In vivo determination of mouse olfactory mucus cation concentrations in normal and inflammatory States

Objective

Olfaction is impaired in chronic rhinosinusitis (CRS). The study has two aims: (1) to determine whether changes in cation concentration occur in the olfactory mucus of mice with CRS, which may affect chemo-electrical transduction, (2) and to examine whether these alterations are physiologically significant in humans.

Study Design

Animal study in mice and translational study in humans.

Methods

Inflammation was induced by sensitization and chronic exposure of 16 C57BL/6 mice to Aspergillus fumigatus. The control group included 16 untreated mice. Ion-selective microelectrodes were used to measure free cation concentrations in the olfactory mucus of 8 mice from each treatment group, while the remaining mice were sacrificed for histology. To validate the findings in the animal model, olfactory threshold was measured in 11 healthy human participants using Sniffin’ Sticks before and after nasal irrigation with solutions that were composed of either of the cation concentrations.

Results

In 8 mice, olfactory mucus of chronically inflamed mice had lower [Na⁺] (84.8±4.45 mM versus 93.73±3.06 mM, p = 0.02), and higher [K⁺] (7.2±0.65 mM versus 5.7±0.20 mM, p = 0.04) than controls. No difference existed in [Ca²⁺] (0.50±0.12 mM versus 0.54±0.06 mM, p = 0.39). In humans, rinsing with solutions replicating ion concentrations of the mouse mucosa with chronic inflammation caused a significant elevation in the median olfactory threshold (9.0 to 4.8, p = 0.003) but not with the control solution (8.3 to 7.8, p = 0.75).

Conclusion

Chronic inflammation elevates potassium and lowers sodium ion concentration in mice olfactory mucus. Nasal irrigation with a corresponding solution induced olfactory threshold shift in humans.     

Spatial differences in the presence of FOXP3+ and GranzymeB+ T cells between the intra- and extravascular compartments in renal allograft vasculopathy

Background

Allograft vasculopathy (AV) and native atherosclerosis (NA) share the presence of a T-cell mediated inflammatory response, but differ in overall plaque morphology and growth rate. We studied the distribution and frequency of regulatory- and cytotoxic T cells in the arterial intima lesions in both conditions.

Methodology/Principal Findings

The study is based on vessels of 15 explanted human renal allografts with AV and 10 carotid artery plaques obtained at surgery. Distribution and frequency of cytotoxic- and regulatory T cells, as identified by the expression of Granzyme B (GrB) and FOXP3 was established in NA and AV. Furthermore, we compared the distribution of these cells in AV with the perivascular, interstitial renal tissue using immunohistochemistry. The total number of T cells was much higher in AV than in NA lesions (711±135 and 37±8 CD3/mm² respectively, p<0.005, mean, ± SEM). Total numbers of FOXP3⁺ regulatory cells were also significantly increased in AV (36±10 and 0.9±0.3 FOXP3⁺/mm² p<0.05), but relative numbers, expressed as a percentage of the total number of CD3⁺ T cells ((FOXP3⁺/CD3⁺) ×100), were not significantly different (4.6%±0.9 and 2.7%±0.6). GrB⁺ cells were rare in NA, but significantly increased numbers of GrB⁺ cells were found in AV lesions (85±24 and 0.2±0.1 GrB⁺/mm², p<0.05). Perivascular tissues in the allografts showed a higher relative frequency of FOXP3⁺ cells than adjacent intimal lesions (14.0%±2.7 and 4.6%±0.9, respectively, p<0.05), but a lower frequency of GrB⁺ cytotoxic T cells (16.1%±2.7 and 22.6%±3.6, p<0.05).

Conclusions

Similar to NA, AV is characterized by a low frequency of intimal FOXP3⁺ regulatory T cells. Moreover, significant spatial differences exist in the distribution of functional T cell subsets between the intra- and extravascular micro-environments of the graft.     

Liver-derived IGF-I regulates mean life span in mice

Background

Transgenic mice with low levels of global insulin-like growth factor-I (IGF-I) throughout their life span, including pre- and postnatal development, have increased longevity. This study investigated whether specific deficiency of liver-derived, endocrine IGF-I is of importance for life span.

Methods and Findings

Serum IGF-I was reduced by approximately 80% in mice with adult, liver-specific IGF-I inactivation (LI-IGF-I^-/- mice), and body weight decreased due to reduced body fat. The mean life span of LI-IGF-I^-/- mice (n = 84) increased 10% vs. control mice (n = 137) (Cox''s test, p<0.01), mainly due to increased life span (16%) of female mice [LI-IGF-I^-/- mice (n = 31): 26.7±1.1 vs. control (n = 67): 23.0±0.7 months, p<0.001]. Male LI-IGF-I^-/- mice showed only a tendency for increased longevity (p = 0.10). Energy expenditure, measured as oxygen consumption during and after submaximal exercise, was increased in the LI-IGF-I^-/- mice. Moreover, microarray and RT-PCR analyses showed consistent regulation of three genes (heat shock protein 1A and 1B and connective tissue growth factor) in several body organs in the LI-IGF-I^-/- mice.

