Hamid Shegarfi

Review Article: Bone Transplantation and Immune Response

Bone is the second most common transplant tissue after blood, with the iliac crest autologous graft being most used. Bone transplantation induces osteogenesis to repair bone defects. Despite being the most efficient, autogenous bone requires an additional incision and its supply may be inadequate. Deep-frozen allogeneic bone can be an alternative, but is at risk of microbiological contamination, transmission of unrecognised germs, delayed incorporation, and cellular and humoral immune reactions. Synthetic graft substitutes combine scaffolding properties with biological elements to stimulate cell proliferation and differentiation and eventually osteogenesis. However, they generally lack osteoinductive or osteogenic properties and have various effects on bone healing. We present an overview of bone grafts and graft substitutes in clinical use, and the immune responses to allogeneic bone.

Lipopolysaccharide impairs fracture healing An experimental study in rats

Background It has been shown that trauma causes translocation of lipopolysaccharide (LPS) endotoxins from the gut. LPS has been identified as a major bacterial bone resorbing factor. The effects of LPS on bone healing are therefore of clinical interest, as trauma involving fractures followed by sepsis is a clinical scenario. We investigated the effects of systemic and local administration of LPS on the healing of femoral fractures in rats. Animals and methods In 3 groups, each consisting of 9 rats, a mid-diaphyseal osteotomy/fracture of the femoral bone was performed and then nailed. In one group of animals, LPS was applied intraperitoneally (systemically), and in another group, LPS was applied locally at the fracture site. The third group served as a control. The animals were killed after 6 weeks, and the mechanical characteristics of the healing osteotomies were evaluated. Results We found that LPS induced a hypertrophic and immature callus, as evaluated by bone mineral content and density. In the rats given LPS intraperitoneally, the mechanical strength characteristics were reduced, as evaluated by bending moment, rigidity, and energy absorption. Interpretation The rats given LPS intraperitoneally reflect a clinical situation with fracture trauma and endotoxinemia. Our findings indicate that endotoxinemia may impair the fracture healing processes.         ▪

Impact of MHC Mismatch and Freezing on Bone Graft Incorporation: An Experimental Study in Rats

Cortical bone graft failure develops for poorly defined reasons, and the effects of the immune responses on the incorporation of an allograft are less clear. In a rat model of tibial allotransplantation, we have studied biometric and histological changes of the graft and the humoral immune response against it. We have also compared fresh with prefrozen grafts to study putative effects of freezing on the healing of the graft and the immune response against it. Fresh and frozen cortical bone grafts matched or mismatched for major histocompatibility complex antigens (syngeneic and allogeneic grafts) were implanted in an 8‐mm segmental defect in the tibia. The construct was stabilized with intramedullary nailing. Incorporation of the graft was assessed with use of conventional radiography, micro computed tomography (CT(, biomechanical testing and histological examination. The immune response was evaluated by monitoring distribution of leukocytes in the blood and by measuring antibodies in a tailor‐made fluorescence activating cell scanning (FACS( analysis. We found that the fresh syngeneic grafts were well integrated after 8 weeks with intact bone cells. In the fresh allogeneic grafts, all cells were dead with radiological signs of resorption, and mechanical testing indicated failure of incorporation. The frozen grafts showed poorer overall reconstruction than the fresh syngeneic grafts, but the incorporation was better than the fresh allogeneic grafts. A measurable alloantibody response was only detected after fresh allografting. The combined results suggest that freezing of bone allograft impedes the antibody response against major histocompatibility complex (MHC( antigens and improves incorporation, but frozen allografts still perform poorer than do frozen syngeneic grafts.

The role of natural killer cells in resistance to the intracellular bacterium Listeria monocytogenes in rats.

We have investigated the influence of early innate immune resistance mechanisms on infection with the intracellular bacterium Listeria monocytogenes in rats. Rats were injected i.v. with various amounts of Listeria and the number of bacterial colonies in the spleen was determined at different time points after infection. A bacterial dose as low as 2 × 104 cells gave reproducible infection within the spleen. Athymic nude rats lacking normal T cells but with a robust NK cell repertoire for MHC antigens were more resistant to bacterial replication within the spleen than were normal littermate rats and eliminated the infection within 3 days. In vivo depletion of NK cells, or NK subpopulations expressing Ly49 receptors, increased the bacterial load in the spleen, indicating that these cells were important in the initial control of Listeria infection. An increased frequency of Ly49 expressing NK cells in Listeria‐infected rats further supported this notion. As several rat strains, unlike mice, display a large repertoire of MHC‐recognizing activating Ly49 receptors, these observations raise the interesting possibility that NK cells may recognize alterations in the MHC‐I molecules on Listeria‐infected cells leading to their elimination before the adaptive immune system comes into play.

Impact of freezing on immunology and incorporation of bone allograft

With an increasing clinical use of deep frozen allograft for bone reconstruction, it is important to understand the immunological and biological events of allograft incorporation. In this study, we have investigated the impact of deep freezing on immunology and biopotency for incorporation of bone allografts. Deep frozen bone grafts matched or mismatched for major histoscompatibilty complex (MHC) were implanted in an 8‐mm segmental defect in the tibia in rats. The construct was stabilized with intramedullary nailing. The immune response was evaluated by determination of serum antibody against the grafts MHC molecules at day 1 and after 2 and 4 months. Incorporation of the graft was compared with fresh syngeneic grafts and assessed with the use of conventional radiography, biomechanical testing and measurement of bone mineral content and density after 4 months. The analyses revealed no antibody responses in the rats that received grafts from donors differing at histocompatibility loci, and at 4 months the frozen grafts showed an overall reconstruction that was not significantly different from the fresh grafts. This study indicates that in the long run there are no significant consequences; either immunological or biomechanical, of the use of deep frozen allogenous bone as compared to fresh autogenous bone grafts in this animal model.

