Genetic Disruption of All NO Synthase Isoforms Enhances BMD and Bone Turnover in Mice In Vivo: Involvement of the Renin‐Angiotensin System

Introduction : NO is synthesized by three different NO synthase (NOS) isoforms, including neuronal (nNOS), inducible (iNOS) and endothelial NOS (eNOS). The roles of NO in bone metabolism have been extensively investigated in pharmacological studies and in studies with NOS isoform–deficient mice. However, because of the nonspecificity of agents and compensation among the NOS isoforms, the ultimate roles of endogenous NO are still poorly understood. To address this point, we successfully generated mice in which all three NOS genes are completely disrupted. In this study, we examined whether bone metabolism is abnormal in those mice. Materials and Methods : Experiments were performed in 12‐wk‐old male wildtype, singly nNOS^?/?, iNOS^?/?, and eNOS^?/? and triply n/i/eNOS^?/? mice. BMD was assessed by DXA. The kinetics of osteoblastic bone formation and those of osteoclastic bone resorption were evaluated by measurements of morphological and biochemical markers. Results : BMD was significantly higher only in the triply NOS^?/? mice but not in any singly NOS^?/? mice compared with the wildtype mice. Markers of osteoblastic bone formation, including bone formation rate, mineral apposition rate, and serum alkaline phosphatase concentration, were also significantly larger only in the triply NOS^?/? mice compared with wildtype mice. Furthermore, markers of osteoclastic bone resorption, including osteoclast number, osteoclast surface, and urinary deoxypyridinoline excretion, were again significantly greater only in the triply NOS^?/? mice. Importantly, the renin‐angiotensin system in bone was significantly activated in the triply NOS^?/? mice, and long‐term oral treatment with an angiotensin II type 1 (AT₁) receptor blocker normalized this pathological bone remodeling in those mice. Conclusions : These results provide the first direct evidence that genetic disruption of the whole NOS system enhances BMD and bone turnover in mice in vivo through the AT₁ receptor pathway, showing the critical role of the endogenous NO/NOS system in maintaining bone homeostasis.     

肾精亏虚对雄性小鼠及其雄性仔鼠生育力的影响

目的:观察肾精亏虚对雄性小鼠及其雄性仔鼠生育力的影响。方法:将30只6周龄雄性昆明小鼠随机均分为正常对照组、模型组和补肾组3组。模型组和补肾组采用房劳加惊恐的复合伤肾法制造肾精亏虚模型。补肾组每天给予0.16ml/10g体重的补肾填精方浓缩液灌胃。正常对照组及模型组予等量生理盐水灌胃,造模并给药21d。从造模结束次日起,所有小鼠与正常动情期雌鼠配对饲养5d后检测其精子密度及活动率。取出雌鼠待其分娩,分别计算各组配对雌鼠妊娠率和每胎生产数(仔鼠总数/妊娠并分娩的雌鼠只数)以评估父代小鼠生育力。雄性仔鼠饲养至6周龄时每组随机取出10只检测其精子密度及活动率。结果:模型组每胎生产数[(7.00±1.73)只]明显比正常对照组[(9.43±1.27)只]和补肾组[(8.80±1.10)只]低(P<0.05);模型组小鼠精子密度[(9.70±1.45)×106/ml]及活动率[(66.72±10.12)%]均明显低于正常对照组[(14.08±1.15)×106/ml,(81.75±3.56)%]和补肾组[(12.20±1.55)×106/ml,(78.55±4.38)%],P<0.01,而正常对照组与补肾组之间则没有差异(P>0.05);模型组仔鼠精子密度[(10.1±1.79)×106/ml]及活动率[(71.86±7.48)%]均低于正常对照组[(15.30±1.83)×106/ml,(79.86±5.68)%]和补肾组仔鼠[(14.20±2.21)×106/ml,(81.92±2.51)%],P<0.05,正常对照组与补肾组仔鼠之间没有差异(P>0.05)。结论:"惊恐、房劳"可以明显降低小鼠生育力,其仔鼠生育力亦可受累。补肾法可以阻断这种损伤的发生。     

