Donald Regula

Effects of an isogenic Zn-metalloprotease-deficient mutant of Legionella pneumophila in a guinea-pig pneumonia model.

To determine the effects, if any, of the Zn‐metallo‐protease on virulence of Legionella pneumophila infection, an isogenic mutant deficient in protease (encoded by the proA gene) was tested in an Acantha‐moeba cell model, in guinea‐pig macrophages, and in a guinea‐pig pneumonia model. The cloned proA gene was completely inactivated by insertion of a kanamycin‐resistance cassette into the protease gene of L. pneumophila AA100. This mutated gene was then introduced into the L. pneumophila chromosome by allelic exchange to form the isogenic ProA mutant AA200. AA200 showed no difference in its ability to enter, survive, or grow in Acanthamoeba and explanted guinea‐pig macrophages; neither light nor electron microscopy revealed morphological differences in the eukaryotic cells infected with the protease mutant or the wild‐type strains. The proA gene was found to be expressed in L. pneumophila during intracellular growth in amoebae by measuring the light produced from a truncated luxC gene fusion with the proA promoter. Virulence of the protease mutant was attenuated when tested in a guinea‐pig model of infection employing the intratracheal Inoculation method. AA200 was slower to cause death, grew to lower numbers in the lungs, resulted in less necrotic debris and a larger macrophage infiltrate, and was more likely to be found in association with macrophage vacuoles than the parent strain.

Tissue ingrowth and differentiation in the bone-harvest chamber in the presence of cobalt-chromium-alloy and high-density-polyethylene particles.

Particulate wear debris from joint replacements has been implicated in the etiology of periprosthetic bone resorption. However, the effect of high-density-polyethylene or cobalt-chromium-alloy particles on osteoclastic bone resorption in vivo has not been studied previously, to our knowledge. Therefore, we examined the effect of these particles on tissue ingrowth, net bone formation (per cent trabecular bone), and osteoclastic bone resorption (osteoclasts per unit of bone surface) with use of a bone-harvest chamber that had a transverse one-millimeter channel for tissue ingrowth. After an initial six-week period for incorporation of the chamber into the proximal part of the tibia of rabbits, the contents of the channel were harvested repeatedly at three-week intervals. The carrier solution, 1 per cent sodium hyaluronate, was implanted first. In subsequent implantations, the hyaluronate was mixed with high-density-polyethylene or cobalt-chromium particles at concentrations of 10(8) particles per milliliter. The tissue harvested from the chambers that contained no particles was composed of longitudinally oriented trabecular bone in a fibrovascular stroma. Particulate high-density polyethylene evoked a moderate foreign-body reaction and a chronic inflammatory response and decreased net bone formation. When cobalt-chromium particles had been implanted, the tissue exhibited a more florid foreign-body reaction and a chronic inflammatory response, often in a nodular arrangement, in a background of dense connective tissue. Bone was sparse, and areas of cell necrosis and hyaline degeneration were noted. Histomorphometric analyses were carried out to determine the amount of net bone formation and osteoclastic bone resorption in the presence or absence of high-density-polyethylene or cobalt-chromium particles. The amount of bone was greatest in the control specimens, moderately decreased in the presence of high-density-polyethylene particles, and greatly decreased in the presence of cobalt-chromium particles. The number of osteoclasts in Howship lacunae per unit of trabecular bone surface was increased in the presence of high-density polyethylene, indicating that these particles stimulate osteoclastic bone resorption.

Polyethylene and titanium alloy particles reduce bone formation: Dose-dependence in bone harvest chamber experiments in rabbits

Particles similar to those generated from joint replacements affect net bone formation within the Bone Harvest Chamber in rabbits. Whether these effects depend on the concentration of particulate materials is unknown. In this study, we performed a histomorphologic and morphometric analysis of net bone formation in the Bone Harvest Chamber in the presence of different concentrations of phagocytosable particles of high density polyethylene and titanium 6-aluminum 4-vanadium alloy. Chambers were implanted in 9 mature New Zealand white rabbits bilaterally. Concentrations of 10(6), 10(7) and 10(8) polyethylene particles/mL, and 10(8) and 10(9) particles/ mL of titanium alloy in 1% sodium hyaluronate carrier were implanted for 3-week periods in sequence in each of the chambers. 3-week control periods in which nothing was implanted in the chamber were included between the treatments. Increasing concentrations of polyethylene particles were associated with a more marked foreign body response and fibrosis. Net bone formation for the three polyethylene doses was reduced by 11%, 21% and 33% of controls, respectively. For titanium alloy, net bone formation was reduced by 8% and 56% of controls, for concentrations of 10(8) and 10(9) particles/mL, respectively. Our findings suggest possible adverse effects of wear debris on net bone formation and bony remodeling in the prosthetic bed, when concentrations of specific particles reach critical local levels.

