Wiskott–Aldrich Syndrome causing mutation,Pro373Ser restricts conformational changes essential for WASP activity in T-cells

Wiskott–Aldrich Syndrome (WAS) is caused by mutations in Wiskott-Aldrich Syndrome Protein (WASP) and majority of the mutations are found in the WASP Homology ₁ (WH₁) domain which mediates interaction with WIP (WASP Interacting Protein), a WASP chaperone. Two point mutations together in the proline rich region (PRR) domain of WASP (S339Y/P373S) have been reported to cause WAS however the molecular defect has not been characterized. Expression of these mutants separately (WASP_R^S339Y, WASP_R^P373S) or together (WASP_R^SP/YS) did not rescue the chemotaxis defect or membrane projection defect of Jurkat^WKD T-cells (WASP knockdown). This is not due to the inability of WASP-PRR mutants to form functional WASP–WIP complex in growth rescue experiments in las17Δ yeast strain. Expression of WASP_R^S339Y but not WASP_R^P373S or WASP_R^SP/YS rescued the IL-2 expression defect of Jurkat^WKD T-cells, suggesting that Pro373Ser mutation alone is sufficient to inhibit WASP functions in T-cell activation. The diffused localization of WASP-PRR mutants in activated Jurkat T-cells suggests that Ser339 and Pro373 are critical for WASP localization. WASP-PRR mutations either together or individually did not abolish interaction of WASP with sixteen WASP binding proteins including Hck, however they caused reduction in Hck mediated tyrosine phosphorylation of WASP which is critical for WASP activity. The auto-inhibitory conformation of WASP^P373S mutant was not relieved by the binding of Toca-1 or Nck1. Thus, our results suggest that Pro373Ser mutation reduces Tyr291 phosphorylation and prevents conformational changes required for WASP activity in chemotaxis and T-cell activation. Thus Pro3373Ser is probably responsible for all the defects associated with WAS in the patients.     

TNF inhibits production of stromal cell-derived factor 1 by bone stromal cells and increases osteoclast precursor mobilization from bone marrow to peripheral blood

Introduction

The objective of the present study was to investigate the role of the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis in TNF-induced mobilization of osteoclast precursors (OCPs) from bone marrow.

Methods

OCPs were generated from bone marrow cells of TNF-transgenic mice or wild-type mice treated with TNF or PBS. The percentage of CD11b⁺/Gr-1^-/lo OCPs was assessed by fluorescence-activated cell sorting. OCP migration to the SDF-1 gradient and the osteoclast forming potency were assessed in chemotaxis/osteoclastogenic assays. SDF-1 expression was assessed by real-time RT-PCR, ELISA and immunostaining in primary bone marrow stromal cells, in the ST2 bone marrow stromal cell line, and in bones from TNF-injected mice.

Results

OCPs generated in vitro from wild-type mice migrated to SDF-1 gradients and subsequently gave rise to osteoclasts in response to RANKL and macrophage colony-stimulating factor. TNF reduced SDF-1 expression by ST2 cells. Bone marrow stromal cells from TNF-transgenic mice produced low levels of SDF-1. TNF treatment of wild-type mice decreased the SDF-1 concentration in bone marrow extracts and decreased the SDF-1 immunostaining of bone marrow stromal cells, and it also increased the circulating OCP numbers. The percentage of bone marrow CXCR4⁺ OCPs was similar in TNF-transgenic mice and wild-type littermates and in TNF-treated and PBS-treated wild-type mice.

Conclusion

Systemically elevated TNF levels inhibit bone marrow stromal cell production of SDF-1 and increase the release of bone marrow OCPs to the peripheral blood. Disruption of the SDF-1/CXCR4 axis by TNF may play an important role in mediating OCP mobilization from the bone marrow cavity in chronic inflammatory arthritis.     

Upregulation of Stromal Cell–Derived Factor 1 (SDF-1) is Associated with Macrophage Infiltration in Renal Ischemia-Reperfusion Injury

Background

Stromal cell-derived factor-1(SDF-1) is a chemotactic and angiogenic factor that mediates the repair of various tissues. As macrophages are important contributors to ischemic kidney injury, we examine the role of SDF-1 in a rodent model of ischemia-reperfusion (I/R) injury.

