Histological and radiographic evaluations of demineralized bone matrix and coralline hydroxyapatite in the rabbit tibia.

Complex fractures resulting in bone loss or impaired fracture healing remain problematic in trauma and orthopedic surgeries. Many bone graft substitutes have been developed and are commercially available. These products differ in their osteoconductive and osteoinductive properties. Differential enhancement of these properties may optimize the performance of these products for various orthopedic and craniofacial applications. The use of bone graft substitutes offers the ability to lessen the possible morbidity of the harvest site in autografts. The objective of the present study was to compare the ability of two bone graft substitutes, BioSet RT, an allograft demineralized bone matrix formulation, and ProOsteon 500R, a coralline hydroxyapatite, in a rabbit critical tibial defect model. BioSet RT and ProOsteon 500R were implanted into a unicortical proximal metaphyseal tibial defect and evaluated for new bone formation. Samples were analyzed radiographically and histologically at 1 day, 6 weeks, 12 weeks, and 24 weeks post surgery. Both materials were biocompatible and demonstrated significant bone growth and remodeling. At 12 weeks, the BioSet RT implanted sites demonstrated significantly more defect closure and bone remodeling as determined by radiographic analyses with 10 out of 14 defects being completely healed versus 1 out of 14 being completely healed in the ProOsteon 500R implanted sites. At 24 weeks, both materials demonstrated complete closure of the defect as determined histologically. There were no statistical differences in radiographic scores between the two implanted materials. However, there was an observable trend that the BioSet RT material generated higher histological and radiographic scores, although not statistically significant. This study provides evidence that both BioSet RT and ProOsteon 500R are biocompatible and able to induce new bone formation as measured in this rabbit model. In addition, this in vivo study demonstrates the ability of BioSet RT to induce new bone formation in a shorter timeframe than ProOsteon 500R.

Biomechanical and radiographic comparison of demineralized bone matrix, and a coralline hydroxyapatite in a rabbit spinal fusion model.

The use of bone grafts is an essential component in spinal fusion. Autologous bone has been shown to result in long-term stable arthrodesis between spinal motion segments. However, autograft can be associated with significant morbidity and a limited supply. Alternatives, such as allogeneic demineralized bone matrix (DBM), are a potential source and supplement to autograft bone. The current study compares the ability of a DBM product (BioSet RT) and a coralline hydroxyapatite (Pro Osteon 500R), for inducing spinal fusion in a rabbit model. BioSet RT, alone or in combination with autograft, and Pro Osteon 500R were implanted in the posterior lateral inter-transverse process region of the rabbit spine. The spines were evaluated at 18 weeks for fusion of the L4-L5 transverse processes using a total of 33 skeletally mature male rabbits; 4 naïve animals were also included in the study. Samples were evaluated radiographically, histologically, by palpation, and through mechanical strength testing. Radiographical, histological, and palpation measurements demonstrated the ability of BioSet RT to induce new bone formation and bridging fusion comparable to autograft. This material performed well alone or in combination with autograft material. Despite significantly higher biomechanical testing results, minimal bone formation and fusion was recorded for the Pro Osteon 500R-treated group. This in vivo study demonstrates the ability of BioSet RT to induce new bone formation, and there was a clear relationship between bridging bone and mechanical strength.

Development of immunoglobulin lambda-chain-positive B cells, but not editing of immunoglobulin kappa-chain, depends on NF-kappaB signals.

By genetically ablating IkappaB kinase (IKK)-mediated activation of the transcription factor NF-kappaB in the B cell lineage and by analyzing a mouse mutant in which immunoglobulin lambda-chain-positive B cells are generated in the absence of rearrangements in the locus encoding immunoglobulin kappa-chain, we define here two distinct, consecutive phases of early B cell development that differ in their dependence on IKK-mediated NF-kappaB signaling. During the first phase, in which NF-kappaB signaling is dispensable, predominantly kappa-chain-positive B cells are generated, which undergo efficient receptor editing. In the second phase, predominantly lambda-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding kappa-chain. This second phase of development is dependent on NF-kappaB signals, which can be substituted by transgenic expression of the prosurvival factor Bcl-2.

Gram's stain of peritoneal fluid is rarely helpful in the evaluation of the ascites patient.

