants which are modified with respect to oxide thickness and surface topography. However, the result with the smooth (electropolished) implants indicates that a reduction of surface roughness, in the initial phase, decreases the rate of bone formation in rabbit cortical bone.     

Antiretroviral treatment: when to start and with what?

Quantitative ultrasound (QUS) of the calcaneus is a widely utilized method for bone densitometry. However, the meaning of measured parameters, such as speed of sound (SOS) and broadband attenuation (BUA), is not well established. We performed in vitro measurement of dissected human calcaneus (n = 60; male 29, female 31; mean age 81 years) using two QUS machines and also measured bone densities using SXA, DXA and pQCT. Finally, we investigated breaking strength of the calcaneus and studied the correlation with QUS parameters and other assessed parameters. The two QUS measurements were in good agreement in most experiments. SOS correlated most closely with bone mineral densities assessed by pQCT and did not correlate with factors related to bone size, while BUA showed the highest correlation with BMC and association with parameters related with bone size such as bone area and bone width. With maximal breaking stress of the calcaneus, correlations were almost equal among QUS parameters and bone mineral density. We conclude that SOS is the parameter most closely associated with true bone mineral density (g/cm3), whereas BUA represents both bone mineral density and bone size, mimicing BMD assessed by DXA or SXA.     

Multiple joint infections with Enterobacter cloacae

An infant with multiple joint infections from Enterobacter cloacae seems not to have been reported previously. The infant survived, incurred a pathologic dislocation of the left hip, but at 18 months of age was well with all other joints functioning normally.     

A new scanning electron microscopy approach to the quantification of bone mineral distribution: backscattered electron image grey-levels correlated to calcium K alpha-line intensities

The introduction of backscattered electron (BSE) imaging in scanning electron microscopy (SEM) has led to new possibilities for the evaluation of mineral distributions in bone on a microscopic level. The different grey-levels seen in the BSE-images can be used as a measure for the local mineral content of bone. In order to calibrate these BSE-grey-levels (BSE-GL) and correlate them to mineral contents, various attempts, using reference samples with known weighted mean atomic number and/or using simulated bone tissues with known hydroxyapatite concentrations, have been made. In contrast, a new approach is presented here based on measurements of the X-ray intensities of the calcium K alpha-line on selected areas of real bone samples; the measured intensities are then related to the corresponding BSE-GL. A linear positive correlation between weight percent (wt%) calcium and BSE-GL was found. When the BSE-mode is standardized using carbon and aluminum as references, the different mineral contents in bone samples can be recorded as BSE-GL, calibrated to wt% of calcium or hydroxyapatite (HA), respectively. The resulting mineral concentration histograms have a dynamic range from 0 to 89 wt% HA and have a binwidth resolution of 0.45 wt% HA. The presented modifications of the BSE method strongly enhance its feasibility in the field of bone research and its application as a special diagnostic tool for bone diseases.     

Adaptation of bone to physiological stimuli

The ability of bone to alter its morphology in response to local physical stimuli is predicated upon the appropriate recruitment of bone cell populations. In turn, the ability to initiate cellular recruitment is influenced by numerous local and systemic factors. In this paper, we discuss data from three ongoing projects from our laboratory that examine how physiological processes influence adaptation and growth in the skeleton. In the first study, we recorded in vivo strains to quantify the locomotion-induced distribution of two parameters closely related to bone fluid flow strain rate and strain gradients. We found that the magnitude of these parameters (and thus the implied fluid flow) varies substantially within a given cross-section, and that while strain rate magnitude increases uniformly with elevated speed, strain gradients increase focally as gait speed is increased. Secondly, we examined the influence of vascular alterations on bone adaptation by assessing bone blood flow and bone mechanical properties in an in vivo model of trauma-induced joint laxity. A strong negative correlation (r2 = 0.8) was found between increased blood flow (76%) in the primary and secondary spongiosa and decreased stiffness (-34%) following 14 weeks of joint laxity. These data suggest that blood flow and/or vascular adaptation may interact closely with bone adaptation initiated by trauma. Thirdly, we examined the effect of a systemic influence upon skeletal health. After 4 weeks old rats were fed high fat-sucrose diets for 2 yr, their bone mechanical properties were significantly reduced. These changes were primarily due to interference with normal calcium absorption. In the aggregate, these studies emphasize the complexity of bone's normal physical environment, and also illustrate the potential interactions of local and systemic factors upon the process by which bone adapts to physical stimuli.     