Methods

Male wild-type (WT) (C57BL/6) mice were subjected to bilateral I/R injury or sham operation in the presence or absence of macrophage depletion (liposomal clodronate [0.2 ml/20–25 g body weight i.p.]). Macrophage accumulation was assessed by immunohistochemistry. Tissue levels of SDF-1 (ELISA) and SDF-1 mRNA expression (real-time PCR) were measured. The cellular location of SDF-1 was assessed using immunohistochemical staining.

Results

Immunofluorescence staining of renal tissue sections confirmed macrophage depletion by liposomal clodronate. SDF-1 production was elevated in response to I/R injury and was significantly increased upon macrophage depletion. SDF-1 positive cells initially appeared initially in the cortex, and subsequently diffused to the outer medulla after I/R injury.

Conclusions

Our study demonstrates that SDF-1 is significantly upregulated during renal I/R. We hypothesize that SDF-1 upregulation may be an important macrophage effector mechanism during I/R injury.     

Airflow limitation or static hyperinflation: which is more closely related to dyspnea with activities of daily living in patients with COPD?

Background

Dyspnea while performing the activities of daily living has been suggested to be a better measurement than peak dyspnea during exercise. Furthermore, the inspiratory capacity (IC) has been shown to be more closely related to exercise tolerance and dyspnea than the FEV₁, because dynamic hyperinflation is the main cause of shortness of breath in patients with COPD. However, breathlessness during exercise is measured in most studies to evaluate this relationship.

Purpose

To evaluate the correlation between breathlessness during daily activities and airflow limitation or static hyperinflation in COPD.

Methods

We examined 167 consecutive outpatients with stable COPD. The Baseline Dyspnea Index (BDI) was used to evaluate dyspnea with activities of daily living. The relationship between the BDI score and the clinical measurements of pulmonary function was then investigated.

Results

The Spearman rank correlation coefficients (Rs) between the BDI score and the FEV₁(L), FEV₁(%pred) and FEV₁/FVC were 0.60, 0.56 and 0.56, respectively. On the other hand, the BDI score also correlated with the IC, IC/predicted total lung capacity (TLC) and IC/TLC (Rs = 0.45, 0.46 and 0.47, respectively). Although all of the relationships studied were strongly correlated, the correlation coefficients were better between dyspnea and airflow limitation than between dyspnea and static hyperinflation. In stepwise multiple regression analyses, the BDI score was most significantly explained by the FEV₁ (R² = 26.2%) and the diffusion capacity for carbon monoxide (R² = 14.4%) (Cumulative R² = 40.6%). Static hyperinflation was not a significant factor for clinical dyspnea on the stepwise multiple regression analysis.

Conclusion

Both static hyperinflation and airflow limitation contributed greatly to dyspnea in COPD patients.     

Roots affect the response of heterotrophic soil respiration to temperature in tussock grass microcosms

Aims and Background

While the temperature response of soil respiration (R_S) has been well studied, the partitioning of heterotrophic respiration (R_H) by soil microbes from autotrophic respiration (R_A) by roots, known to have distinct temperature sensitivities, has been problematic. Further complexity stems from the presence of roots affecting R_H, the rhizosphere priming effect. In this study the short-term temperature responses of R_A and R_H in relation to rhizosphere priming are investigated.

Methods

Temperature responses of R_A, R_H and rhizosphere priming were assessed in microcosms of Poa cita using a natural abundance δ¹³C discrimination approach.

Results

The temperature response of R_S was found to be regulated primarily by R_A, which accounted for 70 % of total soil respiration. Heterotrophic respiration was less sensitive to temperature in the presence of plant roots, resulting in negative priming effects with increasing temperature.

Conclusions

The results emphasize the importance of roots in regulating the temperature response of R_S, and a framework is presented for further investigation into temperature effects on heterotrophic respiration and rhizosphere priming, which could be applied to other soil and vegetation types to improve models of soil carbon turnover.     

miR-27b Represses Migration of Mouse MSCs to Burned Margins and Prolongs Wound Repair through Silencing SDF-1a

Background

Interactions between stromal cell-derived factor-1α (SDF-1α) and its cognate receptor CXCR4 are crucial for the recruitment of mesenchymal stem cells (MSCs) from bone marrow (BM) reservoirs to damaged tissues for repair during alarm situations. MicroRNAs are differentially expressed in stem cell niches, suggesting a specialized role in stem cell regulation. Here, we gain insight into the molecular mechanisms involved in regulating SDF-1α.