Molecular and Genetic Aspects of Odontogenic Lesions

In this article we outline the molecular findings of select odontogenic tumors. In each section, we briefly review selected the clinicoradiographic, histologic, immunologic features, focusing on the molecular findings and their applications in practice. The understanding of molecular pathobiology at various other organ sites has developed quite rapidly in recent years, however much remains unknown about the genetic profile of odontogenic tumors. Improved understanding of mutations in odontogenic tumors may clarify classification schema and elucidate targets for novel therapies. Molecular testing will no doubt improve our understanding of odontogenic tumor pathogenesis and will likely be, someday, an important component of routine clinical practice and its role will only increase in the coming years.     

Impact of Genetic and Pharmacologic Inhibition of Myostatin in a Murine Model of Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a genetic connective tissue disorder characterized by compromised skeletal integrity, altered microarchitecture, and bone fragility. Current OI treatment strategies focus on bone antiresorptives and surgical intervention with limited effectiveness, and thus identifying alternative therapeutic options remains critical. Muscle is an important stimulus for bone formation. Myostatin, a TGF-β superfamily myokine, acts through ActRIIB to negatively regulate muscle growth. Recent studies demonstrated the potential benefit of myostatin inhibition with the soluble ActRIIB fusion protein on skeletal properties, although various OI mouse models exhibited variable skeletal responses. The genetic and clinical heterogeneity associated with OI, the lack of specificity of the ActRIIB decoy molecule for myostatin alone, and adverse events in human clinical trials further the need to clarify myostatin's therapeutic potential and role in skeletal integrity. In this study, we determined musculoskeletal outcomes of genetic myostatin deficiency and postnatal pharmacological myostatin inhibition by a monoclonal anti-myostatin antibody (Regn647) in the G610C mouse, a model of mild–moderate type I/IV human OI. In the postnatal study, 5-week-old wild-type and +/G610C male and female littermates were treated with Regn647 or a control antibody for 11 weeks or for 7 weeks followed by a 4-week treatment holiday. Inhibition of myostatin, whether genetically or pharmacologically, increased muscle mass regardless of OI genotype, although to varying degrees. Genetic myostatin deficiency increased hindlimb muscle weights by 6.9% to 34.4%, whereas pharmacological inhibition increased them by 13.5% to 29.6%. Female +/mstn +/G610C (Dbl.Het) mice tended to have similar trabecular and cortical bone parameters as Wt showing reversal of +/G610C characteristics but with minimal effect of +/mstn occurring in male mice. Pharmacologic myostatin inhibition failed to improve skeletal bone properties of male or female +/G610C mice, although skeletal microarchitectural and biomechanical improvements were observed in male wild-type mice. Four-week treatment holiday did not alter skeletal outcomes. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Heterogeneity in the Evolution and Mechanisms of the Lesions of Kidney Allograft Rejection in Mice

The natural history and pathogenesis of the pathologic lesions that define rejection of kidney transplants have not been well characterized. We studied the evolution of the pathology of rejection in mouse kidney allografts, using four strain combinations across full major histocompatibility complex (MHC) plus nonMHC disparities, to permit more general conclusions. Interstitial infiltrate, MHC induction, and venulitis appeared by day 5, peaked at day 7-10, then stabilized or regressed by day 21. In contrast, tubulitis, arteritis, and glomerulitis were absent or mild at days 5 and 7, but progressed through day 21, indicating separate regulation and homeostatic control of these lesions. Edema, hemorrhage, and necrosis also increased through day 21. All lesions were T-dependent, failing to develop in T-cell-deficient hosts. Allografts into immunoglobulin-deficient hosts manifested typical infiltration, MHC induction, and tubulitis at days 7 and 21, indicating that these lesions are alloantibody-independent. However at day 21 kidneys rejecting in immunoglobulin-deficient hosts showed decreased edema, arteritis, venulitis, and necrosis. Thus the three groups of lesions are: T-cell-mediated interstitial infiltration, MHC induction, and venulitis, which develops rapidly then stabilizes; slower but progressive T-cell-mediated tubulitis and arteritis; and late antibody-mediated endothelial injury, which contributes to late edema, arteritis, and venulitis.     

