New perspectives in regenerative medicine and surgery: the bioactive composite therapies (BACTs).

Regenerative medicine and surgery is a rapidly expanding branch of translational research in tissue engineering, cellular and molecular biology. To date, the methods to improve cell intake, survival, and isolation need to comply with a complex and still unclear regulatory frame, becoming everyday more restrictive and often limiting the effectiveness and outcome of the therapeutic choices. Thus, the authors developed a novel 360° regenerative strategy based on the synergic action of several new components called the bioactive composite therapies (BACTs) to improve grafted cells intake, and survival in total compliance with the legal and ethical limits of the current regulatory frame. The rationale at the origin of this new technology is based on the evidence that cells need supportive substrate to survive in vitro and this observation, applying the concept of translational medicine, is true also in vivo. Bioactive composite mixtures (BACMs) are tailor-made bioactive mixtures containing several bioactive components that support cells' survival and induce a regenerative response in vivo by stimulating the recipient site to act as an in situ real bioreactor. Many different tissues have been used in the past for the isolation of cells, molecules, and growth factors, but the adipose tissue and its stromal vascular fraction (SVF) remains the most valuable, abundant, safe, and reliable source of regenerative components and particularly of adipose-derived stems cells (ADSCs). The role of plastic surgeons as the historical experts in all the most advanced techniques for harvesting, manipulating, and grafting adipose tissue is fundamental in this constant process of expansion of regenerative procedures. In this article, we analyze the main causes of cell death and the strategies for preventing it, and we present all the technical steps for preparing the main components of BACMs and the different mixing modalities to obtain the most efficient regenerative action on different clinical and pathological conditions. The second section of this work is dedicated to the logical and sequential evolution from simple bioactive composite grafts (BACGs) that distinguished our initial approach to regenerative medicine, to BACTs where many other fundamental technical steps are analyzed and integrated for supporting and enhancing the most efficient regenerative activity. Level of Evidence: Not gradable.

Disease-related malnutrition in outpatients with systemic sclerosis.

BACKGROUND & AIM: Disease-related malnutrition is known to negatively affect clinical outcomes. The aim of the present study was to evaluate the prevalence of malnutrition in a cohort of outpatients affected by Systemic Sclerosis (SSc) and its association with clinical variables. METHODS: One hundred sixty SSc patients were consecutively evaluated. The following clinical variables were assessed: disease duration, activity and severity, treatments, functional status, gastrointestinal involvement. Nutritional assessment included: body mass index (BMI), weight loss (WL) history, nutritional intakes and serum prealbumin. Malnutrition was defined as BMI <20 kg/m² and/or previous 6-month WL ≥ 10%. RESULTS: Prevalence of malnutrition was 15% (10-21%). Logistic regression showed that malnutrition was independently associated with disease activity (OR 3.72; p < 0.001) and low serum prealbumin (OR 8.58; p < 0.001). The association with gastrointestinal involvement was not statistically significant, although a trend was detected (OR 1.88). CONCLUSION: Malnutrition is common in SSc outpatients. It appears associated with disease activity and not influenced by nutritional intakes; gastrointestinal involvement might contribute to its development over time. Serum prealbumin could be an early marker of malnutrition in SSc, whose role should be confirmed by further longitudinal investigations. Prospective studies are also required to clarify the clinical significance of the association between malnutrition and disease activity in SSc.

A proposal of simple calculation (ERI calculator) to predict the early response to TNF-α blockers therapy in rheumatoid arthritis.

Increasing evidence has been accumulated for treating rheumatoid arthritis (RA) with TNF-α blocking agents. The formulation and definition of an early indicator of patient's reactivity during therapy may be extremely simplified by a mathematical model of clinical response. We analyzed the most significant clinical and laboratory parameters of response of 35 homogeneous patients (30 women, 5 men mean age ± SD: 52.31 ± 12.30 years) treated with adalimumab 40 mg every 2 weeks associated with methotrexate (MTX) 10-15 mg/week and with a stable dosage of steroids for 30 weeks. The over time trend of the studied parameters showed a linear response, which has allowed the realization of a simple mathematical model. The formula derived from this mathematical model was then applied and therefore validated in a group of 121 patients previously treated with several anti-TNF-alpha agents for at least 6 months. We drafted a mathematical model (early response indicator, ERI) that, by using a simple calculation, allows us to identify a high percentage of responder patients after only 2 weeks of treatment. ERI identified a high percentage (88%) of patients responding after only 2 weeks, as was confirmed at weeks 30; the use of ERI calculation after 6 weeks increases the proportion of responding patients to 92% with a percentage of false negatives of only 12%. ERI could be a useful tool to early differentiate the responder from the non-responder patients.

