Serum Immunoglobulin G (IgG) Subclasses in a Cohort of Systemic Sclerosis Patients.

OBJECTIVES: To assess serum immunoglobulin G (IgG) subclasses in a cohort of systemic sclerosis (SSc) patients and to evaluate the influence of IgG subclasses in the main complications of the disease. METHODS: The serum level of IgG subclasses was evaluated in 67 SSc patients and 48 healthy controls (HC), matched for sex and age. Serum samples were collected and measured IgG1-4 subclasses by turbidimetry. RESULTS: SSc patients had lower median total IgG [9.88 g/l (IQR 8.18-11.42 g/l) vs. 12.09 g/l (IQR 10.24-13.54 g/l), p < 0.001], IgG1 [5.09 g/l (IQR 4.25-6.38 g/l) vs. 6.03 g/l (IQR 5.39-7.90 g/l), p < 0.001], and IgG3 [0.59 g/l (IQR 0.40-0.77 g/l) vs. 0.80 g/l (IQR 0.46-1 g/l), p < 0.05] serum levels compared to HC. The logistic regression analysis showed IgG3 as the only variable associated with the diffusing capacity of the lung for carbon monoxide (DLco) ≤60% of the predicted [OR 9.734 (CI 95%: 1.312-72.221), p < 0.05] and modified Rodnan skin score (mRSS) [OR 1.124 (CI 95%: 1.019-1.240), p < 0.05], anti-topoisomerase I [OR 0.060 (CI 95%: 0.007-0.535), p < 0.05], and IgG3 [OR 14.062 (CI 95%: 1.352-146.229), p < 0.05] as variables associated with radiological interstitial lung disease (ILD). CONCLUSION: SSc patients have reduced levels of total IgG and an altered IgG subclass distribution compared to HC. Moreover, SSc patients show different serum IgG subclasses profiles according to the main involvement of the disease.

Circulating and Salivary NGF and BDNF Levels in SARS-CoV-2 Infection: Potential Predictor Biomarkers of COVID-19 Disease-Preliminary Data.

COVID-19 continues to afflict the global population, causing several pathological diseases and exacerbating co-morbidities due to SARS-CoV-2's high mutation. Recent interest has been devoted to some neuronal manifestations and to increased levels of Nerve Growth Factor (NGF) and Brain-derived Neurotrophic Factor (BDNF) in the bloodstream during SARS-CoV-2 infection, neurotrophins that are well-known for their multifactorial actions on neuro-immune-endocrine and visual functions. Nineteen (19) patients were enrolled in this monocentric prospective study and subjected to anamnesis and biosamples collection (saliva and blood) at hospitalization (acute phase) and 6 months later (remission phase). NGF and BDNF were quantified by ELISA, and biochemical data were related to biostrumental measurements. Increased NGF and BDNF levels were quantified in saliva and serum during the acute phase of SARS-CoV-2 infection (hospitalized patients), and reduced levels were observed in the next 6 months (remission phase), never matching the baseline values. Salivary and circulating data would suggest the possibility of considering sera and saliva as useful matrices for quickly screening neurotrophins, in addition to SARS-CoV2 antigens and RNA. Overall, the findings described herein highlight the importance of NGF and BDNF as dynamic biomarkers for monitoring disease and reinforces the possibility of using saliva and sera for quick, non-invasive COVID-19 screening.

Increased Complement Activation in Systemic Sclerosis Patients with Skin and Lung Fibrosis.