Impaired Cortical Bone Acquisition and Osteoblast Differentiation in Mice with Osteoblast-Targeted Disruption of Glucocorticoid Signaling

To determine the role of endogenous glucocorticoids in bone, we previously developed transgenic mice in which a 2.3 kb fragment of the Col1a1 promoter drives 11beta-hydroxysteroid dehydrogenase 2 expression in mature osteoblasts. This transgene should inactivate glucocorticoids upstream of all receptor signaling pathways. In the present study, we show that femoral cortical bone area and thickness were approximately 10-15% lower in transgenic mice than in wild-type littermates. Femur length was unchanged, indicating that bone elongation was not affected in this model. Expression of osteocalcin mRNA, pOBCol2.3-GFP (a green fluorescent protein marker of mature osteoblasts), and the formation of mineralized nodules were impaired in ex vivo transgenic primary calvarial cultures. The extent of crystal violet staining in bone marrow cultures, indicative of the number of adherent stromal cells, was also decreased. These data suggest that endogenous glucocorticoids are required for cortical bone acquisition and full osteoblast differentiation. It appears that blocking glucocorticoid signaling in vivo leads to a decrease in the commitment and/or expansion of progenitors entering the osteoblast lineage.     

Altered Skeletal Muscle Mitochondrial Proteome As the Basis of Disruption of Mitochondrial Function in Diabetic Mice

Insulin plays pivotal role in cellular fuel metabolism in skeletal muscle. Despite being the primary site of energy metabolism, the underlying mechanism on how insulin deficiency deranges skeletal muscle mitochondrial physiology remains to be fully understood. Here we report an important link between altered skeletal muscle proteome homeostasis and mitochondrial physiology during insulin deficiency. Deprivation of insulin in streptozotocin-induced diabetic mice decreased mitochondrial ATP production, reduced coupling and phosphorylation efficiency, and increased oxidant emission in skeletal muscle. Proteomic survey revealed that the mitochondrial derangements during insulin deficiency were related to increased mitochondrial protein degradation and decreased protein synthesis, resulting in reduced abundance of proteins involved in mitochondrial respiration and β-oxidation. However, a paradoxical upregulation of proteins involved in cellular uptake of fatty acids triggered an accumulation of incomplete fatty acid oxidation products in skeletal muscle. These data implicate a mismatch of β-oxidation and fatty acid uptake as a mechanism leading to increased oxidative stress in diabetes. This notion was supported by elevated oxidative stress in cultured myotubes exposed to palmitate in the presence of a β-oxidation inhibitor. Together, these results indicate that insulin deficiency alters the balance of proteins involved in fatty acid transport and oxidation in skeletal muscle, leading to impaired mitochondrial function and increased oxidative stress.     

Imatinib Mesylate Reduces Endoplasmic Reticulum Stress and Induces Remission of Diabetes in db/db Mice

