Absence of Dap12 and the αvβ3 integrin causes severe osteopetrosis

In vitro, ligand occupancy of αvβ3 integrin induces phosphorylation of Dap12, which is essential for osteoclast function. Like mice deleted of only αvβ3, Dap12^−/− mice exhibited a slight increase in bone mass, but Dap12^−/− mice, lacking another ITAM protein, FcRγ, were severely osteopetrotic. The mechanism by which FcRγ compensates for Dap12 deficiency is unknown. We find that co-deletion of FcRγ did not exacerbate the skeletal phenotype of β3^−/− mice. In contrast, β3/Dap12 double-deficient (DAP/β3^−/−) mice (but not β1/Dap12 double-deficient mice) were profoundly osteopetrotic, reflecting severe osteoclast dysfunction relative to those lacking αvβ3 or Dap12 alone. Activation of OSCAR, the FcRγ co-receptor, rescued Dap12^−/− but not DAP/β3^−/−osteoclasts. Thus, the absence of αvβ3 precluded compensation for Dap12 deficiency by FcRγ. In keeping with this, Syk phosphorylation did not occur in OSCAR-activated DAP/β3^−/− osteoclasts. Thus, FcRγ requires the osteoclast αvβ3 integrin to normalize the Dap12-deficient skeleton.     

TGF-β1 Down-Regulation of NKG2D/DAP10 and 2B4/SAP Expression on Human NK Cells Contributes to HBV Persistence

The mechanism underlying persistent hepatitis B virus (HBV) infection remains unclear. We investigated the role of innate immune responses to persistent HBV infection in 154 HBV-infected patients and 95 healthy controls. The expression of NKG2D- and 2B4-activating receptors on NK cells was significantly decreased, and moreover, the expression of DAP10 and SAP, the intracellular adaptor proteins of NKG2D and 2B4 (respectively), were lower, which then impaired NK cell-mediated cytotoxic capacity and interferon-γ production. Higher concentrations of transforming growth factor-beta 1 (TGF-β1) were found in sera from persistently infected HBV patients. TGF-β1 down-regulated the expression of NKG2D and 2B4 on NK cells in our in vitro study, leading to an impairment of their effector functions. Anti-TGF-β1 antibodies could restore the expression of NKG2D and 2B4 on NK cells in vitro. Furthermore, TGF-β1 induced cell-cycle arrest in NK cells by up-regulating the expression of p15 and p21 in NK cells from immunotolerant (IT) patients. We conclude that TGF-β1 may reduce the expression of NKG2D/DAP10 and 2B4/SAP, and those IT patients who are deficient in these double-activating signals have impaired NK cell function, which is correlated with persistent HBV infection.     

Progressive Muscular Dystrophy in α-Sarcoglycan–deficient Mice

Limb-girdle muscular dystrophy type 2D (LGMD 2D) is an autosomal recessive disorder caused by mutations in the α-sarcoglycan gene. To determine how α-sarcoglycan deficiency leads to muscle fiber degeneration, we generated and analyzed α-sarcoglycan– deficient mice. Sgca-null mice developed progressive muscular dystrophy and, in contrast to other animal models for muscular dystrophy, showed ongoing muscle necrosis with age, a hallmark of the human disease. Sgca-null mice also revealed loss of sarcolemmal integrity, elevated serum levels of muscle enzymes, increased muscle masses, and changes in the generation of absolute force. Molecular analysis of Sgca-null mice demonstrated that the absence of α-sarcoglycan resulted in the complete loss of the sarcoglycan complex, sarcospan, and a disruption of α-dystroglycan association with membranes. In contrast, no change in the expression of ε-sarcoglycan (α-sarcoglycan homologue) was observed. Recombinant α-sarcoglycan adenovirus injection into Sgca-deficient muscles restored the sarcoglycan complex and sarcospan to the membrane. We propose that the sarcoglycan–sarcospan complex is requisite for stable association of α-dystroglycan with the sarcolemma. The Sgca-deficient mice will be a valuable model for elucidating the pathogenesis of sarcoglycan deficient limb-girdle muscular dystrophies and for the development of therapeutic strategies for this disease.     