STUDY OBJECTIVE: We determine the sensitivity, specificity, and clinical utility of the Gram's stain of peritoneal fluid in patients undergoing paracentesis. METHODS: We conducted a retrospective chart review of all peritoneal fluid analyses in a 3-year period in an urban 3-hospital system. Peritoneal dialysis and diagnostic peritoneal lavage patients were excluded. Data collected included Gram's stain, cell count, and culture results. Spontaneous bacterial peritonitis was defined as an absolute neutrophil count greater than 250 cells/mm(3). In patients with a positive Gram's stain result, charts were reviewed for antibiotic changes. Primary outcome measures were the sensitivity, specificity, and predictive values of the Gram's stain for the detection of spontaneous bacterial peritonitis. RESULTS: Of 796 fluid samples, Gram's stain demonstrated an organism in 31 (3.9%; 95% confidence interval [CI] 2.6% to 5.4%). Gram stain had a sensitivity of 10% (95% CI 6% to 15%), specificity of 97.5% (95% CI 96.7% to 98.3%), positive predictive value of 48% (95% CI 32% to 65%), and negative predictive value of 81.3% (95% CI 80.7% to 82.0%) in the detection of spontaneous bacterial peritonitis. Antibiotic treatment of spontaneous bacterial peritonitis was changed after Gram's stain results in only 1 case, and 16 of 31 positive Gram's stain results occurred in patients without spontaneous bacterial peritonitis, likely representing contaminants. CONCLUSION: The Gram's stain result was rarely positive in patients undergoing paracentesis, and when positive, it rarely provided clinically useful information.

Multiple signaling pathways promote B lymphocyte stimulator dependent B-cell growth and survival.

We investigated the mechanism by which B lymphocyte stimulator (BLyS)/BAFF, a tumor necrosis factor superfamily ligand, promotes B-cell survival and resistance to atrophy. BLyS stimulation activates 2 independent signaling pathways, Akt/mTOR and Pim 2, associated with cell growth and survival. BLyS blocks the cell volume loss (atrophy) that freshly isolated B cells normally undergo when maintained in vitro while concurrently increasing glycolytic activity and overall metabolism. This atrophy resistance requires Akt/mTOR. We used a genetic approach to resolve the contributions of Akt/mTOR and Pim kinase pathways to BLyS-mediated survival. Pim 2-deficient B cells are readily protected from death by BLyS stimulation, but this protection is completely abrogated by treatment with the mTOR inhibitor rapamycin. Furthermore, rapamycin treatment in vivo significantly reduces both follicular and marginal zone B cells in Pim-deficient but not healthy hosts. BLyS-dependent survival requires the antiapoptotic protein Mcl-1. Mcl-1 protein levels rise and fall in response to BLyS addition and withdrawal, respectively, and conditional deletion of the Mcl-1 gene renders B cells refractory to BLyS-mediated protection. Because BlyS is required for the normal homeostasis of all B cells, these data suggest a therapeutic strategy simultaneously inhibiting mTOR and Pim 2 could target pathogenic B cells.

Targeting PIM kinases impairs survival of hematopoietic cells transformed by kinase inhibitor-sensitive and kinase inhibitor-resistant forms of Fms-like tyrosine kinase 3 and BCR/ABL.

Previous studies have shown that activation of the signal transducer and activator of transcription 5 (STAT5) plays an essential role in leukemogenesis mediated through constitutive activated protein tyrosine kinases (PTK). Because PIM-1 is a STAT5 target gene, we analyzed the role of the family of PIM serine/threonine kinases (PIM-1 to PIM-3) in PTK-mediated transformation of hematopoietic cells. Ba/F3 cells transformed to growth factor independence by various oncogenic PTKs (TEL/JAK2, TEL/TRKC, TEL/ABL, BCR/ABL, FLT3-ITD, and H4/PDGFbetaR) show abundant expression of PIM-1 and PIM-2. Suppression of PIM-1 activity had a negligible effect on transformation. In contrast, expression of kinase-dead PIM-2 mutant (PIM-2KD) led to a rapid decline of survival in Ba/F3 cells transformed by FLT3-ITD but not by other oncogenic PTKs tested. Coexpression of PIM-1KD and PIM-2KD abrogated growth factor-independent growth of Ba/F3 transformed by several PTKs, including BCR/ABL. Targeted down-regulation of PIM-2 by RNA interference (RNAi) selectively abrogated survival of Ba/F3 cells transformed by various Fms-like tyrosine kinase 3 (FLT3)-activating mutants [internal tandem duplication (ITD) and kinase domain] and attenuated growth of human cell lines containing FLT3 mutations. Interestingly, cells transformed by FLT3 and BCR/ABL mutations that confer resistance to small-molecule tyrosine kinase inhibitors were still sensitive to knockdown of PIM-2, or PIM-1 and PIM-2 by RNAi. Our observations indicate that combined inactivation of PIM-1 and PIM-2 interferes with oncogenic PTKs and suggest that PIMs are alternative therapeutic targets in PTK-mediated leukemia. Targeting the PIM kinase family could provide a new avenue to overcome resistance against small-molecule tyrosine kinase inhibitors.

