Nonimmune IgG Research Articles

Targeted drug delivery to the brain endothelium dominates over passive delivery via vascular leak in experimental intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is one of the most common causes of fatal stroke, yet has no specific drug therapies. Many attempts at passive intravenous (IV) delivery in ICH have failed to deliver drugs to the salvageable area around the hemorrhage. The passive delivery method assumes vascular leak through the ruptured blood-brain barrier will allow drug accumulation in the brain. Here we tested this assumption using intrastriatal injection of collagenase, a well-established experimental model of ICH. Fitting with hematoma expansion in clinical ICH, we showed that collagenase-induced blood leak drops significantly by 4h after ICH onset and is gone by 24h. We observed passive-leak brain accumulation also declines rapidly over ∼4h for 3 model IV therapeutics (non-targeted IgG; a protein therapeutic; PEGylated nanoparticles). We compared these passive leak results with targeted brain delivery by IV monoclonal antibodies (mAbs) that actively bind vascular endothelium (anti-VCAM, anti-PECAM, anti-ICAM). Even at early time points after ICH induction, where there is high vascular leak, brain accumulation via passive leak is dwarfed by brain accumulation of endothelial-targeted agents: At 4h after injury, anti-PECAM mAbs accumulate at 8-fold higher levels in the brain vs. non-immune IgG; anti-VCAM nanoparticles (NPs) deliver a protein therapeutic (superoxide dismutase, SOD) at 4.5-fold higher levels than the carrier-free therapeutic at 24h after injury. These data suggest that relying on passive vascular leak provides inefficient delivery of therapeutics even at early time points after ICH, and that a better strategy might be targeted delivery to the brain endothelium, which serves as the gateway for the immune attack on the peri-hemorrhage inflamed brain region.

Blocking RAGE expression after injury reduces inflammation in mouse model of acute lung injury

BackgroundReceptor for Advanced Glycated Endproducts (RAGE) plays a major role in the inflammatory response to infectious and toxin induced acute lung injury. We tested the hypothesis that a RAGE blocking antibody when administered after the onset of injury can reduce lung inflammation compared to control antibody.MethodsMale and female C57BL/6 (WT) mice were used. Forty-six received lipopolysaccharide (LPS) and 26 PBS by nasal instillation on day one, repeated on day three. On day 2, 36 mice receiving LPS were divided into two groups of 18, one treated with 200 μg of non-immune isotype control IgG and the second group treated with 200 μg of anti-RAGE Ab, each dose divided between IV and IP. Ten of the 46 were not treated. On day 4, before euthanasia, mice were injected with fluorescein isothiocyanate (FITC) labelled albumen. BALF and serum samples were collected as well as lung tissue for immunohistochemistry (IHC). BALF was analyzed for cell (leukocyte) counts, for FITC BALF/serum ratios indicating pulmonary vascular leak, and for cytokines/chemokines using bead based multiplex assays. Quantitative IHC was performed for MPO and RAGE.ResultsTen LPS mice showed minimal inflammation by all measures indicating poor delivery of LPS and were excluded from analysis leaving n = 11 in the LPS + IgG group and n = 12 in the LPS + anti-RAGE group. BALF cell counts were low in the PBS administered mice (4.9 ± 2.1 × 105/ml) and high in the LPS injured untreated mice (109 ± 34) and in the LPS + IgG mice (91 ± 54) while in comparison, LPS + anti-RAGE ab mice counts were significantly lower (51.3 ± 18 vs. LPS + IgG, P = 0.03). The BALF/serum FITC ratios were lower for the LPS + anti-RAGE mice than for the LPS + IgG mice indicating less capillary leakiness. Quantitative IHC RAGE staining was lower in the LPS + anti-RAGE ab mice than in the LPS + IgG treated mice (P = 0.02).ConclusionsThese results describe a four-day LPS protocol to sustain lung injury and allow for treatment and suggests that treatment aimed at blocking RAGE when given after onset of injury can reduce lung inflammation.

Blocking RAGE improves wound healing in diabetic pigs.