Conclusions

Adult inactivation of liver-derived, endocrine IGF-I resulted in moderately increased mean life span. Body weight and body fat decreased in LI-IGF-I^-/- mice, possibly due to increased energy expenditure during exercise. Genes earlier reported to modulate stress response and collagen aging showed consistent regulation, providing mechanisms that could underlie the increased mean life span in the LI-IGF-I^-/- mice.     

Zoledronate inhibits ischemia-induced neovascularization by impairing the mobilization and function of endothelial progenitor cells

Background

Bisphosphonates are a class of pharmacologic compounds that are commonly used to treat postmenopausal osteoporosis and malignant osteolytic processes. Studies have shown that bone marrow-derived endothelial progenitor cells (EPCs) play a significant role in postnatal neovascularization. Whether the nitrogen-containing bisphosphonate zoledronate inhibits ischemia-induced neovascularization by modulating EPC functions remains unclear.

Methodology/Principal Findings

Unilateral hindlimb ischemia was surgically induced in wild-type mice after 2 weeks of treatment with vehicle or zoledronate (low-dose: 30 μg/kg; high-dose: 100 μg/kg). Doppler perfusion imaging demonstrated that the ischemic limb/normal side blood perfusion ratio was significantly lower in wild-type mice treated with low-dose zoledronate and in mice treated with high-dose zoledronate than in controls 4 weeks after ischemic surgery (control vs. low-dose vs. high-dose: 87±7% vs. *61±18% vs. **49±17%, *p<0.01, **p<0.005 compared to control). Capillary densities were also significantly lower in mice treated with low-dose zoledronate and in mice treated with high-dose zoledronate than in control mice. Flow cytometry analysis showed impaired mobilization of EPC-like cells (Sca-1⁺/Flk-1⁺) after surgical induction of ischemia in mice treated with zoledronate but normal levels of mobilization in mice treated with vehicle. In addition, ischemic tissue from mice that received zoledronate treatment exhibited significantly lower levels of the active form of MMP-9, lower levels of VEGF, and lower levels of phosphorylated eNOS and phosphorylated Akt than ischemic tissue from mice that received vehicle. Results of the in vitro studies showed that incubation with zoledronate inhibited the viability, migration, and tube-forming capacities of EPC.

Conclusions/Significance

Zoledronate inhibited ischemia-induced neovascularization by impairing EPC mobilization and angiogenic functions. These findings suggest that administration of zoledronate should be withheld in patients with ischemic events such as acute limb ischemia.     

Monocyte-platelet interaction induces a pro-inflammatory phenotype in circulating monocytes

Background

Activated platelets exert a pro-inflammatory action that can be largely ascribed to their ability to interact with leukocytes and modulate their activity. We hypothesized that platelet activation and consequent formation of monocyte-platelet aggregates (MPA) induces a pro-inflammatory phenotype in circulating monocytes.

Methodology/Principal Findings

CD62P⁺ platelets and MPA were measured, and monocytes characterized, by whole blood flow cytometry in healthy subjects, before and two days after receiving influenza immunization. Three monocytic subsets were identified: CD14⁺CD16⁻, CD14^highCD16⁺and CD14^lowCD16⁺. The increase in high sensitivity C-reactive protein post-immunization was accompanied by increased platelet activation and MPA formation (25.02±12.57 vs 41.48±16.81; p = 0.01), along with enhancement of circulating CD14^highCD16⁺ cells (4.7±3.6 vs 10.4±4.8; p = 0.003), their percentage being linearly related to levels of CD62P⁺-platelets (r² = 0.4347; p = 0.0008). In separate in vitro experiments, co-incubation of CD14⁺CD16⁻ cells, isolated from healthy donor subjects, with autologous platelets gave rise to up-regulation of CD16 on monocytes as compared with those maintained in medium alone (% change in CD14⁺CD16⁺ cells following 48 h co-incubation of monocytes with platelets was +106±51% vs monocytes in medium alone; p<0.001). This effect correlated directly with degree of MPA formation (r² = 0.7731; p<0.0001) and was associated with increased monocyte adhesion to endothelial cells. P-selectin glycoprotein ligand-1 (PSGL-1) blocking antibody, which abrogates MPA formation, abolished these effects, as did the cyclooxygenase (COX)-2 selective inhibitor NS-398, aspirin and the EP1/EP2-selective antagonist AH6809.

Conclusions/Significance

These data suggest that MPA formation, as occurs in the blood under pro-inflammatory conditions, expands the pool of circulating CD14^highCD16⁺ monocytes in a COX-2 dependent manner, and these monocytes exhibit increased adhesion to endothelium. Our findings delineate a novel mechanism underlying the pro-inflammatory effect of platelet activation.     

The type of responder T-cell has a significant impact in a human in vitro suppression assay

Background

In type 1 diabetes (T1D), a prototypic autoimmune disease, effector T cells destroy beta cells. Normally, CD4⁺CD25^+high, or natural regulatory T cells (Tregs), counter this assault. In autoimmunity, the failure to suppress CD4⁺CD25^low T cells is important for disease development. However, both Treg dysfunction and hyperactive responder T-cell proliferation contribute to disease.