INTRODUCTION: The involvement of complement system in the phenotypic expression of systemic sclerosis (SSc) is a debated topic. We aimed to assay complement fractions in SSc patients and to correlate their levels with the clinical course of disease. KEY POINTS: 1. CH50 is increased in SSc patients compared to HC; 2. Serum C2 levels are increased in SSc patients compared to HC; 3. CH50 may represent a biomarker of skin and lung fibrosis severity in SSc patients. METHOD: Complement hemolysis 50% (CH50), C2, C3 and C4 levels have been assessed in 85 SSc patients and 47 healthy controls (HC). RESULTS: SSc patients displayed a statistically significant higher value of CH50 [76.3 U/mL (IQR 65.8-89.4 U/mL) vs. 29.6 U/mL (IQR 24.7-34 U/mL); p < 0.0001] and of C2 [26.1 mg/L (IQR 24.1-32.1 mg/L) vs. 22.7 mg/L (IQR 20.6-24.4 mg/L); p < 0.0001] if compared to HC. Patients with diffuse cutaneous SSc (dcSSc) had higher levels of CH50 than patients with limited cutaneous SSc (lcSSc) [83.6 U/mL (IQR 72.3-102.7 U/mL) vs. 71.3 U/mL (IQR 63.7-83.6 U/mL); p = 0.003]. SSc patients with interstitial lung disease (ILD) had higher CH50 levels if compared to SSc patients without ILD [79.6 U/mL (IQR 68.3-97.4 U/mL) vs. 69.7 U/mL (54.6-85.7 U/mL); p = 0.042]. A positive linear correlation existed between CH50 and the modified Rodnan Skin Score (mRSS) (r = 0.285, p = 0.008) and disease severity scale (DSS) (r = 0.285, p = 0.005); a negative linear correlation was demonstrated between CH50 and the diffusing capacity of carbon monoxide (DLco) (r = -0.252, p = 0.012). In multiple linear regression analysis, only DSS was significant (p = 0.01, beta coefficient 2.446). CONCLUSIONS: Our results show an increment of CH50 and serum C2 levels in SSc patients in comparison to HC; we retain that CH50 may represent a biomarker of disease severity and of skin and lung fibrosis in these patients.

Cytokines and Hepatocellular Carcinoma: Biomarkers of a Deadly Embrace.

Hepatocellular carcinoma (HCC) represents a worldwide health matter with a major care burden, high prevalence, and poor prognosis. Its pathogenesis mainly varies depending on the underlying etiological factors, although it develops from liver cirrhosis in the majority of cases. This review summarizes the role of the most interesting soluble factors as biomarkers for early diagnosis and as recommended targets for treatment in accordance with the new challenges in precision medicine. In the premalignant environment, inflammatory cells release a wide range of cytokines, chemokines, growth factors, prostaglandins, and proangiogenic factors, making the liver environment more suitable for hepatocyte tumor progression that starts from acquired genetic mutations. A complex interaction of pro-inflammatory (IL-6, TNF-α) and anti-inflammatory cytokines (TGF-α and -ß), pro-angiogenic molecules (including the Angiopoietins, HGF, PECAM-1, HIF-1α, VEGF), different transcription factors (NF-kB, STAT-3), and their signaling pathways are involved in the development of HCC. Since cytokines are expressed and released during the different stages of HCC progression, their measurement, by different available methods, can provide in-depth information on the identification and management of HCC.

Salivary Biomarkers in COVID-19 Patients: Towards a Wide-Scale Test for Monitoring Disease Activity.

The ongoing outbreak of coronavirus disease 2019 (COVID-19), which impairs the functionality of several organs, represents a major threat to human health. One of the hardest challenges in the fight against COVID-19 is the development of wide-scale, effective, and rapid laboratory tests to control disease severity, progression, and possible sudden worsening. Monitoring patients in real-time is highly demanded in this pandemic era when physicians need reliable and quantitative tools to prioritize patients' access to intensive care departments. In this regard, salivary biomarkers are extremely promising, as they allow for the fast and non-invasive collection of specimens and can be repeated multiple times. METHODS: We compare salivary levels of immunoglobulin A subclasses (IgA1 and IgA2) and free light chains (kFLC and λFLC) in a cohort of 29 SARS-CoV-2 patients and 21 healthy subjects. RESULTS: We found that each biomarker differs significantly between the two groups, with p-values ranging from 10-8 to 10-4. A Receiving Operator Curve analysis shows that λFLC level is the best-suited candidate to discriminate the two groups (AUC = 0.96), with an accuracy of 0.94 (0.87-1.00 95% CI), a precision of 0.91 (0.81-1.00 95% CI), a sensitivity of 1.00 (0.96-1.00 95% CI), and a specificity of 0.86 (0.70-1.00 95% CI). CONCLUSION: These results suggest λFLC as an ideal indicator of patient conditions. This hypothesis is strengthened by the consideration that the λFLC half-life (approximately 6 h) is significantly shorter than the IgA one (21 days), thus confirming the potential of λFLC for effectively monitoring patients' fluctuation in real-time.