Receptor for Advanced Glycated End-products (RAGE) is highly expressed in diabetes and impairs wound healing. We proposed that administering an antibody that blocks RAGE will hasten the healing of dorsal wounds in diabetic pigs compared with a non-immune IgG. Two purpose-bred diabetic (D) Yucatan minipigs (Sinclair, Auxvasse MO) each underwent 12 2 × 2 cm full thickness dorsal wounds: four wounds received decellularized porcine skin patches (Xylyx Bio, Bklyn NY): four anti-RAGE Ab (CR-3) infused patches, four saline infused patches and four wounds were left open. One pig received anti-RAGE Ab (CR-3) 1mg/kg IM q 10 days and other received non-immune IgG. Wounds were measured at 2 and 4 weeks followed by euthanasia and wound harvesting. At 2 weeks few of the patches appeared to be incorporated into the wound. By 4 weeks all patches in pigs treated systemically with CR-3 were detached and the wounds almost healed. For all 24 wounds for both pigs regardless of presence of patch or type of patch, the average IgG treated pig wound size at 4 weeks was 69.2 ± 14.6% of initial size and the average CR-3 treated pig wound size was 40.9 ± 11.3% of initial size (P=0.0002). Quantitative immunohistology showed greater staining for collagen in the CR-3 treated wounds compared with IgG treated. Staining was positive for RAGE, Mac, and IL-6 in the IgG treated wounds and negative in the CR-3 treated wounds. From these pilot experiments, we conclude that a RAGE blocking antibody given parenterally improved wound healing in a diabetic pig while patches were not effective.

CD39 and CD73 Activity Are Protective in a Mouse Model of Antiphospholipid Antibody-Induced Miscarriage

Antiphospholipid antibodies (aPL-ab) including lupus anticoagulant, anticardiolipin (aCL) antibodies and anti-β2-glycoprotein I (β2GP1) antibodies initiate inflammation, tissue factor-activated coagulation, oxidative stress and complement activation. These pathways contribute to the pathophysiology of thrombotic and obstetric manifestations in antiphospholipid syndrome (APS). Not all patients with aPL-ab develop disease phenotype and hence a ‘second hit’ is postulated to be necessary. Extracellular purines (ATP, ADP, and adenosine) are important signalling molecules with the general acceptance that ATP promotes inflammation, ADP is pro platelet-aggregatory, while adenosine has vasodilatory, antithrombotic and anti-inflammatory effects. Extracellular ATP/ADP levels are tightly regulated by CD39 and CD73. CD39 hydrolyses ATP and ADP to AMP, which is then hydrolysed to adenosine by the ecto-5'-nucleotidase CD73.We hypothesised that CD39 and CD73 mitigate both vascular thrombosis as well as inflammatory responses in APS. We developed a murine model of APS-miscarriages by passive infusion of aPL-ab harvested from patients into pregnant mice. Comparison made with infusion of non-immune IgG. We applied this model to wild type (WT), CD39-transgenic mice as well as CD39-null and CD73-null mice. Non-immune IgG administration to WT or CD39-/- C57BL/6 mice did not cause miscarriages, whereas aPL-ab-treated WT mice displayed a mean resorption frequency of 3.67%. In comparison, aPL-ab-treated CD39-/- mice showed a significantly higher mean resorption frequency of 20.1% (p<0.001 vs aPL-ab-treated WT; Fig. 1a). aPL-ab-treated CD73-/- mice also had a significantly increased mean resorption frequency of 10.5% (p<0.05 vs aPL-ab-treated WT).We have previously demonstrated that CD39 over expression in BALB/c transgenic mice is associated with enhanced adenosine generation under prothrombotic conditions. WT BALB/c mice injected with non-immune IgG had a mean resorption frequency of 21.2%, which doubled with aPL-ab injection (39.7%; p<0.05 vs non-immune IgG-treated WT). In transgenic BALB/c mice over-expressing CD39, mean resorption frequencies were significantly lower in the aPL-ab-treated group (11.4% vs 39.7% in aPL-ab-treated WT, p<0.01).QRT-PCR showed 1.6-fold and 2.4-fold increase in TF expression in aPL-ab administered CD39-/- and CD73-/- mice relative to both WT C57BL/6 mice and littermates injected with non-immune sera and. CD39 over expression suppressed TF expression compared with WT BALB/c mice after both non-immune IgG and aPL-ab challenge particularly with 6.3 fold lower TF mRNA levels after aPL-ab infusion. Decidua from aPL-ab injected CD39-/- and CD73-/- C57BL/6 mice showed increased TF immunoreactivity compared to aPL-ab-treated or nonimmune IgG-treated WT mice. C3d deposition was increased by aPL-ab treatment relative to non-immune IgG treatment in all groups except the CD39 transgenic mice (Fig. 3). C3d deposition was most marked in aPL-ab-treated CD39-/- and CD73-/- mice.Placental expression of inflammatory cytokines C57BL/6was measured at the mRNA level by qRT-PCR. TNF-α expression was doubled in CD39-/- and CD73-/- mice relative to non-immune IgG controls. CD39 Tg mice showed suppression of TNF- α after aPL-ab challenge. Consistent with the previously documented role for IL-10 in exacerbating lupus/auto-immune diseases, we noted a significant reduction in IL-10 mRNA in aPL-ab-treated CD39 Tg mice compared to aPL-ab-treated WT mice.Therefore, we demonstrate a protective role for CD39 in APS and provide rationale for the development of soluble CD39 as a novel therapeutic for APS and analysis of perturbations in the purinergic pathway to explain human disease DisclosuresDwyer:Kyowa Kirin Pharmaceutical Development, Inc.: Employment.