Methods/Principal Findings

We investigated human CD4⁺CD25^low T cells and compared them to CD4⁺CD25^- T cells in otherwise equivalent in vitro proliferative conditions. We then asked whether these differences in suppression are exacerbated in T1D. In both single and co-culture with Tregs, the CD4⁺CD25^low T cells divided more rapidly than CD4⁺CD25^- T cells, which manifests as increased proliferation/reduced suppression. Time-course experiments showed that this difference could be explained by higher IL-2 production from CD4+CD25^low compared to CD4+CD25- T cells. There was also a significant increase in CD4+CD25^low T-cell proliferation compared to CD4+CD25- T cells during suppression assays from RO T1D and at-risk subjects (n = 28, p = 0.015 and p = 0.024 respectively).

Conclusions/Significance

The in vitro dual suppression assays proposed here could highlight the impaired sensitivity of certain responder T cells to the suppressive effect of Tregs in human autoimmune diseases.     

Influence of short-term glucocorticoid therapy on regulatory T cells in vivo

Background

Pre- and early clinical studies on patients with autoimmune diseases suggested that induction of regulatory T(T_reg) cells may contribute to the immunosuppressive effects of glucocorticoids(GCs).

Objective

We readdressed the influence of GC therapy on T_reg cells in immunocompetent human subjects and naïve mice.

Methods

Mice were treated with increasing doses of intravenous dexamethasone followed by oral taper, and T_reg cells in spleen and blood were analyzed by FACS. Sixteen patients with sudden hearing loss but without an inflammatory disease received high-dose intravenous prednisolone followed by stepwise dose reduction to low oral prednisolone. Peripheral blood T_reg cells were analyzed prior and after a 14 day GC therapy based on different markers.

Results

Repeated GC administration to mice for three days dose-dependently decreased the absolute numbers of T_reg cells in blood (100 mg dexamethasone/kg body weight: 2.8±1.8×10⁴ cells/ml vs. 33±11×10⁴ in control mice) and spleen (dexamethasone: 2.8±1.9×10⁵/spleen vs. 95±22×10⁵/spleen in control mice), which slowly recovered after 14 days taper in spleen but not in blood. The relative frequency of FOXP3⁺ T_reg cells amongst the CD4⁺ T cells also decreased in a dose dependent manner with the effect being more pronounced in blood than in spleen. The suppressive capacity of T_reg cells was unaltered by GC treatment in vitro. In immunocompetent humans, GCs induced mild T cell lymphocytosis. However, it did not change the relative frequency of circulating T_reg cells in a relevant manner, although there was some variation depending on the definition of the T_reg cells (FOXP3⁺: 4.0±1.5% vs 3.4±1.5%*; AITR⁺: 0.6±0.4 vs 0.5±0.3%, CD127^low: 4.0±1.3 vs 5.0±3.0%* and CTLA4+: 13.8±11.5 vs 15.6±12.5%; * p<0.05).

Conclusion

Short-term GC therapy does not induce the hitherto supposed increase in circulating T_reg cell frequency, neither in immunocompetent humans nor in mice. Thus, it is questionable that the clinical efficacy of GCs is achieved by modulating T_reg cell numbers.     

FcγRIIIa expression on monocytes in rheumatoid arthritis: role in immune-complex stimulated TNF production and non-response to methotrexate therapy

Objective

The expression of FcγRIIIa/CD16 may render monocytes targets for activation by IgG-containing immune complexes (IC). We investigated whether FcγRIIIa/CD16 was upregulated in rheumatoid arthritis (RA), associated with TNF production in response to IC-stimulation, and if this predicted response to methotrexate therapy.

Methods

FcγRIIIa/CD16 expression on CD14^low and CD14⁺⁺ monocytes was measured by flow cytometry in healthy controls and RA patients (early and long-standing disease). Intracellular TNF-staining was carried out after in vitro LPS or heat-aggregated immunoglobulin (HAG) activation. FcγRIIIa/CD16 expression pre- and post-steroid/methotrexate treatment was examined.

Results

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in long-standing RA patients compared to controls was demonstrated (p = 0.002) with intermediate levels in early-RA patients. HAG-induced TNF-production in RA patients was correlated with the percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 (p<0.001). The percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 at baseline in early DMARD-naïve RA patients was negatively correlated with DAS28-ESR improvement 14-weeks post-methotrexate therapy (p = 0.003) and was significantly increased in EULAR non-responders compared to moderate (p = 0.01) or good responders (p = 0.003). FcγRIIIa/CD16 expression was not correlated with age, presence of systemic inflammation or autoantibody titers.

Conclusion

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in RA may result in a cell that has increased responsiveness to IC-stimulation. This monocyte subset may contribute to non-response to methotrexate therapy.     

Augmented atherogenesis in LDL receptor deficient mice lacking both macrophage ABCA1 and ApoE

Aim

ABCA1 protects against atherosclerosis by facilitating cholesterol efflux from macrophage foam cells in the arterial wall to extracellular apolipoprotein (apo) A-I. In contrast to apoA-I, apoE is secreted by macrophages and can, like apoA-I, induce ABCA1-mediated cholesterol efflux. Yet, the combined effect of macrophage ABCA1 and apoE on lesion development is unexplored.