Methods

MSCs from green fluorescent protein transgenic male mice were transfused to irradiated recipient female C57BL/6 mice, and skin burn model of bone marrow-chimeric mice were constructed. Six miRNAs with differential expression in burned murine skin tissue compared to normal skin tissue were identified using microarrays and bioinformatics. The expression of miR-27b and SDF-1α was examined in burned murine skin tissue using quantitative real-time PCR (qPCR) and immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA). The Correlation of miR-27b and SDF-1α expression was analyzed by Pearson analysis Correlation. miRNAs suppressed SDF-1α protein expression by binding directly to its 3′UTR using western blot and luciferase reporter assay. The importance of miRNAs in MSCs chemotaxis was further estimated by decreasing SDF-1α in vivo and in vitro.

Results

miR-23a, miR-27a and miR-27b expression was significantly lower in the burned skin than in the normal skin (p<0.05). We also found that several miRNAs suppressed SDF-1α protein expression, while just miR-27a and miR-27b directly bound to the SDF-1α 3′UTR. Moreover, the forced over-expression of miR-27a and miR-27b significantly reduced the directional migration of mMSCs in vitro. However, only miR-27b in burn wound margins significantly inhibited the mobilization of MSCs to the epidermis.

Conclusion

miR-27b may be a unique signature of the stem cell niche in burned mouse skin and can suppress the directional migration of mMSCs by targeting SDF-1α by binding directly to its 3′UTR.     

Predicting total, abdominal, visceral and hepatic adiposity with circulating biomarkers in caucasian and Japanese american women

Background

Characterization of abdominal and intra-abdominal fat requires imaging, and thus is not feasible in large epidemiologic studies.

Objective

We investigated whether biomarkers may complement anthropometry (body mass index [BMI], waist circumference [WC], and waist-hip ratio [WHR]) in predicting the size of the body fat compartments by analyzing blood biomarkers, including adipocytokines, insulin resistance markers, sex steroid hormones, lipids, liver enzymes and gastro-neuropeptides.

Methods

Fasting levels of 58 blood markers were analyzed in 60 healthy, Caucasian or Japanese American postmenopausal women who underwent anthropometric measurements, dual energy X-ray absorptiometry (DXA), and abdominal magnetic resonance imaging. Total, abdominal, visceral and hepatic adiposity were predicted based on anthropometry and the biomarkers using Random Forest models.

Results

Total body fat was well predicted by anthropometry alone (R² = 0.85), by the 5 best predictors from the biomarker model alone (leptin, leptin-adiponectin ratio [LAR], free estradiol, plasminogen activator inhibitor-1 [PAI1], alanine transaminase [ALT]; R² = 0.69), or by combining these 5 biomarkers with anthropometry (R² = 0.91). Abdominal adiposity (DXA trunk-to-periphery fat ratio) was better predicted by combining the two types of predictors (R² = 0.58) than by anthropometry alone (R² = 0.53) or the 5 best biomarkers alone (25(OH)-vitamin D₃, insulin-like growth factor binding protein-1 [IGFBP1], uric acid, soluble leptin receptor [sLEPR], Coenzyme Q10; R² = 0.35). Similarly, visceral fat was slightly better predicted by combining the predictors (R² = 0.68) than by anthropometry alone (R² = 0.65) or the 5 best biomarker predictors alone (leptin, C-reactive protein [CRP], LAR, lycopene, vitamin D₃; R² = 0.58). Percent liver fat was predicted better by the 5 best biomarker predictors (insulin, sex hormone binding globulin [SHBG], LAR, alpha-tocopherol, PAI1; R² = 0.42) or by combining the predictors (R² = 0.44) than by anthropometry alone (R² = 0.29).

Conclusion

The predictive ability of anthropometry for body fat distribution may be enhanced by measuring a small number of biomarkers. Studies to replicate these data in men and other ethnic groups are warranted.     

7 Tesla Magnetic Resonance Imaging to Detect Cortical Pathology in Multiple Sclerosis

Background

Neocortical lesions (NLs) are an important pathological component of multiple sclerosis (MS), but their visualization by magnetic resonance imaging (MRI) remains challenging.