Epstein-Barr virus-associated natural killer-large granular lymphocyte leukemia

We describe the first case of an Epstein-Barr virus (EBV)-associated natural killer-large granular lymphocyte (NK-LGL) leukemia in the United States to the best of our knowledge. A 29-year-old woman of Japanese descent developed EBV infection after a blood transfusion as indicated by a rise in serum antibody titers. Peripheral blood and bone marrow aspirate smears demonstrated increased LGLs. Flow cytometry showed that these cells expressed NK-associated surface antigens. Cytogenetic analysis of the bone marrow aspirate showed two distinct but related clones with multiple copies of a modified 7 marker chromosome. Death followed colonic perforation. Findings at necropsy included bone marrow lymphocytosis and erythrophagocytosis, a mononucleosis-like lymphadenitis, atypical hepatitis with a mixed, predominantly T-cell infiltrate, interstitial pneumonitis, and multiorgan system vasculitis with perforation of the transverse colon. Epstein-Barr virus transcripts were identified in lymphocytes infiltrating liver and peripheral nerve by in situ hybridization. In addition, Southern blot analyses showed monoclonal bands superimposed on oligoclonal ladders of EBV termini in liver and lymph node. The identical episomal form of EBV was found in the bone marrow, lymph node, and liver. No immunoglobulin (Ig), T-cell receptor beta, or T-cell receptor gamma chain gene rearrangements were identified. These studies support the hypothesis that the LGL population was a neoplastic EBV-related clonal proliferation of NK cells.

The characterization of macrophages and osteoclasts in tissues harvested from revised total hip prostheses

The differentiation and maturation of macrophages and osteoclasts at the prosthetic interface in cases of implant loosening are poorly understood. Using histochemical and immunohistochemical staining methods, we compare macrophage differentiation in tissues from revised hip replacements in patients with specific clinical-radiological appearances. Periprosthetic tissues were harvested from 12 cemented acetabular and 12 cemented femoral components in 24 patients undergoing revision hip replacement. The prostheses were all radiographically and clinically loose. Six acetabular and six femoral components demonstrated radiographic ballooning osteolysis. Serial 6 microm frozen sections of the periprosthetic tissues were processed with hematoxylin and eosin for general tissue morphology, and analyzed for the presence of tartrate resistant acid phosphatase (TRAP, an osteoclast marker). Immunoperoxidase staining using monoclonal antibodies to CD68 (macrophages and osteoclasts) and CD51 (the alpha chain of the vitronectin receptor, an osteoclast marker) was also performed. Approximately 8-30% of the total cells in the tissues were positive for TRAP and the vitronectin receptor, and comprised a subset of the CD68 positive macrophages and macrophage polykaryons. However, there were no statistically significant differences between specific groups (femoral vs. acetabular, osteolysis vs. no osteolysis) for the numbers or percentages of macrophages or osteoclast-like cells. Once prosthetic loosening has occurred, few differences in the macrophage-osteoclast profile of tissues from different periprosthetic locations, with and without osteolysis, are noted. This suggests a final common biologic pathway for periprosthetic bone resorption, once implant loosening has transpired.

Effects of TGFβ on bone ingrowth in the presence of polyethylene particles

We implanted bone harvest chambers (BHCs) bilaterally in ten mature male New Zealand white rabbits. Polyethylene particles (0.3+/-0.1 microm in diameter, 6.4 x 10(12) particles/ml) were implanted for two, four or six weeks bilaterally in the BHCs, with subsequent removal of the ingrown tissue after each treatment. In addition to the particles, one side also received 1.5 microg of recombinant transforming growth factor beta1 (TGFbeta1). At two weeks, the bone area as a percentage of total area was less in chambers containing TGFbeta compared with those with particles alone (7.8+/-1.3% v 16.9+/-2.7% respectively; 95% confidence interval (CI) for difference -14.0 to -4.30; p = 0.002). At four weeks, the percentage area of bone was greater in chambers containing TGFbeta compared with those with particles alone (31.2+/-3.4% v 22.5+/-2.0% respectively; 95% CI for difference 1.0 to 16.4; p = 0.03). There were no statistical differences at six weeks, despite a higher mean value with TGFbeta treatment (38.2+/-3.9% v 28.8 +/-3.5%; 95% CI for difference -4.6 to 23.3; p = 0.16). The number of vitronectin-receptor-positive cells (osteoclast-like cells) was greater in the treatment group with TGFbeta compared with that with particles alone; most of these positive cells were located in the interstitium, rather than adjacent to bone. TGFbeta1 is a pleotropic growth factor which can modulate cellular events in the musculoskeletal system in a time- and concentration-dependent manner. Our data suggest that there is an early window at between two and six weeks, in which TGFbeta may favourably affect bone ingrowth in the BHC model. Exogenous growth factors such as TGFbeta may be a useful adjunct in obtaining osseointegration and bone ingrowth, especially in revisions when there is compromised bone stock and residual particulate debris.