Methods and Results

LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCA1/apoE double KO (dKO) mice, their respective single KO''s, and wild-type (WT) controls and were challenged with a high-fat/high-cholesterol diet for 9 weeks. In vitro cholesterol efflux experiments showed no differences between ABCA1 KO and dKO macrophages. The serum non-HDL/HDL ratio in dKO transplanted mice was 1.7-fold and 2.4-fold (p<0.01) increased compared to WT and ABCA1 KO transplanted mice, respectively. The atherosclerotic lesion area in dKO transplanted animals (650±94×10³ µm²), however, was 1.9-fold (p<0.01) and 1.6-fold (p<0.01) increased compared to single knockouts (ABCA1 KO: 341±20×10³ µm²; apoE KO: 402±78×10³ µm², respectively) and 3.1-fold increased (p<0.001) compared to WT (211±20×10³ µm²). When normalized for serum cholesterol exposure, macrophage ABCA1 and apoE independently protected against atherosclerotic lesion development (p<0.001). Moreover, hepatic expression levels of TNFα and IL-6 were highly induced in dKO transplanted animals (3.0-fold; p<0.05, and 4.3-fold; p<0.001, respectively). In agreement, serum IL-6 levels were also enhanced in ABCA1 KO transplanted mice (p<0.05) and even further enhanced in dKO transplanted animals (3.1-fold as compared to ABCA1 KO transplanted animals; p<0.05).

Conclusions

Combined deletion of macrophage ABCA1 and apoE results in a defect in cholesterol efflux and, compared to ABCA1 KO transplanted mice, elevated serum total cholesterol levels. Importantly, these mice also suffer from enhanced systemic and hepatic inflammation, together resulting in the observed augmented atherosclerotic lesion development.     

Role of heparanase on hepatic uptake of intestinal derived lipoprotein and fatty streak formation in mice

Background

Heparanase modulates the level of heparan sulfate proteoglycans (HSPGs) which have an important role in multiple cellular processes. Recent studies indicate that HSPGs have an important function in hepatic lipoprotein handling and processes involving removal of lipoprotein particles.

Principal Findings

To determine the effects of decreased HSPGs chain length on lipoprotein metabolism and atherosclerosis, transgenic mice over-expressing the human heparanase gene were studied.Hepatic lipid uptake in hpa-Tg mice were evaluated by giving transgenic mice oral fat loads and labeled retinol. Sections of aorta from mice over-expressing heparanase (hpa-Tg) and controls (C57/BL6) fed an atherogenic diet were examined for evidence of atherosclerosis. Heparanase over-expression results in reduced hepatic clearance of postprandial lipoproteins and higher levels of fasting and postprandial serum triglycerides. Heparanase over-expression also induces formation of fatty streaks in the aorta. The mean lesion cross-sectional area in heparanase over-expressing mice was almost 6 times higher when compared to control mice (23,984 µm²±5,922 vs. 4,189 µm²±1,130, p<0.001).

Conclusions

Over-expression of heparanase demonstrates the importance of HSPGs for the uptake of intestinal derived lipoproteins and its role in the formation of fatty streaks.     

Coreceptor and cytokine concentrations may not explain differences in disease progression observed in HIV-1 clade A and D infected Ugandans

Background

The use of cellular coreceptors and modulation of cytokine concentrations by HIV to establish a productive infection is well documented. However, it is unknown whether the expression of these proteins affects the course of HIV clade A and D disease, reported to have different progression rates.

Methodology/Principal Findings

We investigated whether the number of CD4⁺ T-cells expressing CCR5 or CXCR4, the density of these coreceptors and concentrations of specific immune proteins linked to HIV pathogenesis vary between individuals infected with HIV clade A or D. We undertook additional analyses stratifying participants by early (CD4>500 cells/µl) or late (CD4<200 cells/µl) disease stage. Whole blood samples were taken from 50 HIV-1 infected individuals drawn from cohorts in rural south-west Uganda. Late stage participants had less than half the number of CD4⁺/CCR5⁺ T-cells (p = 0.0113) and 5.6 times fewer CD4⁺/CXCR4⁺ cells (p<0.0001) than early stage participants. There was also a statistically significant difference in the density of CXCR4 on CD4⁺ cells between clade A and D infected early stage participants (142 [A] vs 84 [D]; p = 0.0146). Across all participants we observed significantly higher concentration of Th₁ cytokines compared to Th₂ (66.4 vs 23.8 pg/ml; p<0.0001). Plasma concentrations of IFNγ and IL-2 were 1.8 and 2.4 fold lower respectively in Late-D infected participants compared to Late-A participants. MIP-1β levels also decreased from 118.0 pg/ml to 47.1 pg/ml (p = 0.0396) as HIV disease progressed.

Conclusions/Significance

We observed specific alterations in the abundance of CD4⁺/CCR5⁺ and CD4⁺/CXCR4⁺ T-cells, and concentrations of immune proteins across different HIV clades and as infection progresses. Our results suggest that these changes are unlikely to explain the observed differences in disease progression between subtype A and D infections. However, our observations further the understanding of the natural progression of non-clade B HIV infection and how the virus adapts to exploit the host environment.     

