Correction to: Impact of smoking habit on adult-onset Still’s disease prognosis,findings from a multicentre observational study

Clinical Rheumatology -     

HLA-C1 ligands are associated with increased susceptibility to systemic lupus erythematosus

Recently, the role of killer cell immunoglobulin-like receptor (KIR) in autoimmune diseases has received increasing attention. The present study was undertaken to determine the association of KIR genes and the human leukocytes antigen (HLA) ligands with Systemic Lupus Erythematosus (SLE) and accompanying oxidative stress. Presence or absence of 17 KIR and 5 HLA loci was performed using the polymerase chain reaction-sequence specific primer (PCR-SSP) method by case-control study. A total of 45 SLE patients, and 60 healthy controls, all of Sicilian descent, were enrolled. Plasma values of the anti-oxidant molecule Taurine were determined in all subjects by capillary electrophoresis UV detection. The carrier frequency of the KIR2DS2 gene was significantly increased in SLE patients compared to healthy controls (73.3 versus 45.0%; OR?=?3.36; 95% CI?=?1.46–7.74; p?=?.005) suggesting a role of KIR2DS2 gene in the susceptibility to disease. We also observed a strong positive association between the presence of HLA-C1 ligands group and the disease (82.2% in SLE patients versus 41.7% in controls; OR?=?6.47, 95% CI?=?2.58–16.26; p?<?.0001). Stepwise logistic regression analysis supported the effect of the HLA-C1 ligands in SLE patients (OR?=?7.06, 95% CI?=?0.07–2.19; p?=?.002), while the KIR genes were no longer significant. Interestingly, we found that SLE patients HLA-C1 positive showed significantly decreased plasma levels of antioxidant activity marker Taurine (69.38?±?28.49?μmol/L) compared to SLE patients HLA-C1 negative (108.37?±?86.09?μmol/L) (p?=?.03). In conclusion, HLA-C1 ligands group was significantly associated with an increased risk of SLE as well as an increased oxidative stress status overall in SLE patients.     

H‐ferritin and proinflammatory cytokines are increased in the bone marrow of patients affected by macrophage activation syndrome

Macrophage activation syndrome (MAS) is hyperinflammatory life‐threatening syndrome, associated typically with high levels of serum ferritin. This is an iron storage protein including heavy (H) and light (L) subunits, categorized on their molecular weight. The H‐/L subunits ratio may be different in tissues, depending on the specific tissue and pathophysiological status. In this study, we analysed the bone marrow (BM) biopsies of adult MAS patients to assess the presence of: (i) H‐ferritin and L‐ferritin; (ii) CD68⁺/H‐ferritin⁺ and CD68⁺/L‐ferritin⁺; and (iii) interleukin (IL)‐1β, tumour necrosis factor (TNF) and interferon (IFN)‐γ. We also explored possible correlations of these results with clinical data. H‐ferritin, IL‐1β, TNF and IFN‐γ were increased significantly in MAS. Furthermore, an increased number of CD68⁺/H‐ferritin⁺ cells and an infiltrate of cells co‐expressing H‐ferritin and IL‐12, suggesting an infiltrate of M1 macrophages, were observed. H‐ferritin levels and CD68⁺/H‐ferritin⁺ cells were correlated with haematological involvement of the disease, serum ferritin and C‐reactive protein. L‐ferritin and CD68⁺/L‐ferritin⁺ cells did not correlate with these parameters. In conclusion, during MAS, H‐ferritin, CD68⁺/H‐ferritin⁺ cells and proinflammatory cytokines were increased significantly in the BM inflammatory infiltrate, pointing out a possible vicious pathogenic loop. To date, H‐ferritin and CD68⁺/H‐ferritin⁺ were associated significantly with haematological involvement of the disease, suggesting biomarkers assessing severity of clinical picture.     