The Role of Natural Killer Cells in Alzheimer's Disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive dysfunction and selective neuronal death in the brain. The aetiology of AD is not clear but environmental factors and heritable predisposition may play a role in the disease emergence. It has also been suggested that neural–immune interaction has a role in disease appearance. However, the underlying mechanisms are still unknown. Natural killer (NK) cells play an important role in the host defence, which is related to their ability to secrete a variety of cytokines and chemokines, as well as killing infected host cells. Moreover, there is some evidence that imply the involvement of NK cells in immunopathogenesis of AD. In this review, we have attempted to clarify the role of NK cells in the immunopathogenesis of AD.

Short-term treatment with COX-2 inhibitors does not impair fracture healing.

ABSTRACT Background: The effects of cyclooxygenase (COX) inhibition on fracture healing are insufficiently documented, and the aim of this study was to evaluate the effects of nonspecific and specific COX-2 inhibition in the early phase of fracture healing. Methods: Thirty rats were randomized in three groups. A diaphyseal fracture was performed and stabilized by intramedullary nailing. In group A parecoxib in a dose of 1 mg/kg body weight/day was given prior to surgery and daily for seven days; in group B diclofenac 2 mg/kg body weight/day was given; and in group C the same amount of saline was given. Blood samples were harvested at 7 and 30 days postoperatively and analyzed for active medications. At 30 days the rats were sacrificed, and the fractures were examined for bone mineralization and tested mechanically. Results: The fractures healed by the production of callus. Plasma concentrations at seven days of medication revealed therapeutic levels of parecoxib, valdecoxib, and diclofenac. There were no significant differences in bone mineralization or mechanical characteristics between the three groups at 30 days postfracture. Conclusion: This study indicates that nonspecific or specific COX-2 inhibitors in therapeutic doses during seven days after fracture do not significantly influence bone healing.

The Role of Natural Killer Cells in the Defense against Listeria monocytogenes Lessons from a Rat Model

Ly49 receptors in rodents, like killer cell immunoglobulin-like receptors in humans, regulate natural killer (NK) cell activity. Although inhibitory Ly49 receptors clearly recognize classical major histocompatibility complex class I (MHC-I) molecules, the role for the activating Ly49 receptors has been less well understood. Here, we discuss recent data from a rat model for listeriosis. Rats depleted of NK cells, or more specifically the Ly49 receptor-bearing cells, showed increased bacterial loads in their spleen. Athymic nude rats with no functional T cells but increased numbers of Ly49-expressing NK cells were more resistant to infection, indicating a central role of NK cells in early immune defense against Listeria in this species. Listeria infection of macrophages or enteric epithelial cells led to upregulation of MHC-I, including nonclassical (Ib) molecules not regularly recognized by T cells. We have shown that activating Ly49 receptors are more efficiently stimulated when binding to upregulated class Ib antigens on infected cells. From this we postulate that activating Ly49 receptors may have a sentinel function in the early immune response against Listeria in detecting diseased cells ‘flagged’ by increased MHC-Ib expression.

The activating rat Ly49s5 receptor responds to increased levels of MHC class Ib molecules on Listeria monocytogenes-infected enteric epithelial cells.

We have investigated whether rat Ly49 receptors can monitor Listeria‐infected intestinal epithelial cells through altered expression of MHC class I molecules. The rat colon carcinoma epithelial cell line CC531 infected with Listeria expressed higher levels of both classical and nonclassical MHC‐I molecules. Reporter cells expressing the activating Ly49s5 receptor displayed increased stimulatory responses when incubated with Listeria‐infected CC531 cells in vitro, which could be blocked with mAb 8G10 specific for nonclassical MHC‐I molecules of the RT1u haplotype, but not with mAb OX18 reacting with classical MHC‐I molecules in this haplotype. Similar responses were observed against IFN‐γ‐treated cells that also upregulated their expression of MHC‐I molecules. Thus, the Ly49s5 receptor can respond to increased levels of nonclassical MHC‐I molecules induced on target cells by either bacterial infection or cytokine stimulation. We furthermore found that splenic NK and NKT cells produced IFN‐γ in response to Listeria‐infected CC531 cells, and that this was not limited to Ly49‐expressing cells, since similar levels of IFN‐γ production were observed in Ly49+ and Ly49− NK cell subsets. Therefore, NK cells may recognize Listeria‐infected cells through both MHC‐I‐dependent and ‐independent innate immune receptor systems.

The rat NK cell receptors Ly49s4 and Ly49i4 recognize nonclassical MHC-I molecules on Listeria monocytogenes-infected macrophages.

Ly49 receptors in rodents, like KIRs in humans, regulate NK cell activity. Although inhibitory Ly49 receptors clearly recognize MHC‐I molecules, ligands for the activating Ly49 receptors are less well defined. Here, we show that the activating Ly49s4 and the inhibitory Ly49i4 receptors recognize nonclassical MHC‐I molecules on the rat macrophage cell line R2 (RT1d). Listeria infection of R2 macrophages led to increased expression of classical and nonclassical MHC‐I molecules. Coincubation of these infected cells with reporter cells expressing Ly49i4 or Ly49s4 increased the reporter cell responses. These responses were blocked by mAb OX18 (anti‐MHC‐I) and AAS1 (anti‐nonclassical MHC‐I). IFN‐γ treatment of normal R2 cells also increased the MHC‐I expression and enhanced the reporter cell responses. These results suggest that activating and inhibitory Ly49 receptors monitor MHC‐I expression on Listeria‐infected cells.