Plasma lipids and betaine are related in an acute coronary syndrome cohort

Background

Low plasma betaine has been associated with unfavorable plasma lipid profiles and cardiovascular risk. In some studies raised plasma betaine after supplementation is associated with elevations in plasma lipids. We aimed to measure the relationships between plasma and urine betaine and plasma lipids, and the effects of lipid-lowering drugs on these.

Methodology

Fasting plasma samples were collected from 531 subjects (and urine samples from 415) 4 months after hospitalization for an acute coronary syndrome episode. In this cross-sectional study, plasma betaine and dimethylglycine concentrations and urine excretions were compared with plasma lipid concentrations. Subgroup comparisons were made for gender, with and without diabetes mellitus, and for drug treatment.

Principal Findings

Plasma betaine negatively correlated with triglyceride (Spearman''s r_s = −0.22, p<0.0001) and non-high-density lipoprotein cholesterol (r_s = −0.27, p<0.0001). Plasma betaine was a predictor of BMI (p<0.05) and plasma non-high-density lipoprotein cholesterol and triglyceride (p<0.001) independently of gender, age and the presence of diabetes. Using data grouped by plasma betaine decile, increasing plasma betaine was linearly related to decreases in BMI (p = 0.008) and plasma non-HDL cholesterol (p = 0.002). In a non-linear relationship betaine was negatively associated with elevated plasma triglycerides (p = 0.004) only for plasma betaine >45 µmol/L. Subjects taking statins had higher plasma betaine concentrations (p<0.001). Subjects treated with a fibrate had lower plasma betaine (p = 0.003) possibly caused by elevated urine betaine loss (p<0.001). The ratio of coenzyme Q to non-high-density lipoprotein cholesterol was higher in subjects with higher plasma betaine, and in subjects taking a statin.

Conclusion

Low plasma betaine concentrations correlated with an unfavourable lipid profile. Betaine deficiency may be common in the study population. Controlled clinical trials of betaine supplementation should be conducted in appropriate populations to determine whether correction affects cardiovascular risk.     

Identification of a New Functional Domain in Angiopoietin-like 3 (ANGPTL3) and Angiopoietin-like 4 (ANGPTL4) Involved in Binding and Inhibition of Lipoprotein Lipase (LPL)

Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are secreted proteins that regulate triglyceride (TG) metabolism in part by inhibiting lipoprotein lipase (LPL). Recently, we showed that treatment of wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4, recapitulated the Angptl4 knock-out (-/-) mouse phenotype of reduced serum TG levels. In the present study, we mapped the region of mouse ANGPTL4 recognized by mAb 14D12 to amino acids Gln²⁹–His⁵³, which we designate as specific epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL, consistent with its ability to neutralize the LPL-inhibitory activity of ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino acids Glu³²–His⁵⁵. We produced a mouse mAb against this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL activity in vitro. Treatment of wild-type as well as hyperlipidemic mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the lipid phenotype found in Angptl3^-/- mice. These results show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain important for binding LPL and inhibiting its activity in vitro and in vivo. Moreover, these results demonstrate that therapeutic antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of some forms of hyperlipidemia.Lipoprotein lipase (LPL)⁵ plays a pivotal role in lipid metabolism by catalyzing the hydrolysis of plasma triglycerides (TGs). LPL is likely to be regulated by mechanisms that depend on nutritional status and on the tissue in which it is expressed (1–3). Two secreted proteins, angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4), play important roles in the regulation of LPL activity (4, 5). ANGPTL3 and ANGPTL4 consist of a signal peptide, an N-terminal segment containing coiled-coil domains, and a C-terminal fibrinogen-like domain. The N-terminal segment as well as full-length ANGPTL3 and ANGPTL4 have been shown to inhibit LPL activity, and deletion of the N-terminal segment of ANGPTL3 and ANGPTL4 resulted in total loss of LPL-inhibiting activity (6, 7). These observations clearly indicate that the N-terminal region of ANGPTL4 contains the functional domain that inhibits LPL and affects plasma lipid levels. The coiled-coil domains have been proposed to be responsible for oligomerization (8); however, it is not known whether the coiled-coil domains directly mediate the inhibition of LPL activity.To define the physiological role of ANGPTL4 more clearly, we characterized the pharmacological consequences of ANGPTL4 inhibition in mice treated with the ANGPTL4-neutralizing monoclonal antibody (mAb) 14D12 (9). Injection of mAb 14D12 significantly lowered fasting TG levels in C57BL/6J mice relative to levels in C57BL/6J mice treated with an isotype-matched anti-KLH control (KLH) mAb (9). These reduced TG values were similar to decreases in fasting plasma TG levels measured in Angptl4 knock-out (-/-) mice. This study demonstrated that mAb 14D12 is a potent ANGPTL4-neutralizing antibody that is able to inhibit systemic ANGPTL4 activity and thereby recapitulate the reduced lipid phenotype found in Angptl4^-/- mice. The readily apparent pharmacological effect of mAb 14D12 prompted new questions about the epitope recognized by mAb 14D12 and how this antibody-antigen binding event affected ANGPTL4 function as an LPL inhibitor.Although ANGPTL4 is able to interact directly with LPL (10), it is not clear which amino acids within ANGPTL4 mediate this interaction. Here we show that amino acids Gln²⁹–His⁵³ of mANGPTL4 contain the epitope for mAb 14D12. This region, hereby designated specific epitope 1 (SE1), also defines a domain that mediates the interaction between ANGPTL4 and LPL and the subsequent inactivation of LPL. With this information we present evidence that ANGPTL3 also contains an SE1 region, and with antibodies specifically reactive with ANGPTL3 SE1 we examine whether the ANGPTL3 SE1 region is involved in LPL binding and inhibition. We also determined whether treatment of C57BL/6 mice with an anti-ANGPTL3 SE1 mAb can recapitulate the phenotype of lower serum TG and cholesterol levels found in Angptl3^-/- mice. Finally we tested the therapeutic potential of an anti-ANGPTL3 SE1 mAb for treatment of hyperlipidemia in apolipoprotein E^-/- (ApoE^-/-) or low density lipoprotein receptor^-/- (LDLr^-/-) mice.     