Adhesion molecule L1 inhibition increases infarct size in cerebral ischemia-reperfusion without change in blood-brain barrier disruption

ABSTRACT Objective Neural cell adhesion molecule L1CAM (L1) is involved in neuroprotection. To investigate a possible neuroprotective effect of L1 during ischemia, we determined whether blocking L1 with an antagonistic antibody would worsen the outcome of focal cerebral ischemia-reperfusion and increase blood-brain barrier (BBB) disruption. Methods Transient middle cerebral artery occlusion (MCAO) was performed in anesthetized rats. Five µg of antagonistic mouse IgG monoclonal L1 antibody 324 or non-immune control mouse IgG was applied on the ischemic-reperfused cortex during one hour of MCAO and two hours of reperfusion. At two hours of reperfusion, BBB permeability, size of infarct using tetrazolium staining, number of TUNEL-labeled apoptotic cells, and immunohistochemistry for expression of PTEN and p53 were studied. Results The antagonistic L1 antibody 324 increased the percentage of cortical infarct area (+36%), but did not affect BBB permeability in the ischemic-reperfused cortex. The antagonistic L1 antibody increased number of apoptotic neurons and p53 expression, but decreased PTEN expression. Conclusion Functional antagonism of L1 increases infarct size by increasing numbers of apoptotic neurons without affecting BBB permeability during the early stage of cerebral ischemia-reperfusion. Our data suggest that L1 affects primarily the brain parenchyma rather than BBB during early stages of cerebral ischemia-reperfusion and that endogenous brain L1 may be neuroprotective.

Human cytomegalovirus antagonizes activation of Fcγ receptors by distinct and synergizing modes of IgG manipulation.

Human cytomegalovirus (HCMV) is endowed with multiple highly sophisticated immune evasion strategies. This includes the evasion from antibody mediated immune control by counteracting host Fc-gamma receptor (FcγR) mediated immune control mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). We have previously shown that HCMV avoids FcγR activation by concomitant expression of the viral Fc-gamma-binding glycoproteins (vFcγRs) gp34 and gp68. We now show that gp34 and gp68 bind IgG simultaneously at topologically different Fcγ sites and achieve efficient antagonization of host FcγR activation by distinct but synergizing mechanisms. While gp34 enhances immune complex internalization, gp68 acts as inhibitor of host FcγR binding to immune complexes. In doing so, gp68 induces Fcγ accessibility to gp34 and simultaneously limits host FcγR recognition. The synergy of gp34 and gp68 is compelled by the interfering influence of excessive non-immune IgG ligands and highlights conformational changes within the IgG globular chains critical for antibody effector function.

Novel Receptor for Advanced Glycation End Products-Blocking Antibody to Treat Diabetic Peripheral Artery Disease.