Objectives

We aimed at assessing the sensitivity of multi echo gradient echo (ME-GRE) T₂ ^*-weighted MRI at 7.0 Tesla in depicting NLs compared to myelin and iron staining.

Methods

Samples from two MS patients were imaged post mortem using a whole body 7T MRI scanner with a 24-channel receive-only array. Isotropic 200 micron resolution images with varying T₂ ^* weighting were reconstructed from the ME-GRE data and converted into R₂ ^* maps. Immunohistochemical staining for myelin (proteolipid protein, PLP) and diaminobenzidine-enhanced Turnbull blue staining for iron were performed.

Results

Prospective and retrospective sensitivities of MRI for the detection of NLs were 48% and 67% respectively. We observed MRI maps detecting only a small portion of 20 subpial NLs extending over large cortical areas on PLP stainings. No MRI signal changes suggestive of iron accumulation in NLs were observed. Conversely, R₂ ^* maps indicated iron loss in NLs, which was confirmed by histological quantification.

Conclusions

High-resolution post mortem imaging using R₂ ^* and magnitude maps permits detection of focal NLs. However, disclosing extensive subpial demyelination with MRI remains challenging.     

High Dynamic Range Processing for Magnetic Resonance Imaging

Purpose

To minimize feature loss in T₁- and T₂-weighted MRI by merging multiple MR images acquired at different T_R and T_E to generate an image with increased dynamic range.

Materials and Methods

High Dynamic Range (HDR) processing techniques from the field of photography were applied to a series of acquired MR images. Specifically, a method to parameterize the algorithm for MRI data was developed and tested. T₁- and T₂-weighted images of a number of contrast agent phantoms and a live mouse were acquired with varying T_R and T_E parameters. The images were computationally merged to produce HDR-MR images. All acquisitions were performed on a 7.05 T Bruker PharmaScan with a multi-echo spin echo pulse sequence.

Results

HDR-MRI delineated bright and dark features that were either saturated or indistinguishable from background in standard T₁- and T₂-weighted MRI. The increased dynamic range preserved intensity gradation over a larger range of T₁ and T₂ in phantoms and revealed more anatomical features in vivo.

Conclusions

We have developed and tested a method to apply HDR processing to MR images. The increased dynamic range of HDR-MR images as compared to standard T₁- and T₂-weighted images minimizes feature loss caused by magnetization recovery or low SNR.     

Optical and NMR dose response of N-isopropylacrylamide normoxic polymer gel for radiation therapy dosimetry

Background

Application of less toxic normoxic polymer gel of N-isopropyl acrylamide (NIPAM) for radiation therapy has been studied in recent years.

Aim

In the current study the optical and NMR properties of NIPAM were studied for radiation therapy dosimetry application.

Materials and methods

NIPAM normoxic polymer gel was prepared and irradiated by 9 MV photon beam of a medical linac. The optical absorbance was measured using a conventional laboratory spectrophotometer in different wavelengths ranging from 390 to 860 nm. R₂ measurements of NIPAM gels were performed using a 1.5 T scanner and R₂–dose curve was obtained.

Results

Our results showed R₂ dose sensitivity of 0.193 ± 0.01 s⁻¹ Gy⁻¹ for NIPAM gel. Both R₂ and optical absorbance showed a linear relationship with dose from 1.5 to 11 Gy for NIPAM gel dosimeter. Moreover, absorbance–dose response varied considerably with light wavelength and highest sensitivity was seen for the blue part of the spectrum.

Conclusion

Our results showed that both optical and NMR approaches have acceptable sensitivity and accuracy for dose determination with NIPAM gel. However, for optical reading of the gel, utilization of an optimum optical wavelength is recommended.     

Mutation analysis of NR5A1 encoding steroidogenic factor 1 in 77 patients with 46, XY disorders of sex development (DSD) including hypospadias

Background

Mutations of the NR5A1 gene encoding steroidogenic factor-1 have been reported in association with a wide spectrum of 46,XY DSD (Disorder of Sex Development) phenotypes including severe forms of hypospadias.