Adipocytokines and CD34 progenitor cells in Alzheimer's disease

Background

Alzheimer''s disease (AD) and atherosclerosis share common vascular risk factors such as arterial hypertension and hypercholesterolemia. Adipocytokines and CD34⁺ progenitor cells are associated with the progression and prognosis of atherosclerotic diseases. Their role in AD is not adequately elucidated.

Methods and Findings

In the present study, we measured in 41 patients with early AD and 37 age- and weight-matched healthy controls blood concentrations of adiponectin and leptin by enzyme linked immunoabsorbent assay and of CD34⁺ progenitor cells using flow cytometry. We found significantly lower plasma levels of leptin in AD patients compared with the controls, whereas plasma levels of adiponectin did not show any significant differences (AD vs. control (mean±SD): leptin:8.9±5.6 ng/mL vs.16.3±15.5 ng/mL;P = 0.038; adiponectin:18.5±18.1 µg/mL vs.16.7±8.9 µg/mL;P = 0.641). In contrast, circulating CD34⁺ cells were significantly upregulated in AD patients (mean absolute cell count±SD:253±51 vs. 203±37; P = 0.02) and showed an inverse correlation with plasma levels of leptin (r = −0.248; P = 0.037).In logistic regression analysis, decreased leptin concentration (P = 0.021) and increased number of CD34⁺ cells (P = 0.036) were both significantly associated with the presence of AD. According to multifactorial analysis of covariance, leptin serum levels were a significant independent predictor for the number of CD34⁺ cells (P = 0.002).

Conclusions

Our findings suggest that low plasma levels of leptin and increased numbers of CD34⁺ progenitor cells are both associated with AD. In addition, the results of our study provide first evidence that increased leptin plasma levels are associated with a reduced number of CD34⁺ progenitor cells in AD patients. These findings point towards a combined involvement of leptin and CD34⁺ progenitor cells in the pathogenesis of AD. Thus, plasma levels of leptin and circulating CD34⁺ progenitor cells could represent an important molecular link between atherosclerotic diseases and AD. Further studies should clarify the pathophysiological role of both adipocytokines and progenitor cells in AD and possible diagnostic and therapeutic applications.     

Circulating vascular progenitor cells and central arterial stiffness in polycystic ovary syndrome

Objective

Subjects with Polycystic ovarian syndrome (PCOS) are at increased risk of Type 2 diabetes mellitus (T2DM). The mechanism of this enhanced risk is unclear. Circulating vascular progenitor cells (VPC) are immature bone marrow derived cells capable of differentiating into mature endothelial cells. VPC number/function and central arterial stiffness predict cardio-metabolic disease in at-risk populations.

Design

We studied VPC and arterial stiffness measures in non-obese PCOS subjects as compared to age and body mass index (BMI) matched healthy controls in a cross–sectional study.

Methods

Fourteen subjects with PCOS and 12 controls of similar age, BMI (all <30 kg/m²) and metabolic profile were studied. VPC number and in vitro function were studied by flow cytometry and tube formation assays respectively. Augmentation index (AIx), a measure of central arterial stiffness, and central (aortic) blood pressures (BP) were measured by applanation tonometry.

Results

Subjects with PCOS had a reduced number, mean±SEM, of circulating CD34⁺133⁺ VPCs (317.5±51.0 vs. 558.3±101.2, p = 0.03) and impaired in vitro tube formation (completed tube area 1.0±0.06 vs. 1.2±0.05×10⁶ µm² p = 0.02). PCOS subjects had significantly higher AIx (18.4±1.9% vs. 4.9±2.0%) and this difference remained significant even after adjustments for age, BMI and smoking (p = 0.003) in multivariate analyses. Central systolic and pulse pressure were higher in PCOS subjects but these differences were not statistically significant after adjustment for age. Brachial systolic and pulse pressures were similar. VPC number/function and arterial stiffness or BP measures were not correlated.

Conclusions

Non-obese PCOS is characterized by a reduced VPC number, impaired VPC function and increased central arterial stiffness. These changes in novel vascular risk markers may explain the enhanced risk of T2DM and CVD in PCOS.     