BackgroundExpression of receptor for advanced glycation end products (RAGE) plays an important role in diabetic peripheral artery disease. We proposed to show that treatment with an antibody blocking RAGE would improve hind limb perfusion and muscle viability in diabetic pig with femoral artery (FA) ligation.Methods and ResultsPurpose‐bred diabetic Yucatan minipigs with average fasting blood sugar of 357 mg/dL on insulin to maintain a glucose range of 300 to 500 mg/dL were treated with either a humanized monoclonal anti‐RAGE antibody (CR‐3) or nonimmune IgG. All pigs underwent intravascular occlusion of the anterior FA. Animals underwent (201Tl) single‐photon emission computed tomography/x‐ray computed tomography imaging on days 1 and 28 after FA occlusion, angiogenesis imaging with [99mTc]dodecane tetra‐acetic acid–polyethylene glycol–single chain vascular endothelial growth factor (scVEGF), muscle biopsies on day 7, and contrast angiogram day 28. Results showed greater increases in perfusion to the gastrocnemius from day 1 to day 28 in CR‐3 compared with IgG treated pigs (P=0.0024), greater uptake of [99mTc]dodecane tetra‐acetic acid‐polyethylene glycol‐scVEGF (scV/Tc) in the proximal gastrocnemius at day 7, confirmed by tissue staining for capillaries and vascular endothelial growth factor A, and less muscle loss and fibrosis at day 28. Contrast angiograms showed better reconstitution of the distal FA from collaterals in the CR‐3 versus IgG treated diabetic pigs (P=0.01). The gastrocnemius on nonoccluded limb at necropsy had higher 201Tl uptake (percentage injected dose per gram) and reduced RAGE staining in arterioles in CR‐3 treated compared with IgG treated animals (P=0.04).ConclusionsA novel RAGE‐blocking antibody improved hind limb perfusion and angiogenesis in diabetic pigs with FA occlusion. Contributing factors are increased collaterals and reduced vascular RAGE expression. CR‐3 shows promise for clinical treatment in diabetic peripheral artery disease.

Inhibition of interleukin-6 signaling attenuates aortitis, left ventricular hypertrophy and arthritis in interleukin-1 receptor antagonist deficient mice.

The aim of the present study was to examine whether inhibition of Interleukin (IL)-6 signaling by MR16-1, an IL-6 receptor antibody, attenuates aortitis, cardiac hypertrophy, and arthritis in IL-1 receptor antagonist deficient (IL-1RA KO) mice. Four weeks old mice were intraperitoneally administered with either MR16-1 or non-immune IgG at dosages that were adjusted over time for 5 weeks. These mice were stratified into four groups: MR16-1 treatment groups, KO/MR low group (first 2.0 mg, following 0.5 mg/week, n=14) and KO/MR high group (first 4.0 mg, following 2.0 mg/week, n=19) in IL-1RA KO mice, and IgG treatment groups, KO/IgG group (first 2.0 mg, following 1.0 mg/week, n=22) in IL-1RA KO mice, and wild/IgG group (first 2.0 mg, following 1.0 mg/week, n=17) in wild mice. Aortitis, cardiac hypertrophy and arthropathy were histologically analyzed. Sixty-eight percent of the KO/IgG group developed aortitis (53% developed severe aortitis). In contrast, only 21% of the KO/MR high group developed mild aortitis, without severe aortitis (P<0.01, vs KO/IgG group). Infiltration of inflammatory cells, such as neutrophils, T cells, and macrophages, was frequently observed around aortic sinus of the KO/IgG group. Left ventricle and cardiomyocyte hypertrophy were observed in IL-1RA KO mice. Administration of high dosage of MR16-1 significantly suppressed cardiomyocyte hypertrophy. MR16-1 attenuated the incidence and severity of arthritis in IL-1RA KO mice in a dose-dependent manner. In conclusion, blockade of IL-6 signaling may exert a beneficial effect to attenuate severe aortitis, left ventricle hypertrophy, and arthritis.

Abstract 520: Therapeutic antibodies against parathyroid hormone-related peptide (PTHrP) inhibit the growth of established skeletal metastasis in mice transplanted with human triple negative breast cancer cell lines