Morphology and matrix composition during early tendon to bone healing

This study outlines the early morphologic phenomenon of tendon to bone healing in the rabbit model. Twelve skeletally mature, male New Zealand White rabbits received transplantation of the hallucis longus tendon into a 2-mm calcaneal bone tunnel. The morphologic characteristics of the healing tendon to bone interface were evaluated at 1, 2, 4, and 6 weeks after surgery by the use of conventional histology and immunohistochemical localization of collagen Types I, II, and III. Histologic analysis illustrated progressive maturation and reorganization of the tendon to bone interface with subsequent development of tissue collagen fiber continuity between the tendon and bone. Initially, diffuse immunolocalization of all three collagen types was observed within the scar tissue filling the space between the tendon and bone. During a 6-week period, reorganization of the scar tissue into an interface occurred, similar to an indirect insertion. Although a definitive fibrocartilage region did not form, Type II collagen was localized at the remodeling insertion site throughout the first 6 weeks of repair. In addition, Type III collagen fibers, resembling Sharpey's fibers, were noted to span this interface. The characterization of the insertion between tendon and bone is important to the understanding of healing in commonly used orthopaedic grafting procedures, such as anterior cruciate ligament reconstructions.     

Sustained response to intravenous alendronate in postmenopausal osteoporosis

We studied the effects of alendronate (amino-hydroxybutylidene bisphosphonate) on biochemical indices of bone turnover and on lumbar spinal bone mineral density in 15 postmenopausal women with vertebral osteoporosis. Alendronate 7.5 mg daily was administered intravenously as a slow infusion for four consecutive days. Treatment was associated with a significant decrease in serum calcium (p < 0.01), fasting urinary calcium excretion (p < 0.01) and hydroxyproline excretion within several days followed a later decrease in serum alkaline phosphatase activity that showed a significant reduction at two months after treatment (p < 0.05). Serum calcium reverted to pretreatment values by the second week after infusion, but the decrease in alkaline phosphatase, urinary calcium, and hydroxyproline excretion persisted to six months after infusion. There was a 3% mean increase in lumbar bone mineral density at six months (p < 0.01). A transient lymphopenia or leucopenia was noted in eight patients and a short-lived fever in six. No other side effects were observed. This study demonstrates that shortterm exposure to high intravenous doses of alendronate induces suppression of bone resorption in osteoporosis that persists for at least 6 months after infusion. We conclude that a short exposure to high intravenous doses induces sustained effects on bone turnover in much the same manner as that observed in Paget's disease of bone.     

The contribution of job satisfaction to the transition from acute to chronic low back pain

The goal of this study was to develop an animal model for the study of acute periodontal disease using silk ligatures in combination with a soft diet in eleven purebred beagle dogs. The silk ligatures were placed subgingivally on the mandible second and fourth premolar on one side of the mouth; the opposite side served as a control. Dogs were monitored during the 16-20 weeks of ligature placement, and for 48 weeks after ligature removal. Development of periodontal disease was evaluated by radiopharmaceutical uptake into bone, radiographic evidence of alveolar bone loss, attachment loss, gingival index and prostaglandin level. Bone loss occurred on the ligatured side during the ligature phase of the study. Radiopharmaceutical uptake was correlated with radiographic evidence of bone loss during the ligature phase. No significant bone loss occurred during the post-ligature phase. Progressive periodontal disease was induced during ligature placement. However, a chronic less aggressive form was not sustained by soft diet alone after ligature removal.     

Quantifying the radiation dosage to individual skeletal lesions treated with samarium-153-EDTMP