Biochemical and nutritional characteristics of buffalo meat and potential implications on human health for a personalized nutrition.

The human consumption of food animal products is the main topic of an important debate among professionals in this sector: dietologists, dietitians and nutritional biologists. The red meat provides all the essential amino acids, bioavailable iron, zinc, calcium, lipids and B-group vitamins. A valid alternative to beef could be the buffalo meat. Italy is the largest European producer of buffalo meat and derivatives. The high nutritional characteristics of buffalo meat make it suitable to be included in the Mediterranean diet to customize it in relation to the needs and conditions of the population. Polyunsaturated/saturated fatty acids ratio can be influenced by diet, breed and type of breeding, but muscle tissue fat percentage is the main factor in determining a favorable fatty acid composition. This review focuses on the biochemical and nutritional characteristics of the buffalo meat (content of fats, cholesterol, amino acids, vitamins and minerals), explaining their variability depending on the different breeds, and the favorable implications on the human health. These results suggest that buffalo meat can be a healthier alternative to beef, not only for healthy people in particular physiological conditions (i.e. pregnancy), but also for persons at risk for cardiovascular and cerebrovascular diseases, thus achieving the goal of a personalized nutrition.

Influences of Different Air-Inhibition Coatings on Monomer Release, Microhardness, and Color Stability of Two Composite Materials.

The aim of this study was to evaluate the effect of light-curing protocols on two modern resin composites using different air-inhibition coating strategies. This was accomplished by assessing the amount of monomer elution, surface microhardness, and composite discoloration in different storage conditions. A total of 120 specimens were prepared using Filtek Supreme XTE (3M ESPE, Seefeld, Germany) and CeramX Universal (Dentsply DeTrey, Konstanz, Germany). Specimens were light-cured in air as per manufacturer's instructions or in the absence of oxygen. This latter condition was achieved using three different approaches: (i) transparent polyester strip; (ii) glycerin; (iii) argon gas. Specimens were assessed for release of monomers, Vickers hardness, and discoloration after storage in different solutions. The results were analyzed with ANOVA one-way test followed by Student-Newman-Keuls test. Moreover, multiple comparisons of means were performed using the Student t-test (p<0.05). The amount of monomers released from the tested specimens was very low in all conditions. The presence of oxygen induced some decrease in microhardness. The highest discoloration values, for both materials, were obtained after ageing in red wine. In case finish and polish procedures are awkward to achieve in posteriors composite restoration, light-curing in the absence of oxygen should be considered, especially when performing composite restoration in esthetic areas.

Quantitative Determination of 18-ß-Glycyrrhetinic Acid in HepG2 Cell Line by High Performance Liquid Chromatography Method.

A reverse phase high performance liquid chromatographic (RP-HPLC) method was developed for identification and estimation of 18-ß-glycyrrhetinic acid (GA) in HepG2 cell line. The analysis was carried out using a JASCO HPLC system with a C-18 (3 µm) Supelco reversed phase column (150 x 4.7 mm) using a mobile phase of 80% CH3OH and 20% of CH3CN: tetrahydrofuran: water (10:80:10, v/v/v). The method was linear in the concentration range of 1.5-120 µg /mL (n = 5). The LOD and LOQ were determined based on standard deviation of the y-intercept and the slope of the calibration curve. The LOD and LOQ values were found to be 11.46 µg/mL and 34.72 µg/mL, respectively. The mean percentage recovery by standard addition experiments of GA is 92.4 % ± 5.2%. The intracellular GA concentration value, obtained as mean of five different determinations, was 45.8 ± 7.45 µg/mL. We have developed a HPLC-UV method for quantitative determination of GA inside cells, with advantages in the cost reduction and economy of the analytical process.

Controlled release of 18-ß-glycyrrhetic acid by nanodelivery systems increases cytotoxicity on oral carcinoma cell line.