Abstract Parathyroid Hormone-related Peptide (PTHrP)is a potent regulator of bone turnover and is thought to play a major role in the progression of skeletal metastasis through its activation of growth factors in the bone microenvironment. Our previous studies using genetically engineered mouse models in which PTHrP was ablated in the mammary epithelium have demonstrated that PTHrP also plays a direct role on tumor progression by enhancing tumor initiation, growth and metastasis outside the skeleton. We therefore proposed a novel mechanism of PTHrP driven bone metastasis by which PTHrP controls both the seed (tumor cells) and the soil (the bone microenvironment). We tested our hypothesis in triple negative breast cancer cell line (TNBC)models which are not responsive to either tamoxifen or Herceptin. Monoclonal antibodies against PTHrP were generated with strong in vitro anti-proliferative and anti-invasive potency in two human PTHrP expressing cell lines: the widely used TNBC MDAMB231 cell line and a patient derived TNBC cell line. Ablation of PTHrP or treatment with anti-PTHrP monoclonal antibodies induced major changes in the phenotype of these cells as determined by flow cytometry with stem cells and EMT markers, invasion assays and mammosphere assays indicating a reversal of their metastatic/invasive phenotype. Next we transplanted these cell lines into 8 weeks old female athymic nude mice to examine the efficacy of the therapeutic monoclonal antibodies on the progression of established bone metastasis.104 tumor cells were injected intra-tibially and animals examined at timed intervals(14, 21, 28, 25 and 42 days) with X-rays and high resolution CTs.At 14 days animals were administered intra-peritoneally with 200 micrograms of therapeutic anti-PTHrP antibodies or non-immune IgG(10 animals per group) and every 3 days thereafter til sacrifice(42 days). At sacrifice PET scan was performed to measure tumor volume and activity and bone were collected for histomorphometry analysis of tumor size and bone turnover activity. A significant reduction in bone lesions (P&amp;lt;0.05) was observed over time by imaging in animals treated with the therapeutic antibodies. At sacrifice PET scan indicates a strong reduction in tumor activity in treated animals (P&amp;lt;0.05). Histomorphometry analysis showed greater than 50% reduction in tumor volume associated with a significant inhibition of bone turnover in treated animals (P&amp;lt;0.05). In summary our data demonstrate that, in two TNBC models, PTHrP blockade can reprogram cancer cells to a reduced tumorigenicity and block the progression of established bone metastasis by targeting both the tumor cell and the bone microenvironment. Our data lend support to the “seed and soil” hypothesis proposed by Paget and the rational of anti-PTHrP therapeutic strategies alone or in combination with bone targeting agents in order to eradicate the development of skeletal metastasis. Citation Format: Jiarong Li, Anne Camirand, Mahvash Zakikhani, Karine Sellin, Richard Kremer. Therapeutic antibodies against parathyroid hormone-related peptide (PTHrP) inhibit the growth of established skeletal metastasis in mice transplanted with human triple negative breast cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 520.

ADAM9 is over-expressed in human ovarian clear cell carcinomas and suppresses cisplatin-induced cell death.

ADAMs (a disintegrin and metalloproteinases) are involved in various biological events such as cell adhesion, migration and invasion, membrane protein shedding and proteolysis. However, there have been no systematic studies on the expression of ADAMs in human ovarian carcinomas. We therefore examined mRNA expression of all the proteolytic ADAM species including ADAM8, 9, 10, 12, 15, 17, 19, 20, 21, 28, 30, 33 and ADAMDEC1 in human ovarian carcinomas, and found that prototype membrane‐anchored ADAM9m, but not secreted isoform ADAM9s, is significantly over‐expressed in carcinomas than in control non‐neoplastic ovarian tissue. Among the histological subtypes of serous, endometrioid, mucinous and clear cell carcinomas, ADAM9m expression was highest in clear cell carcinomas. Immunohistochemistry showed that all the clear cell carcinoma samples displayed ADAM9m primarily on the carcinoma cell membrane. By immunoblotting, ADAM9m was detected mainly in an active form in the clear cell carcinoma tissues. When two clear cell carcinoma cell lines (RMG‐I and TOV21G cells) with ADAM9m expression were treated with cisplatin, viability was significantly reduced and apoptosis increased in ADAM9m knockdown cells compared with mock transfectants. In addition, treatment of the cells with neutralizing anti‐ADAM9m antibody significantly decreased viability compared with non‐immune IgG, whereas ADAM9m over‐expression significantly increased viability compared with mock transfectants. Our data show, to the best of our knowledge, for the first time, that ADAM9m is over‐expressed in an activated form in human ovarian clear cell carcinomas, and suggest that ADAM9m plays a key role in cisplatin resistance.

TSG-6- a double-edged sword for osteoarthritis

TSG-6- a double-edged sword for osteoarthritis

Interaction of p190A RhoGAP with eIF3A and Other Translation Preinitiation Factors Suggests a Role in Protein Biosynthesis

The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr308, but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined.

Antagonism of stem cell factor/c-kit signaling attenuates neonatal chronic hypoxia-induced pulmonary vascular remodeling.