129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant

Mice derived from the 129 strain have a nonsense codon mutation in exon 2 of the polymerase iota (Polι) gene and are therefore considered Polι deficient. When we amplified Polι mRNA from 129/SvJ or 129/Ola testes, only a small fraction of the full-length cDNA contained the nonsense mutation; the major fraction corresponded to a variant Polι isoform lacking exon 2. Polι mRNA lacking exon 2 contains an open reading frame, and the corresponding protein was detected using a polyclonal antibody raised against the C terminus of the murine Polι protein. The identity of the corresponding protein was further confirmed by mass spectrometry. Although the variant protein was expressed at only 5 to 10% of the level of wild-type Polι, it retained de novo DNA synthesis activity, the capacity to form replication foci following UV irradiation, and the ability to rescue UV light sensitivity in Polι^−/− embryonic fibroblasts derived from a new, fully deficient Polι knockout (KO) mouse line. Furthermore, in vivo treatment of 129-derived male mice with Velcade, a drug that inhibits proteasome function, stabilized and restored a substantial amount of the variant Polι in these animals, indicating that its turnover is controlled by the proteasome. An analysis of two xeroderma pigmentosum-variant (XPV) cases corresponding to missense mutants of Polη, a related translesion synthesis (TLS) polymerase in the same family, similarly showed a destabilization of the catalytically active mutant protein by the proteasome. Collectively, these data challenge the prevailing hypothesis that 129-derived strains of mice are completely deficient in Polι activity. The data also document, both for 129-derived mouse strains and for some XPV patients, new cases of genetic defects corresponding to the destabilization of an otherwise functional protein, the phenotype of which is reversible by proteasome inhibition.     

γδ T Cells Are Required for Pulmonary IL-17A Expression after Ozone Exposure in Mice: Role of TNFα

Ozone is an air pollutant that causes pulmonary symptoms. In mice, ozone exposure causes pulmonary injury and increases bronchoalveolar lavage macrophages and neutrophils. We have shown that IL-17A is important in the recruitment of neutrophils after subacute ozone exposure (0.3 ppm for 24–72 h). We hypothesized that γδ T cells are the main producers of IL-17A after subacute ozone. To explore this hypothesis we exposed wildtype mice and mice deficient in γδ T cells (TCRδ^−/−) to ozone or room air. Ozone-induced increases in BAL macrophages and neutrophils were attenuated in TCRδ^−/− mice. Ozone increased the number of γδ T cells in the lungs and increased pulmonary Il17a mRNA expression and the number of IL-17A⁺ CD45⁺ cells in the lungs and these effects were abolished in TCRδ^−/− mice. Ozone-induced increases in factors downstream of IL-17A signaling, including G-CSF, IL-6, IP-10 and KC were also decreased in TCRδ^−/− versus wildtype mice. Neutralization of IL-17A during ozone exposure in wildtype mice mimicked the effects of γδ T cell deficiency. TNFR2 deficiency and etanercept, a TNFα antagonist, also reduced ozone-induced increases in Il17a mRNA, IL-17A⁺ CD45⁺ cells and BAL G-CSF as well as BAL neutrophils. TNFR2 deficient mice also had decreased ozone-induced increases in Ccl20, a chemoattractant for IL-17A⁺ γδ T cells. Il17a mRNA and IL-17A⁺ γδ T cells were also lower in obese Cpe^fat versus lean WT mice exposed to subacute ozone, consistent with the reduced neutrophil recruitment observed in the obese mice. Taken together, our data indicate that pulmonary inflammation induced by subacute ozone requires γδ T cells and TNFα-dependent recruitment of IL-17A⁺ γδ T cells to the lung.     