A phase II,double-blind,randomized, placebo-controlled clinical trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with antrostomies

tgAAVCF, an adeno-associated cystic fibrosis transmembrane conductance regulator (CFTR) viral vector/gene construct, was administered to 23 patients in a Phase II, double-blind, randomized, placebo-controlled clinical trial. For each patient, a dose of 100,000 replication units of tgAAVCF was administered to one maxillary sinus, while the contralateral maxillary sinus received a placebo treatment, thereby establishing an inpatient control. Neither the primary efficacy endpoint, defined as the rate of relapse of clinically defined, endoscopically diagnosed recurrent sinusitis, nor several secondary endpoints (sinus transepithelial potential difference [TEPD], histopathology, sinus fluid interleukin [IL]-8 measurements) achieved statistical significance when comparing treated to control sinuses within patients. One secondary endpoint, measurements of the anti-inflammatory cytokine IL-10 in sinus fluid, was significantly (p < 0.03) increased in the tgAAVCF-treated sinus relative to the placebo-treated sinus at day 90 after vector instillation. The tgAAVCF administration was well tolerated, without adverse respiratory events, and there was no evidence of enhanced inflammation in sinus histopathology or alterations in serum-neutralizing antibody titer to adeno-associated virus (AAV) capsid protein after vector administration. In summary, this Phase II trial confirms the safety of tgAAVCF but provides little support of its efficacy in the within-patient controlled sinus study. Various potentially confounding factors are discussed.     

Correlation between DNA transfer and cystic fibrosis airway epithelial cell correction after recombinant adeno-associated virus serotype 2 gene therapy

Recombinant adeno-associated virus serotype 2 (rAAV2)-based human gene therapy for cystic fibrosis has progressed through a series of preclinical studies and phase I and II clinical trials. This agent has shown an encouraging safety profile, consistent levels of DNA transfer, and positive evidence of short-term clinical improvement in lung function in a prospective, placebo-controlled phase II trial of aerosol administration. Nonetheless, it has been difficult to assess the relationship between its molecular action and the observed clinical improvements, because of the lack of positive results from a highly specific assay for vector mRNA. This issue is further complicated by the fact that the clinical vector utilizes a small cryptic rAAV2 promoter sequence that is less robust for mRNA expression than typical viral promoters. In this paper, we report the results of more sensitive assays performed on primary nasal cells harvested from rAAV2-CFTR gene therapy recipients. These studies demonstrate a correlation between the presence of rAAV2-CFTR vector genomes, CFTR mRNA expression, and cAMP-activated chloride channel function in these cells. The observation of sizeable physiological correction in the face of low mRNA levels may reflect the regulatory role of low levels of CFTR protein as an activator of other chloride channels.     

Expression of a truncated cystic fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates.

Gene therapy using recombinant adeno-associated virus (rAAV2) vectors for cystic fibrosis has shown gene transfer and remarkable safety, yet indeterminate expression. A new construct has been characterized with a powerful exogenous promoter, the cytomegalovirus enhancer/chicken beta-actin promoter, driving a truncated CF transmembrane conductance regulator (CFTR), pseudotyped in an AAV5 viral coat. Our goal is to demonstrate that airway delivery of a pseudotyped rAAV5 vector results in gene transfer as well as expression in non-human primates. Aerosolized pseudotyped rAAV5-DeltaCFTR or rAAV5-GFP (green fluorescent protein) genes were delivered to four and six lungs, respectively. The pseudotyped rAAV5 vector did result in GFP gene transfer (1.005x10(6) copies/mug DNA on average) and quantifiable gene expression. Microscopy confirmed protein expression in airway epithelium. Similarly, the vector also resulted in vector-specific CFTR DNA (1.24x10(5) copies/microg) and mRNA expression. Immunoprecipitation and (32)P phosphoimaging were used to demonstrate CFTR protein expression, as qualitatively enhanced beyond the barely detectable endogenous expression in untreated animals. Based on these promising studies, this CFTR minigene construct is a therapeutic candidate.     