No influence of dabigatran anticoagulation on hemorrhagic transformation in an experimental model of ischemic stroke

Background

Dabigatran etexilate (DE) is a new oral direct thrombin inhibitor. Clinical trials point towards a favourable risk-to-benefit profile of DE compared to warfarin. In this study, we evaluated whether hemorrhagic transformation (HT) occurs after experimental stroke under DE treatment as we have shown for warfarin.

Methods

44 male C57BL/6 mice were pretreated orally with 37.5 mg/kg DE, 75 mg/kg DE or saline and diluted thrombin time (dTT) and DE plasma concentrations were monitored. Ischemic stroke was induced by transient middle cerebral artery occlusion (tMCAO) for 1 h or 3 h. We assessed functional outcome and HT blood volume 24 h and 72 h after tMCAO.

Results

After 1 h tMCAO, HT blood volume did not differ significantly between mice pretreated with DE 37.5 mg/kg and controls (1.5±0.5 µl vs. 1.8±0.5 µl, p>0.05). After 3 h tMCAO, DE-anticoagulated mice did also not show an increase in HT, neither at the dose of 37.5 mg/kg equivalent to anticoagulant treatment in the therapeutic range (1.3±0.9 µl vs. control 2.3±0.5 µl, p>0.05) nor at 75 mg/kg, clearly representing supratherapeutic anticoagulation (1.8±0.8 µl, p>0.05). Furthermore, no significant increase in HT under continued anticoagulation with DE 75 mg/kg could be found at 72 h after tMCAO for 1 h (1.7±0.9 µl vs. control 1.6±0.4 µl, p>0.05).

Conclusion

Our experimental data suggest that DE does not significantly increase hemorrhagic transformation after transient focal cerebral ischemia in mice. From a translational viewpoint, this indicates that a continuation of DE anticoagulation in case of an ischemic stroke might be safe, but clearly, clinical data on this question are warranted.     

Increased bone marrow interleukin-7 (IL-7)/IL-7R levels but reduced IL-7 responsiveness in HIV-positive patients lacking CD4+ gain on antiviral therapy

Background

The bone marrow (BM) cytokine milieu might substantially affect T-lymphocyte homeostasis in HIV-positive individuals. Interleukin-7 (IL-7) is a bone marrow-derived cytokine regulating T-cell homeostasis through a CD4+-driven feedback loop. CD4+ T-lymphopenia is associated with increased free IL-7 levels and reduced IL-7R expression/function, which are only partially reverted by highly active antiretroviral therapy (HAART). We investigated the BM production, peripheral expression and signaling (pStat5+ and Bcl-2+ CD4+/CD8+ T cells) of IL-7/IL-7Rα in 30 HAART-treated HIV-positive patients who did not experience CD4+ recovery (CD4+ ≤200/µl) and who had different levels of HIV viremia; these patients included 18 immunological nonresponders (INRs; HIV-RNA≤50), 12 complete failures (CFs; HIV-RNA>1000), and 23 HIV-seronegative subjects.

Methods

We studied plasma IL-7 levels, IL-7Rα+CD4+/CD8+ T-cell proportions, IL-7Rα mRNA expression in PBMCs, spontaneous IL-7 production by BM mononuclear cells (BMMCs), and IL-7 mRNA/IL-7Rα mRNA in BMMC-derived stromal cells (SCs). We also studied T-cell responsiveness to IL-7 by measuring the proportions of pStat5+ and Bcl-2+ CD4+/CD8+ T cells.

Results

Compared to HIV-seronegative controls, CFs and INRs presented elevated plasma IL-7 levels and lower IL-7Rα CD4+/CD8+ cell-surface expression and peripheral blood production, confirming the most relevant IL-7/IL-7R disruption. Interestingly, BM investigation revealed a trend of higher spontaneous IL-7 production in INRs (p = .09 vs. CFs) with a nonsignificant trend toward higher IL-7-Rα mRNA levels in BMMC-derived stromal cells. However, upon IL-7 stimulation, the proportion of pStat5+CD4+ T cells did not increase in INRs despite higher constitutive levels (p = .06); INRs also displayed lower Bcl-2+CD8+ T-cell proportions than controls (p = .04).

Conclusions

Despite severe CD4+ T-lymphopenia and a disrupted IL-7/IL-7R profile in the periphery, INRs display elevated BM IL-7/IL-7Rα expression but impaired T-cell responsiveness to IL-7, suggesting the activity of a central compensatory pathway targeted to replenish the CD4+ compartment, which is nevertheless inappropriate to compensate the dysfunctional signaling through IL-7 receptor.     

Bile acid sequestration reduces plasma glucose levels in db/db mice by increasing its metabolic clearance rate

Aims/Hypothesis

Bile acid sequestrants (BAS) reduce plasma glucose levels in type II diabetics and in murine models of diabetes but the mechanism herein is unknown. We hypothesized that sequestrant-induced changes in hepatic glucose metabolism would underlie reduced plasma glucose levels. Therefore, in vivo glucose metabolism was assessed in db/db mice on and off BAS using tracer methodology.