OBJECTIVE—Imatinib has been reported to induce regression of type 2 diabetes in chronic leukemia patients. However, the mechanism of diabetes amelioration by imatinib is unknown, and it is uncertain whether imatinib has effects on type 2 diabetes itself without other confounding diseases like leukemia. We studied the effect of imatinib on diabetes in db/db mice and investigated possible mechanism''s underlying improved glycemic control by imatinib.RESEARCH DESIGN AND METHODS—Glucose tolerance and insulin tolerance tests were done after daily intraperitoneal injection of 25 mg/kg imatinib into db/db and C57BL/6 mice for 4 weeks. Insulin signaling and endoplasmic reticulum stress responses were studied by Western blotting. β-Cell mass and apoptotic β-cell number were determined by combined terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) staining and insulin immunohistochemistry. The in vitro effect of imatinib was studied using HepG2 cells.RESULTS—Imatinib induced remission of diabetes in db/db mice and amelioration of insulin resistance. Expression of endoplasmic reticulum stress markers in the liver and adipose tissues of db/db mice, such as phospho-PERK, phospho-eIF2α, TRB3, CHOP, and phospho–c-Jun NH₂-terminal kinase, was reduced by imatinib. Insulin receptor substrate-1 tyrosine phosphorylation and Akt phosphorylation after insulin administration were improved by imatinib. Serum aminotransferase levels and hepatic triglyceride contents were decreased by imatinib. Pancreatic β-cell mass was increased by imatinib, accompanied by decreased TUNEL⁺ β-cell and increased BrdU⁺ β-cell numbers. Imatinib attenuated endoplasmic reticulum stress in hepatoma cells in vitro.CONCLUSIONS—Imatinib ameliorated endoplasmic reticulum stress and induced remission of diabetes in db/db mice. Imatinib or related compounds could be used as therapeutic agents against type 2 diabetes and metabolic syndrome.Type 2 diabetes is a metabolic disease that affects 2.8% of all age-groups worldwide, and this proportion is expected to increase to 4.4% by 2030 (1). The two major components of the pathogenesis of type 2 diabetes are insulin resistance and β-cell failure. However, the biochemical mechanisms underlying these two phenomena are incompletely understood.Regarding the mechanism of insulin resistance, several hypotheses have been proposed, for example, increased circulating free fatty acid level, mitochondrial dysfunction, elevated reactive oxygen species production, and increased levels of proinflammatory mediators (2–4). Downstream of these molecules or events, disturbance in intracellular signaling, such as c-Jun NH₂-terminal kinase (JNK) phosphorylation, IKKβ activation, or endoplasmic reticulum stress responses, may play a role in the development of insulin resistance (5–7).Imatinib mesylate (Gleevec) is a well-known anticancer agent that has a dramatic effect on chronic myelogenous leukemia (CML) and gastrointestinal stromal tumor by specifically inhibiting Bcr-Abl or Kit kinase (8,9). Recently, it was reported that imatinib induced remission of type 2 diabetes in patients having both CML and type 2 diabetes (10,11). The effect of imatinib on animal models of type 1 diabetes resulting from islet injury has also been studied (12,13). However, the effect of imatinib on type 2 diabetes itself in patients or experimental animals without other confounding diseases has not been explored. Furthermore, the mechanism of the improvement of type 2 diabetes by imatinib is unknown. In fact, the possibility that improved glucose tolerance in type 2 diabetic patients with CML by imatinib is due to a positive side effect of leukemic responses leading to the nonspecific generalized improvement of health status cannot be eliminated (11). Thus, it is uncertain whether imatinib could improve glycemic control in type 2 diabetic patients or animals without other confounding diseases. To address this question, we investigated whether imatinib could improve glycemic control in diabetic db/db mice.     

WNT5A Has Anti‐Prostate Cancer Effects In Vitro and Reduces Tumor Growth in the Skeleton In Vivo

Prostate cancer is the most frequent malignancy in men, and a major cause of prostate cancer–related death is attributable to bone metastases. WNT5A is known to influence the clinical outcome of various cancer types, including prostate cancer, but the exact mechanisms remain unknown. The goal of this study was to assess the relevance of WNT5A for the development and progression of prostate cancer. WNT5A expression was determined in a cDNA and tissue microarray of primary tumor samples in well‐defined cohorts of patients with prostate cancer. Compared with benign prostate tissue, the expression of WNT5A and its receptor Frizzled‐5 was higher in prostate cancer, and patients with a WNT5A expression above the median had a higher probability of survival after 10 years. Using different osteotropic human prostate cancer cell lines, the influence of WNT5A overexpression and knock‐down on proliferation, migration, and apoptosis was assessed. In vitro, WNT5A overexpression induced prostate cancer cell apoptosis and reduced proliferation and migration, whereas WNT5A knock‐down showed opposite effects. In vivo, different xenograft models were used to determine the effects of WNT5A on tumor growth. Local tumor growth and tumor growth in the bone microenvironment was considerably diminished after WNT5A overexpression in PC3 cells. WNT5A exhibits antitumor effects in prostate cancer cells and may be suitable as a prognostic marker and therapeutic target for prostate cancer and associated skeletal metastases. © 2014 American Society for Bone and Mineral Research.     