An investigation of the inflammatory cytokine and chemokine network in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is characterised by vasculopathy, an aberrantly activated immune system and excessive extracellular matrix deposition. Inflammatory chemokines control migration of cells to sites of tissue damage; their removal from inflamed sites is essential for resolution of the inflammatory response. The atypical chemokine receptor D6 has a critical role in this physiological balance. To explore potential deregulation of this system in SSc, inflammatory chemokine and D6 expression were compared with that in healthy controls (HC). METHODS: Serum levels of inflammatory mediators were assessed by luminex analysis. Peripheral blood mononuclear cells (PBMCs) were used in molecular and immunocytochemical analysis. Platelet-rich plasma was collected and assessed by western blotting for D6 expression levels. Sex-matched HC were used for comparison. RESULTS: 72 patients with SSc and 30 HC were enrolled in the study. The chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1ß/CCL4 and IL-8/CXCL8 were significantly increased in patients with SSc, regardless of disease subtype and phase. Quantitative PCR analysis revealed a significant 10-fold upregulation of D6 transcripts in patients with SSc compared with controls, and this was paralleled by increased D6 protein expression in the PBMCs of patients with SSc. Platelet lysates also showed strong D6 expression in patients with SSc but not in controls. Importantly, high levels of D6 expression correlated with reduced levels of its ligands in serum. CONCLUSIONS: Inflammatory chemokines and the regulatory receptor D6 are significantly upregulated in SSc and high D6 levels are associated with lower systemic chemokine levels, indicating that some patients control systemic chemokine levels using D6. These results suggest that chemokines may represent a therapeutic target in SSc.

A genetic variation located in the promoter region of the UPAR (CD87) gene is associated with the vascular complications of systemic sclerosis.

OBJECTIVE: The UPAR gene encodes a pleiotropic receptor (urokinase-type plasminogen activator receptor [uPAR]) involved in fibrosis, immunity, angiogenesis, and vascular remodeling. Previous studies have implicated uPAR in systemic sclerosis (SSc) vasculopathy and impaired angiogenesis. We undertook this study to investigate whether UPAR gene promoter polymorphisms might be associated with SSc phenotypes in the European Caucasian population. METHODS: We studied a total population of 1,339 individuals. The Italian discovery cohort comprised 388 SSc patients and 391 healthy controls. The French replication cohort consisted of 344 SSc patients and 216 healthy controls. The UPAR rs344781 and rs4251805 single-nucleotide polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism assay. RESULTS: In the Italian cohort, the rs344781 G allele was associated with SSc-related digital ulceration (odds ratio [OR] 1.39), pulmonary arterial hypertension (PAH) (OR 1.81), anticentromere antibody (ACA) positivity (OR 1.45), and limited cutaneous SSc (lcSSc) (OR 1.37). The rs344781 GG genotype was associated with SSc-related (OR 3.79), ACA-positive SSc (OR 2.17), and lcSSc (OR 1.96). Allelic and genotypic associations with SSc-related digital ulceration and ACA-positive SSc were replicated in the French sample. Combined analyses showed an association of the rs344781 G allele and GG genotype with SSc-related digital ulceration (allele OR 1.41, genotype OR 2.15), SSc-related PAH (allele OR 1.65, genotype OR 3.16), ACA-positive SSc (allele OR 1.47, genotype OR 2.40), and lcSSc (allele OR 1.34, genotype OR 1.77). In a multivariate logistic regression analysis model including the above associated phenotypes of SSc patients, the rs344781 GG genotype remained an independent risk factor for SSc-related digital ulceration (OR 1.96) and SSc-related PAH (OR 2.68). CONCLUSION: The UPAR rs344781 gene variant is associated with the SSc vascular phenotype.

Comparison of brain natriuretic peptide (BNP) and NT-proBNP in screening for pulmonary arterial hypertension in patients with systemic sclerosis.

OBJECTIVE: To compare the performance of brain natriuretic peptide (BNP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) in screening for pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc). METHODS: Between January 2008 and March 2009, outpatients referred to our unit and satisfying LeRoy criteria for SSc were assessed for PAH. Doppler echocardiography, BNP measurement, and NT-proBNP measurement were done concomitantly for a complete clinical, instrumental, and biochemical evaluation. Right-heart catheterization was carried out in cases of suspected PAH [estimated pulmonary arterial pressure (PAP) ≥ 36 mm Hg; diffusion capacity for carbon monoxide (DLCO) ≤ 50% of predicted value; 1-year DLCO decline ≥ 20% in absence of pulmonary fibrosis; unexplained dyspnea]. RESULTS: One hundred thirty-five patients were enrolled (124 women, 11 men; 96 limited SSc, 39 diffuse SSc); precapillary PAH was found in 20 patients (15 limited SSc, 5 diffuse SSc). The estimated PAP correlated with both BNP (R = 0.3; 95% CI 0.14-0.44) and NT-proBNP (R = 0.3, 95% CI 0.14-0.45). BNP [area under the curve (AUC) 0.74, 95% CI 0.59-0.89] was slightly superior to NT-proBNP (AUC 0.63, 95% CI 0.46-0.80) in identification of PAH, with diagnosis cutoff values of 64 pg/ml (sensitivity 60%, specificity 87%) and 239.4 pg/ml (sensitivity 45%, specificity 90%), respectively. BNP (log-transformed, p = 0.032) and creatinine (p = 0.049) were independent predictors of PAH, while NT-proBNP was not (p = 0.50). CONCLUSION: In our single-center study, the performance of BNP was slightly superior to that of NT-proBNP in PAH screening of patients with SSc, although normal levels of these markers do not exclude diagnosis. We observed that impaired renal function is associated with an increased risk of PAH in SSc. Further multicenter studies are needed to confirm our results (ClinicalTrials.gov ID NCT00617487).