Fuel feeds function: energy metabolism and the T-cell response.

Ligation of antigen receptors at the surface of lymphocytes initiates a transcriptional and translational response that is required for cellular proliferation and effector function. By contrast, co-stimulatory-molecule ligation contributes to the immune response by allowing the uptake and utilization of extracellular nutrients to provide energy for cellular proliferation and effector functions. Growth factors also potentiate the ability of lymphocytes to metabolically switch between resting and proliferative states. Lymphocytes that do not receive these signals fail to increase their metabolism to meet the higher bioenergetic demands of cell growth and are either deleted or rendered unresponsive to mitogenic signals. In this Review, we describe how T cells actively acquire metabolic substrates from their environment to meet these energy demands and respond appropriately to pathogens.

Sex-specific effect of insulin-dependent diabetes 4 on regulation of diabetes pathogenesis in the nonobese diabetic mouse.

Many human autoimmune diseases are more frequent in females than males, and their clinical severity is affected by sex hormone levels. A strong female bias is also observed in the NOD mouse model of type I diabetes (T1D). In both NOD mice and humans, T1D displays complex polygenic inheritance and T cell-mediated autoimmune pathogenesis. The identities of many of the insulin-dependent diabetes (Idd) loci, their influence on specific stages of autoimmune pathogenesis, and sex-specific effects of Idd loci in the NOD model are not well understood. To address these questions, we analyzed cyclophosphamide-accelerated T1D (CY-T1D) that causes disease with high and similar frequencies in male and female NOD mice, but not in diabetes-resistant animals, including the nonobese diabetes-resistant (NOR) strain. In this study we show by genetic linkage analysis of (NOD x NOR) x NOD backcross mice that progression to severe islet inflammation after CY treatment was controlled by the Idd4 and Idd9 loci. Congenic strains on both the NOD and NOR backgrounds confirmed the roles of Idd4 and Idd9 in CY-T1D susceptibility and revealed the contribution of a third locus, Idd5. Importantly, we show that the three loci acted at distinct stages of islet inflammation and disease progression. Among these three loci, Idd4 alleles alone displayed striking sex-specific behavior in CY-accelerated disease. Additional studies will be required to address the question of whether a sex-specific effect of Idd4, observed in this study, is also present in the spontaneous model of the disease with striking female bias.

Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and survival.

The Akt kinases promote hematopoietic cell growth and accumulation through phosphorylation of apoptotic effectors and stimulation of mTOR-dependent translation. In Akt-transformed leukemic cells, tumor growth can be inhibited by the mTOR inhibitor rapamycin, and clinical trials of rapamycin analogs for the treatment of leukemia are under way. Surprisingly, nontransformed hematopoietic cells can grow and proliferate in the presence of rapamycin. Here, we show that Pim-2 is required to confer rapamycin resistance. Primary hematopoietic cells from Pim-2- and Pim-1/Pim-2-deficient animals failed to accumulate and underwent apoptosis in the presence of rapamycin. Although animals deficient in Akt-1 or Pim-1/Pim-2 are viable, few animals with a compound deletion survived development, and those that were born had severe anemia. Primary hematopoietic cells from Akt-1/Pim-1/Pim-2-deficient animals displayed marked impairments in cell growth and survival. Conversely, ectopic expression of either Pim-2 or Akt-1 induced increased cell size and apoptotic resistance. However, though the effects of ectopic Akt-1 were reversed by rapamycin or a nonphosphorylatable form of 4EBP-1, those of Pim-2 were not. Coexpression of the transgenes in mice led to additive increases in cell size and survival and predisposed animals to rapid tumor formation. Together, these data indicate that Pim-2 and Akt-1 are critical components of overlapping but independent pathways, either of which is sufficient to promote the growth and survival of nontransformed hematopoietic cells.

The Pim kinases control rapamycin-resistant T cell survival and activation.