Methodology/Principal Findings

We evaluated the frequency of NR5A1 gene mutations in a large series of patients presenting with 46,XY DSD and hypospadias. Based on their clinical presentation 77 patients were classified either as complete or partial gonadal dysgenesis (uterus seen at genitography and/or surgery, n = 11), ambiguous external genitalia without uterus (n = 33) or hypospadias (n = 33). We identified heterozygous NR5A1 mutations in 4 cases of ambiguous external genitalia without uterus (12.1%; p.Trp279Arg, pArg39Pro, c.390delG, c140_141insCACG) and a de novo missense mutation in one case with distal hypospadias (3%; p.Arg313Cys). Mutant proteins showed reduced transactivation activity and mutants p.Arg39Pro and p.Arg313Cys did not synergize with the GATA4 cofactor to stimulate reporter gene activity, although they retained their ability to physically interact with the GATA4 protein.

Conclusions/Significance

Mutations in NR5A1 were observed in 5/77 (6.5%) cases of 46,XY DSD including hypospadias. Excluding the cases of 46,XY gonadal dysgenesis the incidence of NR5A1 mutations was 5/66 (7.6%). An individual with isolated distal hypopadias carried a de novo heterozygous missense mutation, thus extending the range of phenotypes associated with NR5A1 mutations and suggesting that this group of patients should be screened for NR5A1 mutations.     

A Novel Mechanism of Soluble HLA-G Mediated Immune Modulation: Downregulation of T Cell Chemokine Receptor Expression and Impairment of Chemotaxis

Background

In recent years, many immunoregulatory functions have been ascribed to soluble HLA-G (sHLA-G). Since chemotaxis is crucial for an efficient immune response, we have investigated for the first time the effects of sHLA-G on chemokine receptor expression and function in different human T cell populations.

Methodology/Principal Findings

T cell populations isolated from peripheral blood were stimulated in the presence or absence of sHLA-G. Chemokine receptors expression was evaluated by flow cytometry. sHLA-G downregulated expression of i) CCR2, CXCR3 and CXCR5 in CD4⁺ T cells, ii) CXCR3 in CD8⁺ T cells, iii) CXCR3 in Th1 clones iv) CXCR3 in TCR Vδ2γ9 T cells, and upregulated CXCR4 expression in TCR Vδ2γ9 T cells. sHLA-G inhibited in vitro chemotaxis of i) CD4⁺ T cells towards CCL2, CCL8, CXCL10 and CXCL11, ii) CD8⁺ T cells towards CXCL10 and CXCL11, iii) Th1 clones towards CXCL10, and iv) TCR Vδ2γ9 T cells towards CXCL10 and CXCL11. Downregulation of CXCR3 expression on CD4+ T cells by sHLA-G was partially reverted by adding a blocking antibody against ILT2/CD85j, a receptor for sHLA-G, suggesting that sHLA-G downregulated chemokine receptor expression mainly through the interaction with ILT2/CD85j. Follicular helper T cells (T_FH) were isolated from human tonsils and stimulated as described above. sHLA-G impaired CXCR5 expression in T_FH and chemotaxis of the latter cells towards CXCL13. Moreover, sHLA-G expression was detected in tonsils by immunohistochemistry, suggesting a role of sHLA-G in local control of T_FH cell chemotaxis. Intracellular pathways were investigated by Western Blot analysis on total extracts from CD4+ T cells. Phosphorylation of Stat5, p70 s6k, β-arrestin and SHP2 was modulated by sHLA-G treatment.

Conclusions/Significance

Our data demonstrated that sHLA-G impairs expression and functionality of different chemokine receptors in T cells. These findings delineate a novel mechanism whereby sHLA-G modulates T cell recruitment in physiological and pathological conditions.     

Distinct Kinin-Induced Functions Are Altered in Circulating Cells of Young Type 1 Diabetic Patients

Aims/Hypothesis

We aimed to understand early alterations in kinin-mediated migration of circulating angio-supportive cells and dysfunction of kinin-sensitive cells in type-1 diabetic (T1D) patients before the onset of cardiovascular disease.

Methods

Total mononuclear cells (MNC) were isolated from peripheral blood of 28 T1D patients free from cardiovascular complications except mild background retinopathy (age: 34.8±1.6 years, HbA_1C: 7.9±0.2%) and 28 age- and sex-matched non-diabetic controls (H). We tested expression of kinin receptors by flow cytometry and migratory capacity of circulating monocytes and progenitor cells towards bradykinin (BK) in transwell migration assays. MNC migrating towards BK (BK^mig) were assessed for capacity to support endothelial cell function in a matrigel assay, as well as generation of nitric oxide (NO) and superoxide (O₂ ⁻*) by using the fluorescent probes diaminofluorescein and dihydroethidium.