Inhibitory Effect of Diabetes on Proliferation of Vascular Smooth Muscle After Balloon Injury in Rat Aorta

The effect of streptozotocin-induced diabetes on cell proliferation in rat aortic intima-media, as well as on local gene expression of transforming growth factor-β1 (TGF-β1) was studied. TGF-β1 mRNA was measured by solution hybridization and TGF-β1 protein by ELISA. Proliferation was measured by bromodeoxyuridine incorporation into DNA two days after balloon injury. All BrdU-labelled cells observed were smooth muscle cells. After a diabetes duration of 2 and 4 weeks, labelled cells were significantly fewer compared with controls. Circulating levels of total TGF-β1 were lowered in rats with 2 weeks diabetes. Although the balloon injury procedure by itself stimulated the gene expression of TGF-β1, no significant difference in TGF-β1 mRNA content between diabetic and control rats after injury was found. In conclusion: vascular smooth muscle proliferation in vivo is inhibited by the diabetic state in this model of insulin deficient diabetes and this inhibition is not related to an impaired local expression of TGF-β1.     

Both Functional LTβ Receptor and TNF Receptor 2 Are Required for the Development of Experimental Cerebral Malaria

Background

TNF-related lymphotoxin α (LTα) is essential for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The pathway involved has been attributed to TNFR2. Here we show a second arm of LTα-signaling essential for ECM development through LTβ-R, receptor of LTα1β2 heterotrimer.

Methodology/Principal Findings

LTβR deficient mice did not develop the neurological signs seen in PbA induced ECM but died at three weeks with high parasitaemia and severe anemia like LTαβ deficient mice. Resistance of LTαβ or LTβR deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and angiography, associated with lack of microvascular obstruction, while wild-type mice developed distinct microvascular pathology. Recruitment and activation of perforin⁺ CD8⁺ T cells, and their ICAM-1 expression were clearly attenuated in the brain of resistant mice. An essential contribution of LIGHT, another LTβR ligand, could be excluded, as LIGHT deficient mice rapidly succumbed to ECM.

Conclusions/Significance

LTβR expressed on radioresistant resident stromal, probably endothelial cells, rather than hematopoietic cells, are essential for the development of ECM, as assessed by hematopoietic reconstitution experiment. Therefore, the data suggest that both functional LTβR and TNFR2 signaling are required and non-redundant for the development of microvascular pathology resulting in fatal ECM.     

Nitric Oxide Synthase 2 Improves Proliferation and Glycolysis of Peripheral γδ T Cells

γδ T cells play critical roles in host defense against infections and cancer. Although advances have been made in identifying γδ TCR ligands, it remains essential to understand molecular mechanisms responsible for in vivo expansion of γδ T cells in periphery. Recent findings identified the expression of the inducible NO synthase (NOS2) in lymphoid cells and highlighted novel immunoregulatory functions of NOS2 in αβ T cell differentiation and B cell survival. In this context, we wondered whether NOS2 exerts an impact on γδ T cell properties. Here, we show that γδ T cells express NOS2 not only in vitro after TCR triggering, but also directly ex vivo. Nos2 deficient mice have fewer γδ T cells in peripheral lymph nodes (pLNs) than their wild-type counterparts, and these cells exhibit a reduced ability to produce IL-2. Using chemical NOS inhibitors and Nos2 deficient γδ T cells, we further evidence that the inactivation of endogenous NOS2 significantly reduced γδ T cell proliferation and glycolysis metabolism that can be restored in presence of exogenous IL-2. Collectively, we demonstrate the crucial role of endogenous NOS2 in promoting optimal IL-2 production, proliferation and glycolysis of γδ T cells that may contribute to their regulation at steady state.     

Oligomerization of Clostridium perfringens Epsilon Toxin Is Dependent upon Caveolins 1 and 2

Evidence from multiple studies suggests that Clostridium perfringens ε-toxin is a pore-forming toxin, assembling into oligomeric complexes in the plasma membrane of sensitive cells. In a previous study, we used gene-trap mutagenesis to identify mammalian factors contributing to toxin activity, including caveolin-2 (CAV2). In this study, we demonstrate the importance of caveolin-2 and its interaction partner, caveolin-1 (CAV1), in ε-toxin-induced cytotoxicity. Using CAV2-specific shRNA in a toxin-sensitive human kidney cell line, ACHN, we confirmed that cells deficient in CAV2 exhibit increased resistance to ε-toxin. Similarly, using CAV1-specific shRNA, we demonstrate that cells deficient in CAV1 also exhibit increased resistance to the toxin. Immunoprecipitation of CAV1 and CAV2 from ε-toxin-treated ACHN cells demonstrated interaction of both CAV1 and -2 with the toxin. Furthermore, blue-native PAGE indicated that the toxin and caveolins were components of a 670 kDa protein complex. Although ε-toxin binding was only slightly perturbed in caveolin-deficient cells, oligomerization of the toxin was dramatically reduced in both CAV1- and CAV2-deficient cells. These results indicate that CAV1 and -2 potentiate ε-toxin induced cytotoxicity by promoting toxin oligomerization – an event which is requisite for pore formation and, by extension, cell death.     