Methods

Lean and diabetic db/db mice were treated with 2% (wt/wt in diet) Colesevelam HCl (BAS) for 2 weeks. Parameters of in vivo glucose metabolism were assessed by infusing [U-¹³C]-glucose, [2-¹³C]-glycerol, [1-²H]-galactose and paracetamol for 6 hours, followed by mass isotopologue distribution analysis, and related to metabolic parameters as well as gene expression patterns.

Results

Compared to lean mice, db/db mice displayed an almost 3-fold lower metabolic clearance rate of glucose (p = 0.0001), a ∼300% increased glucokinase flux (p = 0.001) and a ∼200% increased total hepatic glucose production rate (p = 0.0002). BAS treatment increased glucose metabolic clearance rate by ∼37% but had no effects on glucokinase flux nor total hepatic or endogenous glucose production. Strikingly, BAS-treated db/db mice displayed reduced long-chain acylcarnitine content in skeletal muscle (p = 0.0317) but not in liver (p = 0.189). Unexpectedly, BAS treatment increased hepatic FGF21 mRNA expression 2-fold in lean mice (p = 0.030) and 3-fold in db/db mice (p = 0.002).

Conclusions/Interpretation

BAS induced plasma glucose lowering in db/db mice by increasing metabolic clearance rate of glucose in peripheral tissues, which coincided with decreased skeletal muscle long-chain acylcarnitine content.     

Fructose modulates cardiomyocyte excitation-contraction coupling and Ca²⁺ handling in vitro

Background

High dietary fructose has structural and metabolic cardiac impact, but the potential for fructose to exert direct myocardial action is uncertain. Cardiomyocyte functional responsiveness to fructose, and capacity to transport fructose has not been previously demonstrated.

Objective

The aim of the present study was to seek evidence of fructose-induced modulation of cardiomyocyte excitation-contraction coupling in an acute, in vitro setting.

Methods and Results

The functional effects of fructose on isolated adult rat cardiomyocyte contractility and Ca²⁺ handling were evaluated under physiological conditions (37°C, 2 mM Ca²⁺, HEPES buffer, 4 Hz stimulation) using video edge detection and microfluorimetry (Fura2) methods. Compared with control glucose (11 mM) superfusate, 2-deoxyglucose (2 DG, 11 mM) substitution prolonged both the contraction and relaxation phases of the twitch (by 16 and 36% respectively, p<0.05) and this effect was completely abrogated with fructose supplementation (11 mM). Similarly, fructose prevented the Ca²⁺ transient delay induced by exposure to 2 DG (time to peak Ca²⁺ transient: 2 DG: 29.0±2.1 ms vs. glucose: 23.6±1.1 ms vs. fructose +2 DG: 23.7±1.0 ms; p<0.05). The presence of the fructose transporter, GLUT5 (Slc2a5) was demonstrated in ventricular cardiomyocytes using real time RT-PCR and this was confirmed by conventional RT-PCR.

Conclusion

This is the first demonstration of an acute influence of fructose on cardiomyocyte excitation-contraction coupling. The findings indicate cardiomyocyte capacity to transport and functionally utilize exogenously supplied fructose. This study provides the impetus for future research directed towards characterizing myocardial fructose metabolism and understanding how long term high fructose intake may contribute to modulating cardiac function.     

Regulatory effects of IFN-β on the development of experimental autoimmune uveoretinitis in B10RIII mice

Background

Experimental autoimmune uveoretinitis (EAU) serves as a model for human intraocular inflammation. IFN-β has been used in the treatment of certain autoimmune diseases. Earlier studies showed that it ameliorated EAU; however, the mechanisms involved in this inhibition are still largely unknown.

Methodology/Principal Findings

B10RIII mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) peptide 161–180 in Complete Freund''s adjuvant. Splenocytes from different time points after immunization were used to evaluate the expression of IFN-β. An increased expression of IFN-β was observed during EAU and its highest expression was observed on day 16, 3 days after the peak of intraocular inflammation. Splenocytes and draining lymph node cells from mice immunized with IRBP_161-180 on day 13 and control mice were activated with anti-CD3/anti-CD28 antibodies or IRBP_161-180 to evaluate the production of IFN-γ and IL-17. The results showed that IFN-γ and IL-17 were significantly higher in immunized mice as compared to the control mice when exposed to anti-CD3/anti-CD28 antibodies. However, the production of IFN-γ and IL-17 was detected only in immunized mice, but not in the control mice when stimulated with IRBP_161-180. Multiple subcutaneous injections of IFN-β significantly inhibited EAU activity in association with a down-regulated expression of IFN-γ, IL-17 and an enhanced IL-10 production. In an in vitro system using cells from mice, IFN-β suppressed IFN-γ production by CD4⁺CD62L⁻ T cells, IL-17 production by CD4⁺CD62L^+/- T cells and proliferation of CD4⁺CD62L^+/- T cells. IFN-β inhibited the secretion of IL-6, but promoted the secretion of IL-10 by monocytes. IFN-β-treated monocytes inhibited IL-17 secretion by CD4⁺CD62L^+/- T cells, but did not influence IFN-γ expression and T cell proliferation.

Conclusions/Significance

IFN-β may exert its inhibitory effect on EAU by inhibiting Th1, Th17 cells and modulating relevant cytokines. IFN-β may provide a potential treatment for diseases mediated by Th1 and Th17 cells.