Samarium-153ethylenediaminetetramethylenephosphonate (EDTMP) is used in the treatment of painful skeletal lesions. This study attempted to quantify the radiation dosage to individual lesions on both the macroscopic and microscopic level. METHODS: A gamma camera-based quantification technique was adapted and refined for 153Sm. The accuracy of the technique was determined by using a realistic phantom. The activity and volume of lesions as well as normal bone were determined and used to estimate the radiation dosages to these regions. Two patients died of unrelated causes shortly after receiving 153Sm-EDTMP. This made it possible to compare the gamma camera results with direct measurements. It also allowed for autoradiographic examination of the lesions. Finally, the microscopic radiation dosages were estimated. RESULTS: The phantom study indicated that the quantification technique was off, on average, by 4.1% (s.d. = 8.1%). The absolute activity concentration of trabecular bone was found to be approximately 0.22 MBq/g, and that of cortical bone was found to be approximately 0.1 MBq/g, regardless of the dosage administered. The corresponding concentrations for lesions were between 3 and 7 times higher than that of normal bone, with no apparent ceiling. From these results, the macroscopic radiation dosage could be estimated. The dosage to normal bone varied between 0.9 and 3.9 cGy x kg/MBq, and that of the lesions varied between 5.2 and 27.1 cGy x kg/MBq. The autopsy results confirmed that the gamma camera technique was accurate. The autoradiography showed clearly that the activity was associated with the surface of the bone. From these findings, the microscopic radiation dosage distribution was estimated for cortical and trabecular bone as well as osteoblastic lesions. The variation in the microscopic dosage compared to the macroscopic dosage was quite large. Microscopic dosages, when compared to the macroscopic dosages, were as high as 965% and as low as 14.9%. CONCLUSION: The techniques used have been proven to be accurate. The activity in normal bone may be at a ceiling value for all the administered doses, which could explain the small variation. This is not true for the lesions. The large variation in dosages on a microscopic scale, combined with the ceiling in normal bone, may explain the lower than expected toxicity and relatively quick relapse of the patients.     

Comparison of hydroxyapatite granules to autogenous bone graft in fusion cages in a goat model

BACKGROUND: With the increased use of fusion cages to achieve lumbar intervertebral fusion, the question arises as to the potential for bone ingrowth from the host bone through the entire cage. Is it even necessary to have an autogenous graft to achieve total bone incorporation? METHODS: Nine adult male goats had fusion cages implanted into three vertebral bodies. The design was Surgical Dynamics/Ray Fusion Cage, measuring 21 mm x 14 mm. In each animal, one fusion cage was filled with autogenous graft, one with hydroxyapatite, porous granules, and the other with nonporous granules. Amount of new bone formation was determined by backscatter electron microscopy at 3 months post implantation in all animals. RESULTS: The histologic section shows that there was total incorporation in all specimens at 3 months. There was slightly more new bone (43%) with the nonporous granules compared with the porous granules (35%). The amount of residual void space was about the same in all specimens, indicating that the amount of new bone formation was similar and not statistically different in cages filled with hydroxyapatite granules versus granules of autogenous bone. CONCLUSION: This study confirms that total incorporation by ingrowth of new bone can be expected in fusion cages. The amount of ingrowth is about the same for autogenous graft versus hydroxyapatite granules. Apparently, it is not necessary to use bone graft to achieve successful bone incorporation if an acceptable biocompatable lattice, such as hydroxyapatite granules, is used.     

Klebsiella and Enterobacter organisms isolated from horses

An account is given of K. pneumoniae capsule types occurring in horses, with particular reference to strains originating from the genital tract in the mare and the external genitalia of the stallion. A survey of the prevalence of K. pneumoniae and E. aerogenes strains in the preputial flora of healthy stallions is described. The majority of horses were found to be carriers of these organisms. The cultural characteristics of these preputial strains are described and compared with those of K. pneumoniae strains associated with epidemic metritis in mares. The epidemiological significance of certain K. pneumoniae capsule types is discussed.     

Prion protein structural features indicate possible relations to signal peptidases

We describe the treatment of 2 patients with extensive midfacial defects by using a customized composite flap. Autogenous bone was transferred from the iliac crest, revascularized by the rectus muscle, and then transferred as a complete unit by microvascular technique to provide a vascularized bone graft to this area of difficult reconstruction. The bone was shaped and customized to fit the osseous defect. Although the rectus muscle was relatively bulky at the time of harvest, its hearty blood supply permitted trimming and contouring to fit the gap. This technique was based on the principles proposed previously by other authors regarding the vascularization of a free bone graft and its transfer as a composite flap. This technique requires more research and refinement, however this clinical demonstration shows the feasibility of revascularizing a composite flap and the extent to which this technique can be performed.     

Biphasic calcium phosphate concept applied to artificial bone, implant coating and injectable bone substitute

The development of calcium phosphate ceramics and other related biomaterials for bone graft involved a better control of the process of biomaterials resorption and bone substitution. The bioactive concept was developed for biphasic calcium phosphate ceramics (BCP). An optimum balance of the more stable phase of HA and more soluble TCP was obtained for controlling gradual dissolution in the body, seeding new bone formation as it releases calcium and phosphate ions into the biological medium. The bone/material interface and the events occurring in the development of this dynamic interface such as cellular response, biodegradation or bioresorption of the materials and their transformation to carbonate hydroxyapatite (CHA) were described. These processes were observed in both bulk samples, implant coating and injectable bone substitute (IBS).     