The Nuclear IκB Family Protein IκBNS Influences the Susceptibility to Experimental Autoimmune Encephalomyelitis in a Murine Model

The nuclear IκB family protein IκB_NS is expressed in T cells and plays an important role in Interferon (IFN)-γ and Interleukin (IL)-2 production. IκB-ζ, the most similar homolog of IκB_NS, plays an important role in the generation of T helper (Th)17 cells in cooperation with RORγt, a master regulator of Th17 cells. Thus, IκB-ζ deficient mice are resistant to Th17-dependent experimental autoimmune encephalomyelitis (EAE). However, IκB-ζ deficient mice develop the autoimmune-like Sjögren syndrome with aging. Here we found that IκB_NS-deficient (Nfkbid^−/−) mice show resistance against developing Th17-dependent EAE. We found that Nfkbid^−/− T cells have decreased expression of IL-17-related genes and RORγt in response to Transforming Growth Factor (TGF)-β1 and IL-6 stimulation. Thus, IκB_NS plays a pivotal role in the generation of Th17 cells and in the control of Th17-dependent EAE.     

Roles of the major, small, acid-soluble spore proteins and spore-specific and universal DNA repair mechanisms in resistance of Bacillus subtilis spores to ionizing radiation from X rays and high-energy charged-particle bombardment

The role of DNA repair by nonhomologous end joining (NHEJ), homologous recombination, spore photoproduct lyase, and DNA polymerase I and genome protection via α/β-type small, acid-soluble spore proteins (SASP) in Bacillus subtilis spore resistance to accelerated heavy ions (high-energy charged [HZE] particles) and X rays has been studied. Spores deficient in NHEJ and α/β-type SASP were significantly more sensitive to HZE particle bombardment and X-ray irradiation than were the recA, polA, and splB mutant and wild-type spores, indicating that NHEJ provides an efficient DNA double-strand break repair pathway during spore germination and that the loss of the α/β-type SASP leads to a significant radiosensitivity to ionizing radiation, suggesting the essential function of these spore proteins as protectants of spore DNA against ionizing radiation.     

LOCATION AND COMPOSITION OF SPORE MUCOPEPTIDE IN BACILLUS SPECIES

Spore integuments of Bacillus coagulans were prepared containing nearly all the hexosamine and α, ε-diaminopimelic acid (DAP) present in intact spores. Subsequent autolytic action resulted in the destruction and removal of the residual cortical structure and "cortical membrane" leaving the appearance of the inner and outer spore coats unchanged in electron micrographs. Concurrently, all the hexosamine and DAP in the preparation was released mainly as non-diffusible mucopeptide containing alanine, glutamic acid, DAP, and all the glucosamine and muramic acid. Some diffusible peptides containing alanine, glutamic acid, and DAP were also present but there was little protein or carbohydrate. Lysozyme digestion of integument preparations from heated spores of Bacillus 636, B. subtilis, B. coagulans, and B. stearothermophilus specifically removed the residual cortex and cortical membrane with the release of the mucopeptide. In B. cereus T, only the residual cortex and part of the mucopeptide were solubilized by lysozyme. The effect of several reagents and enzymes upon the appearance and removal of hexosamine from B. coagul ans spore integuments is reported. The results show that spore mucopeptide is mainly located in the residual cortex and cortical membrane and suggest that these structures consist essentially of mucopeptide. The implications of these results in relation to the "contractile cortex" theory of heat resistance in spores are discussed.     