Although Pim-1 or Pim-2 can contribute to lymphoid transformation when overexpressed, the physiologic role of these kinases in the immune response is uncertain. We now report that T cells from Pim-1(-/-)Pim-2(-/-) animals display an unexpected sensitivity to the immunosuppressant rapamycin. Cytokine-induced Pim-1 and Pim-2 promote the rapamycin-resistant survival of lymphocytes. The endogenous function of the Pim kinases was not restricted to the regulation of cell survival. Like the rapamycin target TOR, the Pim kinases also contribute to the regulation of lymphocyte growth and proliferation. Although rapamycin has a minimal effect on wild-type T cell expansion in vitro and in vivo, it completely suppresses the response of Pim-1(-/-)Pim-2(-/-) cells. Thus, endogenous levels of the Pim kinases are required for T cells to mount an immune response in the presence of rapamycin. The existence of a rapamycin-insensitive pathway that regulates T cell growth and survival has important implications for understanding how rapamycin functions as an immunomodulatory drug and for the development of complementary immunotherapeutics.

Lymphocyte transformation by Pim-2 is dependent on nuclear factor-kappaB activation.

Pim-2 is a transcriptionally regulated oncogenic kinase that promotes cell survival in response to a wide variety of proliferative signals. Deregulation of Pim-2 expression has been documented in several human malignancies, including leukemia, lymphoma, and multiple myeloma. Here, we show that the ability of Pim-2 to promote survival of cells is dependent on nuclear factor (NF)-kappaB activation. Pim-2 activates NF-kappaB-dependent gene expression by inducing phosphorylation of the oncogenic serine/threonine kinase Cot, leading to both augmentation of IkappaB kinase activity and a shift in nuclear NF-kappaB from predominantly p50 homodimers to p50/p65 heterodimers. Blockade of NF-kappaB function eliminates Pim-2-mediated survival in both cell lines and primary cells, and both Cot phosphorylation and expression are required for the prosurvival effects of Pim-2. Although Pim-2 cooperates with Myc to promote growth factor-independent cell proliferation, this feature is abrogated by NF-kappaB blockade. The ability of Pim-2 to serve as an oncogene in vivo depends on sustained NF-kappaB activity. Thus, the transcriptional induction of Pim-2 initiates a novel NF-kappaB activation pathway that regulates cell survival.

The PP2A-associated protein alpha4 is an essential inhibitor of apoptosis.

Despite evidence that protein kinases are regulators of apoptosis, a specific role for phosphatases in regulating cell survival has not been established. Here we show that alpha4, a noncatalytic subunit of protein phosphatase 2A (PP2A), is required to repress apoptosis in murine cells. alpha4 is a nonredundant regulator of the dephosphorylation of the transcription factors c-Jun and p53. As a result of alpha4 deletion, multiple proapoptotic genes were transcribed. Either inhibition of new protein synthesis or Bcl-xL overexpression suppressed apoptosis initiated by alpha4 deletion. Thus, mammalian cell viability depends on repression of transcription-initiated apoptosis mediated by a component of PP2A.

Beginnings of a signal-transduction pathway for bioenergetic control of cell survival.

Two integral components of cellular transformation are increased cellular metabolism and apoptotic resistance. Recent progress in understanding the cellular functions of core apoptotic components and of growth factor-induced apoptotic regulatory proteins has indicated that the control of cellular metabolism and apoptosis are intertwined. There is growing evidence for connections between the regulation of both cellular bioenergetics and apoptosis and, thus, it is intriguing to explore the idea that growth factor regulation of cellular metabolism can directly affect cell survival.

Akt-directed glucose metabolism can prevent Bax conformation change and promote growth factor-independent survival.