Congenic Mice Reveal Sex-Specific Genetic Regulation of Femoral Structure and Strength

Genetic linkage studies in C3H/HeJ (C3H) and C57BL/6J (B6) mice identified several chromosomal locations or quantitative trait loci (QTL) linked to femoral volumetric bone mineral density (vBMD). From QTL identified on chromosomes (chr) 1, 4, 6, 13, and 18, five congenic mouse strains were developed. In each of these mice, genomic DNA from the QTL region of the donor C3H strain was transferred into the recipient B6 strain. Here we report the effects of donated C3H QTL on femoral structure, cortical vBMD and bending strength. Femoral structure was quantified by the polar moment of inertia (I_p) at the mid-diaphysis, which reflects the bending or torsional rigidity of the femur. Although the C3H progenitor mice have a smaller I_p than B6 progenitor mice, the congenic mice carrying the C3H segment at Chr 4 had significantly increased I_p in both males and females, giving these mice stronger femora. In female mice from the congenic Chr 1 strain, I_p was increased whereas male mice from the Chr 1 strain had smaller femoral cross-sections and significantly reduced I_p. This sex-specific effect on femoral structure was seen to a lesser extent in Chr 18 congenic mice. In addition, cortical vBMD was measured using peripheral quantitative computed tomography. Cortical vBMD was similar among most congenic strains except in Chr 6 congenic mice, where cortical vBMD was significantly less in females, but not in males. We conclude that (1) chromosomal QTL from C3H mice, which are genetically linked to total femoral vBMD, also regulate femoral structure; (2) the QTL on Chr 4 improves femoral structure and strength; (3) QTL on Chr 1 and 18 impart sex-specific effects on femoral structure; and (4) the QTL on Chr 6 imparts a sex-specific effect on cortical vBMD and femoral strength.     

Transplantation of Bone Marrow Mononuclear Cells Reduces Mortality and Improves Renal Function on Mercury-Induced Kidney Injury in Mice

Objective: Recent studies have demonstrated the therapeutic effects of bone marrow–derived cells in tissue regeneration. The aim of this study was to investigate the effects of bone marrow mononuclear cell (BMMC) transplantation in a mouse model of acute renal failure (ARF) induced by mercuric chloride. Methods: BMMC was isolated from male BALB/c mice and injected into female mice treated with a lethal dose (LD90) of mercuric chloride. Survival rate, histopathological analysis, and assessment of urea, creatinine, sodium, potassium, and mercury levels were carried out. Results: Cellular therapy with BMMC significantly reduced the mortality induced by mercuric chloride (p < 0.05). This finding correlated with a decrease in serum levels of urea (p = 0.04) and potassium (p < 0.01). However, no differences in renal morphology were observed when BMMC-treated and control group were compared. Conclusion: Transplanted BMMC improve renal function and reduce mortality and, therefore, may represent a new therapeutic alternative to treat ARF.     

Parenteral Vaccination with Heat‐Inactivated Mycobacterium Bovis Reduces the Prevalence of Tuberculosis‐Compatible Lesions in Farmed Wild Boar