Spinophilin Is Indispensable for the α2B Adrenergic Receptor-Elicited Hypertensive Response

The α₂ adrenergic receptor (AR) subtypes are important for blood pressure control. When activated, the α_2A subtype elicits a hypotensive response whereas the α_2B subtype mediates a hypertensive effect that counteracts the hypotensive response by the α_2A subtype. We have previously shown that spinophilin attenuates the α_2AAR-dependent hypotensive response; in spinophilin null mice, this response is highly potentiated. In this study, we demonstrate that spinophilin impedes arrestin-dependent phosphorylation and desensitization of the α_2BAR subtype by competing against arrestin binding to this receptor subtype. The Del301-303 α_2BAR, a human variation that shows impaired phosphorylation and desensitization and is linked to hypertension in certain populations, exhibits preferential interaction with spinophilin over arrestin. Furthermore, Del301-303 α_2BAR-induced ERK signaling is quickly desensitized in cells without spinophilin expression, showing a profile similar to that induced by the wild type receptor in these cells. Together, these data suggest a critical role of spinophilin in sustaining α_2BAR signaling. Consistent with this notion, our in vivo study reveals that the α_2BAR-elicited hypertensive response is diminished in spinophilin deficient mice. In arrestin 3 deficient mice, where the receptor has a stronger binding to spinophilin, the same hypertensive response is enhanced. These data suggest that interaction with spinophilin is indispensable for the α_2BAR to elicit the hypertensive response. This is opposite of the negative role of spinophilin in regulating α_2AAR-mediated hypotensive response, suggesting that spinophilin regulation of these closely related receptor subtypes can result in distinct functional outcomes in vivo. Thus, spinophilin may represent a useful therapeutic target for treatment of hypertension.     

Systemic Administration of Abeta mAb Reduces Retinal Deposition of Abeta and Activated Complement C3 in Age-Related Macular Degeneration Mouse Model

Age-related macular degeneration (AMD) is a leading cause of legal blindness in the Western world. There are effective treatments for the vascular complications of neo-vascular AMD, but no effective therapies are available for the dry/atrophic form of the disease. A previously described transgenic CFH-gene deficient mouse model, (cfh−/−), shows hallmarks of early AMD. The ocular phenotype has been further analysed to demonstrate amyloid beta (Aβ) rich basement membrane deposits associated with activated complement C3. Cfh−/− mice were treated systemically in both prophylactic and therapeutic regimes with an anti-Aβ monoclonal antibody (mAb), 6F6, to determine the effect on the cfh−/− retinal phenotype. Prophylactic treatment with 6F6 demonstrated a dose dependent reduction in the accumulation of both Aβ and activated C3 deposition. A similar reduction in the retinal endpoints could be seen after therapeutic treatment. Serum Aβ levels after systemic administration of 6F6 show accumulation of Aβ in the periphery suggestive of a peripheral sink mechanism. In summary, anti-Aβ mAb treatment can partially prevent or reverse ocular phenotypes of the cfh−/− mouse. The data support this therapeutic approach in humans potentially modulating two key elements in the pathogenesis of AMD – Aβ and activated, complement C3.     

Purification of a beta-Amylase that Accumulates in Arabidopsis thaliana Mutants Defective in Starch Metabolism

Amylase activity is elevated 5- to 10-fold in leaves of several different Arabidopsis thaliana mutants defective in starch metabolism when they are grown under a 12-hour photoperiod. Activity is also increased when plants are grown under higher light intensity. It was previously determined that the elevated activity was an extrachloroplastic β-(exo)amylase. Due to the location of this enzyme outside the chloroplast, its function is not known. The enzyme was purified to homogeneity from leaves of both a starchless mutant deficient in plastid phosphoglucomutase and from the wild type using polyethylene glycol fractionation and cyclohexaamylose affinity chromatography. The molecular mass of the β-amylase from both sources was 55,000 daltons as determined by denaturing gel electrophoresis. Gel filtration studies indicated that the enzyme was a monomer. The specific activities of the purified protein from mutant and wild-type sources, their substrate specificities, and K_m for amylopectin were identical. Based on these results it was concluded that the mutant contained an increased level of β-amylase protein. Enzyme neutralization studies using a polyclonal antiserum raised to purified β-amylase showed that in each of two starchless mutants, one starch deficient mutant and one starch overproducing mutant, the elevated amylase activity was due to elevated β-amylase protein.     