Association of a functional polymorphism in the matrix metalloproteinase-12 promoter region with systemic sclerosis in an Italian population.

OBJECTIVE: To investigate the possible implication of the matrix metalloproteinase-12 (MMP-12) gene in the genetic predisposition to systemic sclerosis (SSc) susceptibility and clinical phenotype. METHODS: The MMP-12 rs2276109 A/G functional polymorphism was selected as a genetic marker and genotyped by polymerase chain reaction-restriction fragment length polymorphism assay in 513 unrelated subjects of Italian white ancestry: 250 patients with SSc [146 limited cutaneous SSc (lcSSc), 104 diffuse cutaneous SSc (dcSSc)] and 263 healthy individuals. RESULTS: A significant difference was observed in MMP-12 rs2276109 genotype distribution between patients with SSc and controls (p = 0.0003), and between lcSSc and dcSSc (p = 0.003). The A allele frequency was significantly higher in patients with SSc than in controls (p = 0.0002), and higher in dcSSc than in lcSSc (p = 0.003). After adjustment for age and sex, the homozygosity for the A allele significantly influenced the predisposition to SSc and to dcSSc (OR 2.44, 95% CI 1.61-3.71, p < 0.0001; OR 4.69, 95% CI 2.36-9.33, p < 0.0001, respectively). A trend toward an association between the AA genotype and lcSSc was observed (p = 0.06). The homozygosity for the A allele was also significantly and independently associated with antitopoisomerase I antibody positivity (OR 6.39, 95% CI 2.18-18.76, p = 0.001) and interstitial lung disease (OR 2.94, 95% CI 1.25-6.95, p = 0.01). CONCLUSION: The MMP-12 rs2276109 gene polymorphism may contribute to susceptibility to SSc, and in particular to dcSSc and pulmonary fibrosis.

Serologic profile and mortality rates of scleroderma renal crisis in Italy.

OBJECTIVE: To analyze clinical and serological characteristics of subjects with scleroderma renal crisis (SRC) in Italian patients with systemic sclerosis (SSc). METHODS: A retrospective analysis of medical records from 9 Italian rheumatologic referral centers was carried out. All patients with SRC and an available serum sample at the time of crisis were included. Antinuclear antibodies (ANA) by indirect immunofluorescence, anti-topoisomerase (topo) I by enzyme-linked assay (ELISA), anti-RNA polymerases (RNAP) by ELISA for the subunit III, and immunoprecipitation (IP) were performed. RESULTS: Forty-six cases (38 female; 40 diffuse cutaneous SSc) were identified. Mean age at SSc and SRC onset was 52.8 years +/- 13.2 and 55.4 years +/- 11.8, respectively. ANA were present in 44 patients (96%). Anti-topo I antibodies were detected in 30 (65%), anti-RNAP I-III in 7 (15%). No differences emerged between these 2 groups for their main clinical characteristics. The proportion of patients in the anti-RNAP I-III group developing SRC early (< 18 mo) in the course of SSc was significantly higher (p = 0.03). Cumulative survival rates were 64%, 53%, and 35% at 1, 2, and 10 years of followup, respectively. Survival rates of SSc patients significantly differed according to their autoantibody profile, being lower in the anti-topo I than in the anti-RNAP I-III group (p = 0.034). CONCLUSION: SRC is a rare manifestation of SSc in Italy but it is still associated with severe prognosis. Anti-topo I reactivity was more frequent than anti-RNAP I-III in our patients with SRC and was associated with delayed onset and high mortality rates.

2-DE and LC-MS/MS for a comparative proteomic analysis of BALf from subjects with different subsets of inflammatory myopathies.

The protein profiles of bronchoalveolar lavage fluid (BALf) of patients belonging to three selected subsets of Polymyositis/Dermatomyositis (PM/DM) have been compared by using a combination of 2-DE and MALDI-TOF/MS or LC-MS/MS. Our study examined the hypothesis that there were distinct differences in protein expression profiles that were related to the phenotype. From among the 323+/-51 protein spots that may represent the most highly expressed proteins in BALf of these patients, 24 unique spots were isolated and proteins identified. In particular, 9 spots were present in BALf of PM/DM patients only; 12 spots were exclusive of Overlap patients and 3 spots of AS patients. From among the proteins identified, a few were classified as cytoskeletal proteins, others were involved in oxidative stress and a number of proteins were associated with general metabolic activity or immunological response and inflammation. This is the first study in which evidence is provided that a number of different proteins are expressed in different subsets of PM/DM and supports our contention that the proteomic approach would be beneficial in discovering molecules which could represent possible prognostic factors of these rare pathologies.