Cement particles inhibit bone growth into titanium chambers implanted in the rabbit

Particles of bone cement have been shown previously to stimulate the resorption of bone. The purpose of this study was to determine whether particles of bone cement (BC) have an adverse effect on bone ingrowth. The bone harvest chamber was implanted bilaterally in the proximal tibial metaphysis of 6 mature rabbits. Both the fixed outer cylinder and the inner removable core of the chamber have a transverse 1 mm wide pore providing a continuous canal for tissue ingrowth. After an initial 6-week period for osseointegration of the outer cylinder, the contents of the inner core were harvested repeatedly at 3 weekly intervals. In the first series of rabbits, the carrier solution, 1% sodium hyaluronate (Healon) was implanted first. In subsequent implantations, Healon was mixed with small fabricated particles of BC (averaging 3.54 mm in diameter) to fill the channel of the core. The contralateral chamber was left empty and served as a control. In the second series of rabbits, implantation was carried out sequentially using the same material bilaterally. The sections from the control harvests, and those with Healon alone contained extensive trabecular bone arranged longitudinally in the canal, in a fibrovascular stroma. The sections containing BC particles were infiltrated by foamy, mononuclear and multinuclear histiocytic cells. Less trabecular bone was seen in the sections containing BC particles compared to the control sections or those containing Healon alone. Previous studies have shown that particles of bone cement stimulate bone resorption. In this study, BC particles have also been shown to diminish the formation of new bone.

Fatal 2009 Influenza A (H1N1) Infection, Complicated by Acute Respiratory Distress Syndrome and Pulmonary Interstitial Emphysema

The authors present an excellently documented case that demonstrates the radiologic-pathologic correlation of 2009 influenza A (H1N1) and its complications and discuss possible reasons for severe complications in patients with compromised immune status.

Continuous intramedullary polymer particle infusion using a murine femoral explant model

In vitro models are important investigative tools in understanding the biological processes involved in wear-particle-induced chronic inflammation and periprosthetic osteolysis. In the clinical scenario, particles are produced and delivered continuously over extended periods of time. Previously, we quantified the delivery of both polystyrene and polyethylene particles over 2- and 4-week time periods using osmotic pumps and collection tubes. In the present study, we used explanted mice femora in organ culture and showed that continuous intramedullary delivery of submicron-sized polymer particles using osmotic pumps is feasible. Furthermore, infusion of 2.60 x 10(11) particles per mL (intermediate concentration) of ultrahigh molecular weight polyethylene (UHMWPE) for 2 weeks and 8.06 x 10(11) particles per mL (high concentration) UHMWPE for 4 weeks both yielded significantly higher scores for bone loss when compared with controls in which only mouse serum was infused.

Different effects of phagocytosable particles during bone formation versus remodeling

Previously, small phagocytosable particles of high density polyethylene (HDPE) but not Ti6-Al4-V alloy, at a concentration of 10(8) particles/mL inhibited net bone formation in vivo after 3 weeks in the bone harvest chamber (BHC). These findings reflected the effects of particles during the phase of bone ingrowth. In this study, we tested whether these effects persisted or were different during the phase of bone maturation and remodeling. BHCs were bilaterally implanted in mature male NZW rabbits. After a 6-week period for osseointegration, the contents of the chamber were harvested and discarded. One percent sodium hyaluronate, the carrier, was then placed within the canal of the chambers bilaterally and the tissue within the chambers was harvested 3 weeks later. HDPE particles were then inserted unilaterally for a 3-week period, followed by Ti6-Al4-V for 3 weeks, HDPE for 6 weeks, and Ti6-Al4-V for 6 weeks. The side chosen for each treatment was switched consecutively; the nonimplanted, contralateral chamber served as a control. At 3 weeks the control treatments yielded trabeculae of woven bone in a fibrovascular stroma. By 6 weeks, the peripheral trabeculae were thicker, and a central marrow cavity was developing. Bone ingrowth was less with HDPE particles at 3 and 6 weeks compared to controls. Ti6-Al4-V particles did not inhibit bone ingrowth at 3 weeks but showed a trend at 6 weeks. The characteristics of particles affect the differentiation, maturation, and remodeling of mesenchymal tissue differently.