BackgroundAccumulating evidence suggests that c-kit positive cells are present in the remodeled pulmonary vasculature bed of patients with pulmonary hypertension (PH). Whether stem cell factor (SCF)/ c-kit regulated pathways potentiate pulmonary vascular remodeling is unknown. Here, we tested the hypothesis that attenuated c-kit signaling would decrease chronic hypoxia-induced pulmonary vascular remodeling by decreasing pulmonary vascular cell mitogenesis.MethodsNeonatal FVB/NJ mice treated with non-immune IgG (PL), or c-kit neutralizing antibody (ACK2) as well as c-kit mutant mice (WBB6F1- Kit W− v/ +) and their congenic controls, were exposed to normoxia (FiO2=0.21) or hypoxia (FiO2=0.12) for two weeks. Following this exposure, right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH), pulmonary vascular cell proliferation and remodeling were evaluated.ResultsAs compared to chronically hypoxic controls, c-kit mutant mice had decreased RVSP, RVH, pulmonary vascular remodeling and proliferation. Consistent with these findings, administration of ACK2 to neonatal mice with chronic hypoxia-induced PH decreased RVSP, RVH, pulmonary vascular cell proliferation and remodeling. This attenuation in PH was accompanied by decreased extracellular signal-regulated protein kinase (ERK) 1/2 activation.ConclusionSCF/c-kit signaling may potentiate chronic hypoxia-induced vascular remodeling by modulating ERK activation. Inhibition of c-kit activity may be a potential strategy to alleviate PH.

Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury.

IntroductionSepsis refers to severe systemic inflammation leading to acute lung injury (ALI) and death. Introducing novel therapies can reduce the mortality in ALI. Osteopontin (OPN), a secretory glycoprotein produced by immune reactive cells, plays a deleterious role in various inflammatory diseases. However, its role in ALI caused by sepsis remains unexplored. We hypothesize that treatment with an OPN-neutralizing antibody (anti-OPN Ab) protects mice against ALI during sepsis.MethodsSepsis was induced in 8-week-old male C57BL/6 mice by cecal ligation and puncture (CLP). Anti-OPN Ab or non-immunized IgG as control, at a dose of 50 μg/mouse, was intravenously injected at the time of CLP. After 20 hours, the expression of OPN and proinflammatory cytokines in tissues and plasma was examined by real-time PCR, Western blot, and ELISA. Plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) and the lung myeloperoxidase (MPO) levels were determined by colorimetric assays. Lung damage and neutrophil infiltrations were determined by histological H&E and Gr-1 staining, respectively. The effect of recombinant mouse OPN (rmOPN) on human neutrophil-like cell (HL-60) migration was performed by Boyden chamber assays and the involvement of intracellular signaling molecules in HL-60 cells was revealed by Western blot.ResultsAfter 20 hours of sepsis, mRNA and protein levels of OPN were significantly induced in lungs, spleen, and plasma. Treatment with an anti-OPN Ab in septic mice significantly reduced the plasma levels of ALT, AST, and LDH, and the proinflammatory cytokines IL-6, IL-1β and the chemokine MIP-2, compared with the vehicle group. Similarly, the lung mRNA and protein expressions of proinflammatory cytokines and chemokine were greatly reduced in anti-OPN Ab-treated animals. The lung histological architecture, MPO and neutrophil infiltration were significantly improved in anti-OPN Ab-treated mice compared with the vehicle animals. Treatment of rmOPN in HL-60 cells significantly increased their migration, in vitro. The neutrophils treated with rmOPN remarkably increased the levels of phospho focal adhesion kinase (pFAK), phospho extracellular signal-regulated kinase (pERK) and phospho p38.ConclusionsOur findings clearly demonstrate the beneficial outcomes of anti-OPN Ab treatment in protecting against ALI, implicating a novel therapeutic strategy in sepsis.