The topical treatment for oral mucosal diseases is often based on products optimized for dermatologic applications; consequently, a lower therapeutic effect may be present. 18-ß-glycyrrhetic acid (GA) is extracted from Glycirrhiza glabra. The first aim of this study was to test the cytotoxicity of GA on PE/CA-PJ15 cells. The second aim was to propose and test two different delivery systems, i.e. nanoparticles and fibers, to guarantee a controlled release of GA in vitro. We used chitosan and poly(lactic-co-glycolic) acid based nanoparticles and polylactic acid fibers. We tested both delivery systems in vitro on PE/CA-PJ15 cells and on normal human gingival fibroblasts (HGFs). The morphology of GA-loaded nanoparticles (GA-NPs) and fibers (GA-FBs) was investigated by electron microscopy and dynamic light scattering; GA release kinetics was studied spectrophotometrically. MTT test was used to assess GA cytotoxicity on both cancer and normal cells. Cells were exposed to different concentrations of GA (20-500 µmol l-1) administered as free GA (GA-f), and to GA-NPs or GA-FBs. ROS production was evaluated using dichlorodihydrofluorescein as a fluorescent probe. Regarding the cytotoxic effect of GA on PE/CA-PJ15 cells, the lowest TC50 value was 200 µmol l-1 when GA was added as GA-NPs. No cytotoxic effects were observed when GA was administered to HGFs. N-acetyl Cysteine reduced mortality induced by GA-f in PE/CA-PJ15 cells. The specific effect of GA on PE/CA-PJ15 cells is mainly due to the different sensitivity of cancer cells to ROS over-production; GA-NPs and GA-FBs formulations increase, in vitro, this toxic effect on oral cancer cells.

Chromographic Analysis and Cytotoxic Effects of Chlorhexidine and Sodium Hypochlorite Reaction Mixtures.

INTRODUCTION: The literature reveals controversies regarding the formation of para-chloroaniline (PCA) when chlorhexidine (CHX) is mixed with sodium hypochlorite (NaOCl). This study aimed to investigate the stability of PCA in the presence of NaOCl and to examine the in vitro cytotoxic effects of CHX/NaOCl reaction mixtures. METHODS: Different volumes of NaOCl were added to CHX (mix 1) or PCA (mix 2). Upon centrifugation, the supernatant and precipitate fractions collected from samples were analyzed using high-performance liquid chromatography. The cytotoxic effects of both fractions were examined on human periodontal ligament and 3T3 fibroblast cell lines. RESULTS: High-performance liquid chromatographic analysis showed no PCA signal when NaOCl was mixed with CHX (mix 1). In mix 2, the intensity of PCA was decreased when NaOCl was added to PCA, and chromatographic signals, similar to that of CHX/NaOCl, were also observed. The mortality of precipitates exerted on both cell lines was lower compared with that of supernatants. CONCLUSIONS: The discrepancy in the data from the literature could be caused by the instability of the PCA in the presence of NaOCl. The CHX/NaOCl reaction mixture exhibits a wide range of cytotoxic effects.

Multicenter evaluation of an enzymatic method for glycated albumin.

BACKGROUND: The use of glycated albumin (GA) has been proposed as an additional glycemic control marker particularly useful in intermediate-term monitoring and in situation when HbA1c test is not reliable. METHODS: We have performed the first multicenter evaluation of the analytical performance of the enzymatic method quantILab Glycated Albumin assay implemented on the most widely used clinical chemistry analyzers (i.e. Abbott Architect C8000, Beckman Coulter AU 480 and 680, Roche Cobas C6000, Siemens ADVIA 2400 and 2400 XPT). RESULTS: The repeatability of the GA measurement (expressed as CV, %) implemented in the participating centers ranged between 0.9% and 1.2%. The within-laboratory CVs ranged between 1.2% and 1.6%. A good alignment between laboratories was found, with correlation coefficients from 0.996 to 0.998. Linearity was confirmed in the range from 7.6 to 84.7%. CONCLUSION: The new enzymatic method for glycated albumin evaluated by our investigation is suitable for clinical use.

Correlation Between Metabolic Syndrome, Periodontitis and Reactive Oxygen Species Production. A Pilot Study.