Results

CD14^hiCD16^neg, CD14^hiCD16^pos and CD14^loCD16^pos monocytes and circulating CD34^pos progenitor cells did not differ between T1D and H subjects in their kinin receptor expression and migration towards BK. T1D BK^mig failed to generate NO upon BK stimulation and supported endothelial cell network formation less efficiently than H BK^mig. In contrast, O₂ ⁻* production was similar between groups. High glucose disturbed BK-induced NO generation by MNC-derived cultured angiogenic cells.

Conclusions/Interpretation

Our data point out alterations in kinin-mediated functions of circulating MNC from T1D patients, occurring before manifest macrovascular damage or progressed microvascular disease. Functional defects of MNC recruited to the vessel wall might compromise endothelial maintenance, initially without actively promoting endothelial damage, but rather by lacking supportive contribution to endothelial regeneration and healing.     

Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1H46R-Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

Background

ALS2/alsin is a guanine nucleotide exchange factor for the small GTPase Rab5 and involved in macropinocytosis-associated endosome fusion and trafficking, and neurite outgrowth. ALS2 deficiency accounts for a number of juvenile recessive motor neuron diseases (MNDs). Recently, it has been shown that ALS2 plays a role in neuroprotection against MND-associated pathological insults, such as toxicity induced by mutant Cu/Zn superoxide dismutase (SOD1). However, molecular mechanisms underlying the relationship between ALS2-associated cellular function and its neuroprotective role remain unclear.

Methodology/Principal Findings

To address this issue, we investigated the molecular and pathological basis for the phenotypic modification of mutant SOD1-expressing mice by ALS2 loss. Genetic ablation of Als2 in SOD1^H46R, but not SOD1^G93A, transgenic mice aggravated the mutant SOD1-associated disease symptoms such as body weight loss and motor dysfunction, leading to the earlier death. Light and electron microscopic examinations revealed the presence of degenerating and/or swollen spinal axons accumulating granular aggregates and autophagosome-like vesicles in early- and even pre-symptomatic SOD1^H46R mice. Further, enhanced accumulation of insoluble high molecular weight SOD1, poly-ubiquitinated proteins, and macroautophagy-associated proteins such as polyubiquitin-binding protein p62/SQSTM1 and a lipidated form of light chain 3 (LC3-II), emerged in ALS2-deficient SOD1^H46R mice. Intriguingly, ALS2 was colocalized with LC3 and p62, and partly with SOD1 on autophagosome/endosome hybrid compartments, and loss of ALS2 significantly lowered the lysosome-dependent clearance of LC3 and p62 in cultured cells.

Conclusions/Significance

Based on these observations, although molecular basis for the distinctive susceptibilities to ALS2 loss in different mutant SOD1-expressing ALS models is still elusive, disturbance of the endolysosomal system by ALS2 loss may exacerbate the SOD1^H46R-mediated neurotoxicity by accelerating the accumulation of immature vesicles and misfolded proteins in the spinal cord. We propose that ALS2 is implicated in endolysosomal trafficking through the fusion between endosomes and autophagosomes, thereby regulating endolysosomal protein degradation in vivo.     

Vibration response imaging: a novel noninvasive tool for evaluating the initial therapeutic effect of noninvasive positive pressure ventilation in patients with acute exacerbation of chronic obstructive pulmonary disease

Background

The popular methods for evaluating the initial therapeutic effect (ITE) of noninvasive positive pressure ventilation (NPPV) can only roughly reflect the therapeutic outcome of a patient’s ventilation because they are subjective, invasive and time-delayed. In contrast, vibration response imaging (VRI) can monitor the function of a patient’s ventilation over the NPPV therapy in a non-invasive manner. This study aimed to investigate the value of VRI in evaluating the ITE of NPPV for patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD).