P-selectin as a potential therapeutic target for endometriosis

To test the hypothesis that P-selectin plays a critical role in the development of endometriosis and that targeting P-selectin has therapeutic potential. Prospective, randomized study. Academic facility. The first experiment used 24 each of female adult P-selectin knockout mice and C57BL/6 wild-type mice, and 96 male green fluorescent protein (GFP) transgenic mice. The second experiment used 16 female adult C57BL/6 mice. In experiment 1, P-selectin knockout and wild-type mice alternately served as donors and recipients for the transplantation of endometrial tissue fragments into the peritoneal cavity to induce endometriosis. Two weeks later all four groups were each randomly divided into two equal-sized subgroups, receiving either green fluorescent protein-expressing platelets or saline infusion. In experiment 2, 2 weeks after the surgical induction of endometriosis, the mice were randomly divided into two groups, one receiving a 2-week treatment with a recombinant P-selectin protein and the other, non-immune IgG, both by intraperitoneal injection. Lesion size, hotplate latency, and immunohistochemistry analysis of platelet aggregation, angiogenesis, transforming growth factor β1, α-smooth muscle actin, collagen I, and the extent of macrophage infiltration in ectopic implants. The extent of fibrosis also was evaluated in experiment 2. P-selectin deficiency significantly retarded the development of endometriosis in a mouse model of endometriosis. In addition, soluble P-selectin treatment markedly reduced the lesion size in mouse through decreased platelet aggregation and angiogenesis, improved general hyperalgesia, and reduced the extent of macrophages infiltration, resulting in reduced fibrotic tissue content. Targeting P-selectin-mediated platelet adhesion onto endometriotic lesions holds promise as a novel therapeutics for treating endometriosis.

Immunoliposomes containing Soluble Leishmania Antigens (SLA) as a novel antigen delivery system in murine model of leishmaniasis

Development of new generation of vaccines against leishmaniasis requires adjuvants to elicit the type and intensity of immune response needed for protection. The coupling of target-specific antibodies to the liposomal surface to create immunoliposomes has appeared as a promising way in achieving a liposome active targeting. In this study, immunoliposomes were prepared by grafting non-immune mouse IgG onto the liposomal surface. The influence of active targeted immunoliposomes on the type and intensity of generated immune response against Leishmania was then investigated and compared with that of liposomes and control groups which received either SLA or HEPES buffer alone. All formulations contained SLA and were used to immunize the mice in the left hind footpad three times in 3-week intervals. Evaluation of lesion development and parasite burden in the foot and spleen after challenge with Leishmania major, evaluation of Th1 cytokine (IFN-γ), and titration of IgG isotypes were carried out to assess the type of generated immune response and the extent of protection. The results indicated that liposomes might be effective adjuvant systems to induce protection against L. major challenge in BALB/c mice, but stronger cell mediated immune responses were induced when immunoliposomes were utilized. Thus, immune modulation using immunoliposomes might be a practical approach to improve the immunization against L. major.

Imaging RAGE expression in atherosclerotic plaques in hyperlipidemic pigs.

BackgroundReceptor for advanced glycated end product (RAGE) expression is a prominent feature of atherosclerosis. We have previously shown in apoE null mice uptake of a radiolabeled anti-RAGE antibody in atherosclerotic plaque and now evaluate RAGE-directed imaging to identify advanced plaques in a large animal model.MethodsNine hyperlipidemic (HL) pigs were injected with 603.1 ± 129.5 MBq of 99mTc-anti-RAGE F(ab′)2, and after 6 h (blood pool clearance), they underwent single-photon emission computed tomography/computed tomography (SPECT/CT) imaging of the neck, thorax, and hind limbs. Two HL pigs received 99mTc non-immune IgG F(ab′)2, and three farm pigs were injected with 99mTc-anti-RAGE F(ab′)2. After imaging, the pigs were euthanized. The aorta from the root to bifurcation was dissected, and the innominates, proximal carotids, and coronaries were dissected and counted, stained for H&E and RAGE, and AHA-classified.ResultsOn pathology, 24% of the arterial segments showed AHA class III or IV lesions, and these lesions were confined almost exclusively to coronaries and carotids with % stenosis from 15% to 65%. Scatter plots of %ID/g for class III/IV vs. I/II lesions showed almost complete separation. Focal vascular uptake of tracer visualized on SPECT scans corresponded to class III/IV lesions in the coronary and carotid vessels. In addition, uptake in the hind limbs was noted in the HL pigs and corresponded to RAGE staining of small arteries in the muscle sections. Correlations for the vascular lesions were r = 0.747, P = 0.001 for %ID vs. %ID/g and r = 0.83, P = 0.002 for %ID/g vs. % RAGE staining.ConclusionsUptake of radiolabeled anti-RAGE antibody in coronary and carotid fibroatheroma and in the small arteries of the hind limbs in a relevant large animal model of atherosclerosis supports the important role of RAGE in atherosclerosis and peripheral artery disease as a target for imaging and treatment.