DNA Polymerase δ Is Required for Early Mammalian Embryogenesis

Background

In eukaryotic cells, DNA polymerase δ (Polδ), whose catalytic subunit p125 is encoded in the Pold1 gene, plays a central role in chromosomal DNA replication, repair, and recombination. However, the physiological role of the Polδ in mammalian development has not been thoroughly investigated.

Methodology/Principal Findings

To examine this role, we used a gene targeting strategy to generate two kinds of Pold1 mutant mice: Polδ-null (Pold1 ^−/−) mice and D400A exchanged Polδ (Pold1 ^exo/exo) mice. The D400A exchange caused deficient 3′–5′ exonuclease activity in the Polδ protein. In Polδ-null mice, heterozygous mice developed normally despite a reduction in Pold1 protein quantity. In contrast, homozygous Pold1 ^−/− mice suffered from peri-implantation lethality. Although Pold1 ^−/− blastocysts appeared normal, their in vitro culture showed defects in outgrowth proliferation and DNA synthesis and frequent spontaneous apoptosis, indicating Polδ participates in DNA replication during mouse embryogenesis. In Pold1 ^exo/exo mice, although heterozygous Pold1 ^exo/+ mice were normal and healthy, Pold1 ^exo/exo and Pold1 ^exo/− mice suffered from tumorigenesis.

Conclusions

These results clearly demonstrate that DNA polymerase δ is essential for mammalian early embryogenesis and that the 3′–5′ exonuclease activity of DNA polymerase δ is dispensable for normal development but necessary to suppress tumorigenesis.     

Omapatrilat,an Angiotensin-Converting Enzyme and Neutral Endopeptidase Inhibitor,Attenuates Early Atherosclerosis in Diabetic and in Nondiabetic Low-Density Lipoprotein Receptor–Deficient Mice

Omapatrilat inhibits both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP). ACE inhibitors have been shown to inhibit atherosclerosis in apoE-deficient mice and in several other animal models but failed in low-density lipoprotein (LDL) receptor– deficient mice despite effective inhibition of the reninangiotensin- aldosterone system. The aim of the present study was to examine the effect of omapatrilat on atherogenesis in diabetic and nondiabetic LDL receptor–deficient mice. LDL receptor–deficient male mice were randomly divided into 4 groups (n = 11 each). Diabetes was induced in 2 groups by low-dose STZ, the other 2 groups served as nondiabetic controls. Omapatrilat (70 mg/kg/day) was administered to one of the diabetic and to one of the nondiabetic groups. The diabetic and the nondiabetic mice were sacrificed after 3 and 5 weeks, respectively. The aortae were examined and the atherosclerotic plaque area was measured. The atherosclerotic plaque area was significantly smaller in the omapatrilat-treated mice, both diabetic and nondiabetic, as compared to nontreated controls. The mean plaque area of omapatrilattreated nondiabetic mice was 9357 ± 7293 μm², versus 71977 ± 34610 μm² in the nontreated mice (P = .002). In the diabetic animals, the plaque area was 8887 ± 5386 μm² and 23220 ± 10400 μm², respectively for treated and nontreated mice (P = .001). Plasma lipids were increased by omapatrilat: Meanplasma cholesterol in treated mice, diabetic and nondiabetic combined, was 39.31 ± 6.00 mmol/L, versus 33.12 ± 7.64 mmol/L in the nontreated animals (P = .008). The corresponding combined mean values of triglycerides were 4.83 ± 1.93 versus 3.00 ± 1.26 mmol/L (P = .02). Omapatrilat treatment did not affect weight or plasma glucose levels. Treatment with omapatrilat inhibits atherogenesis in diabetic as well as nondiabetic LDL receptor–deficient mice despite an increase in plasma lipids, suggesting a direct effect on the arterial wall.