Methods

Thirty-six AECOPD patients received VRI at three time points: before NPPV treatment (T1), at 15 min of NPPV treatment (T2), and at 15 min after the end of NPPV treatment (T4). Blood gas analysis was also performed at T1 and at 2 hours of NPPV treatment (T3). Thirty-nine healthy volunteers also received VRI at T1 and T2. VRI examination at the time point T2 in either the patients or volunteers did not require any interruption of the on-going NPPV. The clinical indices at each time point were compared between the two groups. Moreover, correlations between the PaCO₂ changes (T3 vs T1) and abnormal VRI scores (AVRIS) changes (T2 vs T1) were analyzed.

Results

No significant AVRIS differences were found between T1 and T2 in the healthy controls (8.51 ± 3.36 vs. 8.53 ± 3.57, P > 0.05). The AVRIS, dynamic score, MEF score and EVP score showed a significant decrease in AECOPD patients at T2 compared with T1 (P < 0.05), but a significant increase at T4 compared with T2 (P < 0.05). We also found a positive correlation (R² = 0.6399) between the PaCO₂ changes (T3 vs T1) and AVRIS changes (T2 vs T1).

Conclusions

VRI is a promising noninvasive tool for evaluating the initial therapeutic effects of NPPV in AECOPD patients and predicting the success of NPPV in the early stage.     

Methylprednisolone Stiffens Aortas in Lipopolysaccharide-Induced Chronic Inflammation in Rats

Introduction

Glucocorticoids are commonly used as therapeutic agents in many acute and chronic inflammatory and auto-immune diseases. The current study investigated the effects of methylprednisolone (a synthetic glucocorticoid) on aortic distensibility and vascular resistance in lipopolysaccharide-induced chronic inflammation in male Wistar rats.

Methods

Chronic inflammation was induced by implanting a subcutaneous slow-release ALZET osmotic pump (1 mg kg⁻¹ day⁻¹ lipopolysaccharide) for either 2 or 4 weeks. Arterial wave transit time (τ) was derived to describe the elastic properties of aortas using the impulse response function of the filtered aortic input impedance spectra.

Results

Long-term lipopolysaccharide challenge enhanced the expression of advanced glycation end products (AGEs) in the aortas. Lipopolysaccharide also upregulated the inducible form of nitric oxide synthase to produce high levels of nitric oxide (NO), which resulted in vasodilation, as evidenced by the fall in total peripheral resistance (R_p). However, lipopolysaccharide challenge did not influence the elastic properties of aortas, as shown by the unaltered τ. The NO-mediated vascular relaxation may counterbalance the AGEs-induced arterial stiffening so that the aortic distensibility remained unaltered. Treating lipopolysaccharide-challenged rats with methylprednisolone prevented peripheral vasodilation because of its ability to increase R_p. However, methylprednisolone produced an increase in aorta stiffness, as manifested by the significant decline in τ. The diminished aortic distensibility by methylprednisolone paralleled a significant reduction in NO plasma levels, in the absence of any significant changes in AGEs content.

Conclusion

Methylprednisolone stiffens aortas and elastic arteries in lipopolysaccharide-induced chronic inflammation in rats, for NO activity may be dominant as a counteraction of AGEs.     

Regulation of transplanted mesenchymal stem cells by the lung progenitor niche in rats with chronic obstructive pulmonary disease

Background

Stem cell transplantation is a promising method for the treatment of chronic obstructive pulmonary disease (COPD), and mesenchymal stem cells (MSCs) have clinical potential for lung repair/regeneration. However, the rates of engraftment and differentiation are generally low following MSC therapy for lung injury. In previous studies, we constructed a pulmonary surfactant-associated protein A (SPA) suicide gene system, rAAV-SPA-TK, which induced apoptosis in alveolar epithelial type II (AT II) cells and vacated the AT II cell niche. We hypothesized that this system would increase the rates of MSC engraftment and repair in COPD rats.

Methods

The MSC engraftment rate and morphometric changes in lung tissue in vivo were investigated by in situ hybridization, hematoxylin and eosin staining, Masson’s trichrome staining, immunohistochemistry, and real-time PCR. The expression of hypoxia inducible factor (HIF-1α) and stromal cell-derived factor-1 (SDF-1), and relationship between HIF-1α and SDF-1 in a hypoxic cell model were analyzed by real-time PCR, western blotting, and enzyme-linked immunosorbent assay.