In 2012, a wild boar (Sus scrofa) tuberculosis (TB) control programme was set up in a wild boar farm by means of intramuscular (IM) vaccination with a heat‐inactivated Mycobacterium bovis vaccine (IV). The goal was to assess safety and efficacy of the parenterally administered IV in a large farm setting with natural M. bovis circulation. Based on preceding results under laboratory conditions, we hypothesized that vaccinated piglets would show smaller scores of TB‐compatible lesions (TBCL) than unvaccinated controls. After vaccination, no adverse reactions were detected by visual inspection or at post‐mortem examination (n = 668 and 97, respectively). Post‐mortem data on TBCL were available for 97 vaccinated wild boar and 182 controls. The observed TBCL prevalence was 4.1% (95% CI = 0.2–8%) and 12.1% (95% CI = 7.1–17.1%) for vaccinated and control wild boar, respectively (P < 0.05). Among those animals with TBCL, no difference in the mean lesion score was found (P > 0.05). The results show that IV administered intramuscularly to wild boar piglets is safe and protects vaccinated individuals (66% reduction in TBCL prevalence) against natural challenge in a low‐prevalence setting. In a context of increasing TB prevalence in wild boar in Mediterranean habitats, vaccination achieved a progressive though slow decline in lesion prevalence since the onset of the vaccination scheme. Hence, vaccination might contribute, along with other tools, to TB control in wild boar and in pigs.     

In Vivo Development of Transplant Arteriosclerosis in Humanized Mice Reflects Alloantigen Recognition and Peripheral Treg Phenotype of Lung Transplant Recipients

Experimentally, regulatory T cells inhibit rejection. In clinical transplantations, however, it is not known whether T cell regulation is the cause for, or an epiphenomenon of, long‐term allograft survival. Here, we study naïve and alloantigen‐primed T cell responses of clinical lung transplant recipients in humanized mice. The pericardiophrenic artery procured from human lung grafts was implanted into the aorta of NODrag^?/?/IL‐2rγc^?/? mice reconstituted with peripheral blood mononuclear cells (PBMCs) from the respective lung recipient. Naïve or primed allogeneic PBMCs procured 21 days post–lung transplantation with or without enriching for CD4⁺CD25^high T cells were used. Transplant arteriosclerosis was assessed 28 days later by histology. Mice reconstituted with alloantigen‐primed PBMCs showed significantly more severe transplant arteriosclerosis than did mice with naïve PBMCs (p = 0.005). Transplant arteriosclerosis was equally suppressed by enriching for autologous naïve (p = 0.012) or alloantigen‐primed regulatory T cells (Tregs) (p = 0.009). Alloantigen priming in clinical lung recipients can be adoptively transferred into a humanized mouse model. Transplant arteriosclerosis elicited by naïve or alloantigen‐primed PBMCs can be similarly controlled by potent autologous Tregs. Cellular therapy with expanded autologous Tregs in lung transplantation might be a promising future strategy.     

Administration of COG1410 Reduces Axonal Amyloid Precursor Protein Immunoreactivity and Microglial Activation after Controlled Cortical Impact in Mice

Abstract Traumatic axonal injury (TAI) accounts for at least 35% of the morbidity and mortality in traumatic brain injury (TBI) patients without space-occupying lesions. It is also believed to be a key determinant of adverse outcomes such as cognitive dysfunction across the spectrum of TBI severity. Previous studies have shown that COG1410, a synthetic peptide derived from the apolipoprotein E (apoE) receptor binding region, has anti-inflammatory effects after experimental TBI, with improvements in cognitive recovery. However, the effects of COG1410 on axonal injury following TBI are not known. The current study evaluated the effects of 1?mg/kg daily COG1410 versus saline administered intravenously starting 30?min after controlled cortical impact (CCI) injury on pericontusional TAI in young, wild-type C57BL6/J male mice. We found that COG1410 did not affect the number of amyloid precursor protein (APP)-immunoreactive axonal varicosities in the pericontusional corpus callosum and external capsule at 24?h, but reduced APP-immunoreactive varicosities by 31% at 3 days (p=0.0023), and 36% at 7 days (p=0.0009). COG1410 significantly reduced the number of Iba1-positive cells with activated microglial morphology at all three time points by 21-30%. There was no effect of COG1410 on pericontusional white matter volume or silver staining at any time point. This indicates a possible effect of COG1410 on delayed but not immediate TAI. Future studies are needed to investigate the underlying mechanisms, therapeutic time window, and physiological implications of this effect.     