The serine/threonine kinase Akt is a component of many receptor signal transduction pathways and can prevent cell death following growth factor withdrawal. Here, we show that Akt inhibition of cell death is not dependent on new protein translation. Instead, Akt inhibition of cell death requires glucose hydrolysis through glycolysis. Akt was found to regulate multiple steps in glycolysis via posttranscriptional mechanisms that included localization of the glucose transporter, Glut1, to the cell surface and maintenance of hexokinase function in the absence of extrinsic factors. To test the role of glucose uptake and phosphorylation in growth factor-independent survival, cells were transfected with Glut1 and hexokinase 1 (Glut1/HK1) cells. Glut1/HK1 cells accumulated Glut1 on the cell surface and had high glucose uptake capacity similar to that of cells with constitutively active Akt (mAkt). Unlike mAkt-expressing cells, however, they did not consume more glucose, did not maintain prolonged phosphofructokinase-1 protein levels and activity, and did not maintain pentose phosphate shuttle activity in the absence of growth factor. Nevertheless, expression of Glut1 and HK1 promoted increased cytosolic NADH and NADPH levels relative to those of the control cells upon growth factor withdrawal, prevented activation of Bax, and promoted growth factor-independent survival. These data indicate that Bax conformation is sensitive to glucose metabolism and that maintaining glucose uptake and phosphorylation can promote cell survival in the absence of growth factor. Furthermore, Akt required glucose and the ability to perform glycolysis to prevent Bax activation. The prevention of Bax activation by posttranscriptional regulation of glucose metabolism may, therefore, be a required aspect of the ability of Akt to maintain long-term cell survival in the absence of growth factors.

The serine/threonine kinase Pim-2 is a transcriptionally regulated apoptotic inhibitor.

Growth factor withdrawal results in the termination of factor-dependent transcription. One transcript that declines rapidly following growth factor deprivation of hematopoietic cells is the serine/threonine kinase pim-2. When constitutively expressed, Pim-2 conferred long-term resistance to a variety of apoptotic stimuli including growth factor withdrawal and endogenous levels of Pim-2 contributed to growth factor-mediated apoptotic resistance. Pim-2 expression maintained cell size and mitochondrial potential independently of the PI3K/Akt/TOR pathway. Pim-2-dependent maintenance of cell size and survival correlated with its ability to maintain rapamycin-resistant phosphorylation of the translational repressor 4E-BP1 and phosphorylation of the BH3 protein BAD. These results establish Pim-2 as a direct link between growth factor-induced transcription and a novel, kinase-dependent pathway that promotes cell-autonomous survival.

Bcl-X(L) affects Ca(2+) homeostasis by altering expression of inositol 1,4,5-trisphosphate receptors.

An oligonucleotide-based microarray analysis of 9,500 genes and expressed sequence tags (ESTs) demonstrated that the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R) was significantly down-regulated in Bcl-X(L)-expressing as compared with control cells. This result was confirmed at the mRNA and protein levels by Northern and Western blot analyses of two independent hematopoietic cell lines and murine primary T cells. Bcl-X(L) expression resulted in a dose-dependent decrease in IP(3)R protein. IP(3)R expression is regulated as part of a mitochondrion-to-nucleus stress-responsive pathway. The uncoupling of mitochondrial oxidative phosphorylation resulted in induction of binding of the transcription factor NFATc2 to the IP(3)R promoter and transcriptional activation of IP(3)R. Expression of Bcl-X(L) led to a decreased induction of both NFATc2 DNA binding to the IP(3)R promoter and IP(3)R expression in response to the inhibition of mitochondrial oxidative phosphorylation. The Bcl-X(L)-dependent decrease in IP(3)R expression also correlated with a reduced T cell antigen receptor ligation-induced Ca(2+) flux in Bcl-X(L) transgenic murine T cells, and microsomal vesicles prepared from Bcl-X(L)-overexpressing cells exhibited lower IP(3)-mediated Ca(2+) release capacity. Furthermore, reintroducing IP(3)R into Bcl-X(L)-transfected cells partially reversed Bcl-X(L)-dependent anti-apoptotic activity. These results suggest that even under non-apoptotic conditions, expression of Bcl-2-family proteins influences a signaling network that links changes in mitochondrial metabolism to alterations in nuclear gene expression.

Molecular analysis of mouse T cell receptor expression using PCR.

This unit describes the use of the polymerase chain reaction (PCR) to characterize rearranged murine T cell receptor (TCR) transcripts present in primary lymphoid tissues, peripheral lymphoid tissues, and extra-lymphoid sites of inflammation, and to quantify them either relatively or absolutely. This methodology has been used extensively to characterize TCR variability in murine thymus and liver in T cell clones, and in extralymphoid tissues where the absolute number of T cells is limited. A procedure for cloning and sequencing PCR-amplified cDNA transcripts is provided to examine the junctional diversity of expressed genes. Also included is a method for reverse transcription of RNA into cDNA that is optimal for analysis of tissue samples where the number of T cells is limited. The detailed protocols are followed by a commentary that discusses strategies and artifacts as well as providing troubleshooting suggestions for PCR amplification and TCR analysis.