Results

rAAV-SPA-TK transfection increased the recruitment of MSCs but induced pulmonary fibrosis in COPD rats. HIF-1α and SDF-1 expression were enhanced after rAAV-SPA-TK transfection. Hypoxia increased the expression of HIF-1α and SDF-1 in the hypoxic cell model, and SDF-1 expression was augmented by HIF-1α under hypoxic conditions.

Conclusions

Vacant AT II cell niches increase the homing and recruitment of MSCs to the lung in COPD rats. MSCs play an important role in lung repair and promote collagen fiber deposition after induction of secondary damage in AT II cells by rAAV-SPA-TK, which involves HIF-1α and SDF-1 signaling.     

Asthma and COPD in cystic fibrosis intron-8 5T carriers. A population-based study

Background

Carriers of cystic fibrosis intron-8 5T alleles with high exon-9 skipping could have increased annual lung function decline and increased risk for asthma or chronic obstructive pulmonary disease (COPD).

Methods

We genotyped 9131 individuals from the adult Danish population for cystic fibrosis 5T, 7T, 9T, and F508del alleles, and examined associations between 11 different genotype combinations, and annual FEV₁ decline and risk of asthma or COPD.

Results

5T heterozygotes vs. 7T homozygous controls had no increase in annual FEV₁ decline, self-reported asthma, spirometry-defined COPD, or incidence of hospitalization from asthma or COPD. In 5T/7T heterozygotes vs. 7T homozygous controls we had 90% power to detect an increase in FEV₁ decline of 8 ml, an odds ratio for self-reported asthma and spirometry-defined COPD of 1.9 and 1.7, and a hazard ratio for asthma and COPD hospitalization of 1.8 and 1.6, respectively. Both 5T homozygotes identified in the study showed evidence of asthma, while none of four 5T/F508del compound heterozygotes had severe pulmonary disease. 7T/9T individuals had annual decline in FEV₁ of 19 ml compared with 21 ml in 7T homozygous controls (t-test:P = 0.03). 6.7% of 7T homozygotes without an F508del allele in the cystic fibrosis transmembrane conductance regulator gene reported asthma vs. 11% of 7T/9T individuals with an F508del allele (χ²:P = 0.01) and 40% of 7T homozygotes with an F508del allele (P = 0.04). 7T homozygotes with vs. without an F508del allele also had higher incidence of asthma hospitalization (log-rank:P = 0.003); unadjusted and adjusted equivalent hazard ratios for asthma hospitalization were 11 (95%CI:1.5–78) and 6.3 (0.84–47) in 7T homozygotes with vs. without an F508del allele.

Conclusion

Polythymidine 5T heterozygosity is not associated with pulmonary dysfunction or disease in the adult Caucasian population. Furthermore, our results support that F508del heterozygosity is associated with increased asthma risk independently of the 5T allele.     

Is R2* a New MRI Biomarker for the Progression of Parkinson’s Disease? A Longitudinal Follow-Up

Purpose

To study changes of iron content in basal ganglia in Parkinson’s disease (PD) through a three-year longitudinal follow-up of the effective transverse relaxation rate R₂*, a validated MRI marker of brain iron content which can be rapidly measured under clinical conditions.

Methods

Twenty-seven PD patients and 26 controls were investigated by a first MRI (t₀). Longitudinal analysis was conducted among the 18 controls and 14 PD patients who underwent a second MRI (t₁) 3 years after. The imaging protocol consisted in 6 gradient echo images obtained at different echo-times for mapping R₂*. Quantitative exploration of basal ganglia was performed by measuring the variation of R₂* [R₂*(t₁) – R₂*(t₀)] in several regions of interest.

Results

During the three-year evolution of PD, R₂* increased in Substantia nigra (SN) (by 10.2% in pars compacta, p = 0.001, and 8.1% in pars reticulata, p = 0.013) and in the caudal putamen (11.4%, p = 0.011), without significant change in controls. Furthermore, we showed a positive correlation between the variation of R₂* and the worsening of motor symptoms of PD (p = 0.028).

Conclusion

Significant variation of R₂* was longitudinally observed in the SN and caudal putamen of patients with PD evolving over a three-year period, emphasizing its interest as a biomarker of disease progression. Our results suggest that R₂* MRI follow-up could be an interesting tool for individual assessment of neurodegeneration due to PD, and also be useful for testing the efficiency of disease-modifying treatments.