BACKGROUND AND OBJECTIVE: Metabolic syndrome (MetS) is associated with an increased risk of periodontitis even if the mechanism is unknown. Since both MetS and periodontitis are characterized by an alteration of inflammation status, the aim of this pilot study was to determine if differences in ROS metabolism of phagocytes isolated from (A) patients with MetS, (B) patients with both MetS and mild periodontitis, (C) healthy subjects and (D) normal weight subjects with mild periodontitis, were present. METHODS: ROS metabolism was studied by a Chemiluminescence (CL) technique: the system was made up of luminol (100 nmol/L) and cells (1 × 105) in the presence or absence of stimulus constituted by opsonized zymosan (0.5 mg). The final volume (1.0 mL) was obtained using modified KRP buffer. ROS production was measured at 25°C for 2 h, using an LB 953 luminometer (Berthold, EG & G Co, Germany). All the experiments were performed in triplicate. STATISTICAL ANALYSIS: All results are mean ± standard deviation (SD). The group of means was compared by the analysis of variance "(ANOVA)". A value of p < 0.05 was considered significant. RESULTS: Results showed that basal ROS production (both from PMNs and from PBMs) of groups A, B and D was increased with respect to that obtained from group C (p <0.05). CONCLUSION: These results are congruent with literature data, although the actual clinical relevance of the phenomenon remains to be evaluated.

Dexamethasone-loaded biopolymeric nanoparticles promote gingival fibroblasts differentiation.

Polymer-based nanoparticles (NPs) can be efficiently used for the delivery of bioactive molecules for both in vitro and in vivo applications affording high drug loading and controlled release profiles. Within this framework polylactic-co-glycolic acid (PLGA) NPs with a diameter of 290 ± 41 nm have been fabricated and loaded with dexamethasone (DXM) using a patented procedure. The aim of the project was to setup a controlled delivery system to promote the in vitro differentiation of Human Gingival Fibroblasts (HGFs). First the uptake of fluorescent PLGA NPs by HGFs cells was investigated; then experiments were also addressed to analyze the specific cell response to DXM, in order to evaluate its functional efficiency in comparison with its conventional addition to the culture medium. The results showed that cells treated with DXM-loaded NPs acquired the osteoblast phenotype faster in comparison to those treated with the free drug. The slow and sustained release of DXM from PLGA NPs produced a constant and uniform concentration of drug inside cells with long-term and enhanced biochemical effects.

Homocysteine, cysteine, folate and vitamin B12 status in type 2 diabetic patients with chronic kidney disease.

BACKGROUND: Hyperhomocysteinemia (hHcy) is a risk factor in the progression of chronic kidney disease (CKD). In type 2 diabetes (T2D), hHcy is strongly associated with increased risk of cardiovascular disease. Vitamin B12 and folic acid supplementation have been reported to lower homocysteine (tHcy) levels, but no data on plasma tHcy, cysteine (Cys), folate and vitamin B12 levels in T2D-CKD patients are reported. PROCEDURES: tHcy and Cys levels were analyzed in 178 T2D-CKD patients by high performance liquid chromatography (HPLC) with fluorescence detection. In addition, we determined folate and vitamin B12 levels using a chemiluminescence method. RESULTS: tHcy and Cys levels were increased in T2D patients, and this rise positively correlated with the CKD stage (P < 0.001). Folate levels were comparable to controls at various CKD stages, whereas vitamin B12 levels were lower, except at stage IV. We did not find any correlation between B-vitamins and levels of tHcy and Cys, regardless of the CKD stage. CONCLUSIONS: This is the first study reporting tHcy, Cys and B-vitamins status in T2D-CKD patients. Although limited to our cohort of 178 patients, our findings could be helpful in clarifying the conflicting literature regarding B-vitamins supplementation. Further studies are necessary before any Hcy-lowering therapy can be safely established in T2D-CKD subjects.

Effects of dental methacrylates on oxygen consumption and redox status of human pulp cells.