Lesions of T-Cell-Mediated Kidney Allograft Rejection in Mice Do Not Require Perforin or Granzymes A and B

Organ allograft rejection is strongly associated with the presence of alloreactive cytotoxic T cells but the role of cytotoxicity in the pathologic lesions is unclear. Previous studies showed that the principal lesions of kidney rejection - interstitial infiltration, tubulitis, and endothelial arteritis - are T-cell-dependent and antibody-independent. We studied the role of cytotoxic granule components perforin and granzymes A and B in the evolution of the T-cell-mediated lesions of mouse kidney transplant rejection. By real-time RT-PCR, allografts rejecting in wild-type hosts at days 5, 7, 21, and 42 showed massively elevated and persistent expression of perforin and granzymes A and B, but evolution of tubulitis and arteritis did not correlate with increasing granzyme or perforin expression. Allografts transplanted into hosts with disrupted genes for perforin or granzymes A and B showed no change in tubulitis, arteritis, or MHC induction. Thus the development of the histologic lesions diagnostic of T-cell-mediated kidney transplant rejection are associated with but not mediated by perforin or granzyme A or B. Together with previous graft survival studies, these results indicate that the granule-associated cytotoxic mechanisms of T cells are not the effectors of T-cell-mediated allograft rejection.     

Acid Citrate Dextrose Reduces Platelet Retention During In Vitro and In Vivo Dog Hemoperfusion: A Comparative Study of Different Charcoal Adsorbers

Hemoperfusion through charcoal adsorbers has often been proposed for use in exogenous and endogenous intoxication. A serious drawback of this technique is the often encountered loss of platelets and leucocytes. In vitro hemoperfusion studies with heparinized human blood and in vivo hemoperfusion of heparinized Labrador dogs through different types of charcoal adsorbers (Becton-Dickinson [B-D], Gambro, Haemocol) clearly demonstrated that addition of the anticoagulant acid citrate dextrose (ACD) might reduce the loss of platelets. ACD seemed to have no distinct influence on leucocyte retention.     

Disruption of Four-and-a-Half LIM 2 Decreases Bone Mineral Content and Bone Mineral Density in Femur and Tibia Bones of Female Mice

Four-and-a-half LIM 2 (FHL2) is a member of a family of LIM domain proteins which mediate protein-protein interactions. FHL2 acts as a coactivator and binds to important regulators of bone formation such as insulin-like growth factor binding protein (IGFBP)-5, androgen receptor, and β-catenin. We hypothesized that FHL2 is an important regulator of bone formation. We evaluated growth and skeletal parameters in FHL2 knockout (KO) and wild-type (WT) mice at 4, 8, and 12 weeks of age. At 4 weeks of age, lack of FHL2 reduced femur, tibia, and total bone mineral content (BMC) and body weight in all mice. A gender-by-treatment interaction (P ≤ 0.05) was observed for several parameters due to a greater reduction in females. Specifically, femur BMC was reduced 11–27% at 8 and 12 weeks of age and BMD was reduced 7–13% at all ages in female KO mice (P < 0.05). A similar reduction was observed in the tibias at 8 weeks of age. A 6% reduction (P = 0.07) in femur cortical thickness was observed at 12 weeks of age in female KO mice. Interestingly, a gender-specific reduction in IGFBP-5 expression was observed in the femurs of female KO mice. During differentiation of bone marrow stromal cells into osteoblasts, expression of osteocalcin, alkaline phosphatase, and bone sialoprotein was reduced 47–96% in FHL2 KO cells (P < 0.001). In conclusion, FHL2 is an important regulator of peak bone mass, lack of FHL2 produces gender- and site-specific effects on bone accretion and IGFBP-5 expression, and FHL2 is important for optimal osteoblast differentiation in vitro. The information contained in this publication does not necessarily reflect the position or the policy of the government, and no official endorsement should be inferred. All work was performed in facilities provided by the Department of Veterans Affairs.