HMGB1 neutralization is associated with bacterial translocation during acetaminophen hepatotoxicity

BackgroundAcetaminophen (APAP) hepatotoxicity is associated with a high rate of gram-negative enteric bacterial infection; however, the underlying mechanism is still unknown. APAP overdose induces massive hepatocyte necrosis, necrotic tissue releases high mobility group B1 (HMGB1) and exogenous HMGB1 is able to induce gut bacterial translocation (BT) in normal mice; therefore, it is possible that HMGB1 mediates gut BT in APAP hepatotoxicity. This study aims to test this hypothesis by using anti-HMGB1 neutralizing antibody to treat APAP overdose for 24-48 hours.MethodsMale C57BL/6 mice were intraperitoneally (i.p.) injected with a single dose of APAP (350 mg/kg dissolved in 1 mL sterile saline). 2 hrs after APAP injection, the APAP challenged mice were randomized to receive treatment with either anti-HMGB1 antibody (400 μg per dose) or non-immune (sham) IgG every 24 h for a total of 2 doses.Results24 and 48 hrs after APAP challenge, anti-HMGB1 treatment instead of sham IgG therapy significantly decreased serum HMGB1 concentrations and reduced BT by 85%; serum HMGB1 levels were positively correlated with the amount of BT; anti-HMGB1 therapy decreased hepatic BT at 48 h, which was associated with better recovered liver structure and better restored hepatic immune system that was shown by enhanced hepatic mRNA expression of TNF-α, IL-6 and extensive proliferation of inflammatory and reticuloendothelial cells; however, anti-HMGB1 treatment did not decrease gut mucosal permeability as compared to the sham IgG therapy at either 24 or 48 hrs.ConclusionHMGB1 neutralization is associated with bacterial translocation during APAP hepatotoxicity.

Leishmania donovani Prevents Oxidative Burst-mediated Apoptosis of Host Macrophages through Selective Induction of Suppressors of Cytokine Signaling (SOCS) Proteins

One of the mechanisms for establishment of infection employed by intra-macrophage pathogen-like Leishmania is inhibition of oxidative burst-mediated macrophage apoptosis to protect their niche for survival and replication. We tried to elucidate the underlying mechanism for this by using H2O2 for induction of apoptosis. Leishmania donovani-infected macrophages were much more resistant to H2O2-mediated apoptosis compared with control. Although infected cells were capable of comparable reactive oxygen species production, there was less activation of the downstream cascade consisting of caspase-3 and -7 and cleaved poly(ADP)-ribose polymerase. Suppressors of cytokine signaling (SOCS) 1 and 3 proteins and reactive oxygen species scavenging enzyme thioredoxin, known to be involved in stabilization of protein-tyrosine phosphatases, were found to be induced during infection. Induction of SOCS proteins may be mediated by Egr1, and silencing of Socs1 and -3 either alone or in combination resulted in reduced thioredoxin levels, enhanced activation of caspases, and increased apoptosis of infected macrophages. The induction of protein-tyrosine phosphatases, thioredoxin, SOCS, and Egr1 in L. donovani-infected macrophages was found to be unaffected by H2O2 treatment. SOCS knocked down cells also displayed decreased parasite survival thus marking reduction in disease progression. Taken together, these results suggest that L. donovani may exploit SOCS for subverting macrophage apoptotic machinery toward establishing its replicative niche inside the host.

Early Growth Response-1 (EGR-1) and Nuclear Factor of Activated T Cells (NFAT) Cooperate to Mediate CD40L Expression in Megakaryocytes and Platelets

Increasing evidence implicates circulating platelets as mediators of chronic inflammatory and autoimmune diseases via the expression and release of CD40L, an important modulator of inflammation and adaptive immune responses traditionally associated with activated T cells. Emerging evidence suggests that platelet CD40L is dynamically regulated in several chronic inflammatory and autoimmune diseases and may mediate progression and secondary pathology associated with those disease states. The present study identifies NFATc2 as a key transcriptional modulator of CD40L expression in megakaryocytes and inflammatory activity of platelets. Furthermore, the current data show that EGR-1, a member of the early growth response family of zinc finger transcription factors, modulates NFATc2-dependent regulation of CD40L expression in megakaryocytes. Our novel demonstration that in vivo biochemical or genetic inhibition of NFATc2 activity in megakaryocyte diminishes platelet CD40L implicates the NFATc2/EGR-1 axis as a key regulatory pathway of inflammatory and immunomodulatory activity in platelets and represents a target for the development of therapeutics for the potential treatment of chronic inflammatory and autoimmune diseases.