Ways to feel comfortable: teaching aids to promote children''s comfort

The plant lectin, soybean agglutinin (SBA), has been widely used to separate heterogeneous populations of cells. In the field of bone marrow transplantation, SBA has been used for partial depletion of T cells from bone marrow allografts to reduce graft-vs.-host disease. SBA's high affinity for many different tumor cells has also indicated its use as a tumor purging agent for autologous bone marrow transplants. We have compared two methods of cell separation using either soluble SBA agglutination, or SBA covalently attached to an activated polystyrene surface. The nonbinding SBA-cell populations generated by these two procedures were very similar in terms of cell recovery, light scatter properties, and phenotypic profile. Notably, both SBA- fractions were enriched in cells with the known progenitor markers, CD34, CD33, and HLA-DR, and were relatively depleted of SBA binding cells. In addition, the activity of each SBA- cell population was measured in vitro in short-term progenitor assays. Here, both SBA- populations were significantly enriched for CFU-GM. When device-separated SBA- cell populations were seeded into long-term bone marrow culture, they produced both increased progenitor activity and cell proliferation compared to unseparated BMMCs. The polystyrene technology described here could reduce or eliminate many of the drawbacks of soluble SBA agglutination, making SBA cell separation a viable and convenient technique for clinical application.     

Prevalence of outer membrane porin alteration in beta-lactam-antibiotic-resistant Enterobacter aerogenes

We evaluated the prevalence of impermeability as a mechanism associated with resistance against beta-lactam antibiotics in members of the family Enterobacteriaceae. During a 1-year period, 80 strains were selected from 3,110 routinely isolated strains according to their noticeable cross-resistance pattern to cephalosporins. They were tested for (i) outer membrane nonspecific porins involved in the entry of small hydrophilic molecules; (ii) the MICs of cefepime, cefotaxime, imipenem, and moxalactam; and (iii) beta-lactamase production. Immunological investigations using specific probes showed that 23 of 80 strains presented an alteration of the porin content, most of them expressing an additional resistance mechanism. The prevalence of this porin-deficient phenotype is especially high in Enterobacter aerogenes and concerns 6.4% of the clinical isolates.     

Investigation of cell envelope damage to Pseudomonas aeruginosa and Enterobacter cloacae by dibromopropamidine isethionate

Electron micrographs of log-phase Pseudomonas aeruginosa and Enterobacter cloacae cultured for 4 h in the presence of subinhibitory concentrations of dibromopropamidine isethionate indicate that this antibacterial agent can cause marked damage to the cell envelope structures of both species. This result provides an explanation of how dibromopropamidine can enhance the uptake and thus the activity of a second antibacterial agent used in combination with it.     

Species specificity of monoclonal antibodies to human tartrate-resistant acid phosphatase

Tartrate-resistant acid phosphatase (TRAP) is expressed abundantly by osteoclasts and is required for bone resorption. This enzyme is emerging as an important biomarker in bone pathology, both for histochemical identification of osteoclasts and as a serum marker of osteoclast activity and increased bone turnover. Rat and mouse models are becoming popular systems for studying osteoclast development, bone physiology and morphogenesis, and bone diseases such as osteoporosis. We have developed two unique antibodies to human TRAP purified from hairy cell leukemia spleen. Both antibodies (9C5 and 14G6) are suitable for immunohistochemistry of osteoclasts and macrophages. Only one (14G6) is capable of immunoprecipitating active TRAP from human cell lysates. Antibody 9C5 reacts with a denatured epitope of TRAP while antibody 14G6 probably reacts with a native, conformational determinant. The high degree of homology among TRAPs of various species predicts that these antibodies should be suitable for work in experimental animals as well as humans. Immunohistochemical staining, electrophoretic analyses, immunoprecipitation and immunoblotting assays of human rat and mouse TRAP were carried out to test the validity of these antibodies as cell markers in rodents. Both antibodies were suitable for immunohistochemistry in all species. Antibody 9C5 was suitable for immunoblotting of denatured TRAP of all species tested. Antibody 14G6 reacted with the native TRAP of humans only and failed to immunoprecipitate mouse or rat TRAP activity. Although TRAP is a phylogenetically conserved protein, subtle, species-specific determinants exist. Care should be exercised when anti-TRAP antibodies are used for immunoassay in experimental animals.