Macromolecular interactions and ion transport in cystic fibrosis

Cystic fibrosis (CF) is a genetic disease caused by autosomal recessive mutations of the CF transmembrane regulator, CFTR. CFTR functions in the plasma membrane of epithelial cells lining the lung, pancreas, liver, intestines, sweat duct, and the epididymis. The primary problem in CF is that mutations in CFTR affect its ability to be made, processed, and trafficked to the plasma membrane and/or its function as a Cl(-) channel and conductance regulator. Many proteins and processes normally interact with normal CFTR throughout its life cycle and mutant CFTR during the disease process. Understanding the function of these proteins and processes is expected to provide a clearer understanding of how normal CFTR is involved in salt movement and how mutant CFTR is handled by the cell and leads to the pathophysiology of CF. Recently, efforts to find therapies that correct defective CFTR have been intensifying. To facilitate our understanding of normal and mutant CFTR and the identification of new drug targets for developing novel therapies, a panel of experts was convened by the National Heart, Lung, and Blood Institute to explore the critical questions, challenges, and current opportunities to highlight new areas of research that would facilitate a integrated understanding of the processes and proteins that impact CFTR. The meeting highlighted the multiple pathways and interacting proteins involved in CFTR folding and biosynthesis, processing, and trafficking. A number of critical areas for future study were identified. Although these therapies are promising, a big question remains as to whether simply correcting defective CFTR will lead to significant improvement in patient health or whether the symptoms manifested in CF will require therapies in addition to those that target defective CFTR specifically.     

Impact of immunosuppressive drugs on the therapeutic efficacy of ex vivo expanded human regulatory T cells

Immunosuppressive drugs in clinical transplantation are necessary to inhibit the immune response to donor antigens. Although they are effective in controlling acute rejection, they do not prevent long-term transplant loss from chronic rejection. In addition, immunosuppressive drugs have adverse side effects, including increased rate of infections and malignancies. Adoptive cell therapy with human Tregs represents a promising strategy for the induction of transplantation tolerance. Phase I/II clinical trials in transplanted patients are already underway, involving the infusion of Tregs alongside concurrent immunosuppressive drugs. However, it remains to be determined whether the presence of immunosuppressive drugs negatively impacts Treg function and stability. We tested in vitro and in vivo the effects of tacrolimus, mycophenolate and methylprednisolone (major ISDs used in transplantation) on ex vivo expanded, rapamycin-treated human Tregs. The in vitro results showed that these drugs had no effect on phenotype, function and stability of Tregs, although tacrolimus affected the expression of chemokine receptors and IL-10 production. However, viability and proliferative capacity were reduced in a dose-dependent manner by all the three drugs. The in vivo experiments using a humanized mouse model confirmed the in vitro results. However, treatment of mice with only rapamycin maintained the viability, function and proliferative ability of adoptively transferred Tregs. Taken together, our results suggest that the key functions of ex vivo expanded Tregs are not affected by a concurrent immunosuppressive therapy. However, the choice of the drug combination and their timing and dosing should be considered as an essential component to induce and maintain tolerance by Treg.     

Overcoming the Cystic Fibrosis Sputum Barrier to Leading Adeno-associated Virus Gene Therapy Vectors

Gene therapy has not yet improved cystic fibrosis (CF) patient lung function in human trials, despite promising preclinical studies. In the human CF lung, inhaled gene vectors must penetrate the viscoelastic secretions coating the airways to reach target cells in the underlying epithelium. We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials. Using multiple particle tracking, we found that sputum strongly impeded diffusion of AAV, regardless of serotype, by adhesive interactions and steric obstruction. Approximately 50% of AAV vectors diffused >1,000-fold more slowly in sputum than in water, with large patient-to-patient variation. We thus tested two strategies to improve AAV diffusion in sputum. We showed that an AAV2 mutant engineered to have reduced heparin binding diffused twice as fast as AAV2 on average, presumably because of reduced adhesion to sputum. We also discovered that the mucolytic N-acetylcysteine could markedly enhance AAV diffusion by altering the sputum microstructure. These studies underscore that sputum is a major barrier to CF gene delivery, and offer strategies for increasing AAV penetration through sputum to improve clinical outcomes.