UNLABELLED: Several studies have already demonstrated that the incomplete polymerization of resin-based dental materials causes the release of monomers which might affect cell metabolism. The aim of this study was to investigate the effects of triethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, urethane dimethacrylate, and 2-hydroxyethyl methacrylate on (1) cellular energy metabolism, evaluating oxygen consumption rate, glucose consumption, glucose 6-phosphate dehydrogenase activity, and lactate production, and (2) cellular redox status, through the evaluation of glutathione concentration and of the activities of enzymes regulating glutathione metabolism. METHODS: Human pulp cells were used and oxygen consumption was measured by means of a Clark electrode. Moreover, reactive oxygen species production was quantified. Enzymatic activity and glucose and lactate concentrations were determined through a specific kit. RESULTS: Triethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, and 2-hydroxyethyl methacrylate induced a decrease in oxygen consumption rate, an enhancement of glucose consumption, and lactate production, whilst glucose 6-phosphate dehydrogenase and glutathione reductase activity were not significantly modified. Moreover, the monomers induced an increase of reactive oxygen species production with a consequent increase of superoxide dismutase and catalase enzymatic activities. A depletion of both reduced and total glutathione was also observed. CONCLUSION: The obtained results indicate that dental monomers might alter energy metabolism and glutathione redox balance in human pulp cells.

Physiopathology of Bone Modifications in ß-Thalassemia.

ß-thalassemia major (ßTM) or Cooley anemia is characterized by significantly reduced or absent synthesis of ß-globin chains, which induces important pathologic consequences including hemolytic anemia, altered erythropoiesis, and bone marrow overstimulation. The pathogenesis of bone changes in patients with ßTM is not yet completely understood. However, an unbalance in bone mineral turnover resulting from increased resorption and suppression of osteoblast activity has been detected in ßTM patients. The abnormal regulation of bone metabolism may be related to hormonal and genetic factors, iron overload and iron chelation therapy, nutritional deficits, and decreased levels of physical activity. Here, we review the most recent findings on the physiopathology of bone abnormalities in ßTM. Clinical presentation and radiological features of ßTM-related bone changes are also discussed.

Diagnostic efficacy of conventional as compared to liquid-based cytology in thyroid lesions: evaluation of 10,360 fine needle aspiration cytology cases.

OBJECTIVE: To evaluate the efficacy of thyroid fine needle aspiration cytology (FNAC) processed by liquid-based cytology (LBC) as compared to conventional smears (CS) in 10,360 consecutive cases in 3 reference periods. STUDY DESIGN: In 1997-1998, 2,394 FNACs had CS; in 2001-2002, 3,463 had both CS and LBC; and in 2004-2005, 4,503 had only LBC. Three arbitrary parameters of efficacy were chosen to compare the two different methods: rate of inadequacy (IR), rate of indeterminacy (INDR) and rate of malignancy (MR). In the same biennia 6,184 thyroidectomy specimens were examined. RESULTS: IR was, respectively, 8.4% in 1997-1998, 18.4% in 2001-2002 and 13.3% in 2004-2005. MR was respectively 2.2%, 2.5% and 2.2%. INDR was 16.6% in 1997-1998, 23.3% in 2001-2002 and 15.9% in 2004-2005. In each biennium, respectively, 16.9%, 27.6% and 28% of malignant neoplasms were histologically diagnosed. CONCLUSION: LBC was used as an alternative method in the cytologic diagnosis for the preoperative selection of thyroid neoplasms, decreasing indeterminate diagnoses without MR changes.

Effects of 1,4-butanediol dimethacrylate and urethane dimethacrylate on HL-60 cell metabolism.

The polymerization of methacrylic monomers present in dental composite resins never reaches completion and therefore the leakage of residual monomers into the oral cavity and into biological fluids can cause local and systemic adverse effects. This work was carried out to study the in vitro biochemical interactions of urethane dimethacrylate and 1,4-butanediol dimethacrylate monomers with HL-60 cells, a cell line assumed as an experimental model for simulating granulocyte behaviour. Our main finding was that both monomers induce cell differentiation at toxic concentrations and that cytotoxicity seems to be caused by alterations of glucose metabolism arising from mitochondrial dysfunction rather than from oxidative stress, which could not be altogether verified under our experimental conditions. Our study could be considered as a useful approach to investigate the biochemical mechanisms that contribute to the cytotoxicity of methacrylate compounds and it underlines the importance of assessing such parameters for testing biocompatibility in order to promote the development of better and safer dental materials.