HLA class I expression on human platelets is highly variable and correlates with distinct allele group frequencies.

BACKGROUND: Human leukocyte antigen (HLA) class I molecules are expressed on platelets and can represent a source of alloimmunization in recipients of platelet transfusions. HLA mismatch between donors and recipients may be associated with the induction of anti-HLA antibodies, which can culminate in refractoriness to platelet transfusions. In the present study we analyzed HLA allele group frequencies and HLA expression levels on human platelets from blood donors. MATERIALS AND METHODS: Platelet-rich plasma was collected from 139 donors to monitor platelet HLA class I expression by flow cytometry. DNA from donors with high and low platelet HLA expression was used in the genotype studies. Frequencies of large and normal-sized platelet subpopulations were determined and HLA class I expression was studied. Mean platelet volume (MPV) and platelet large-cell ratio (P-LCR) were analyzed in both groups of donors. RESULTS: The analysis showed variable platelet HLA class I expression with significant differences among donors. HLA class I allele group frequencies in donors with high and low platelet HLA expression showed distinctive genotypic features strictly related to expression level. The main allele groups found in samples with high platelet HLA class I expression were HLA-A*02, -A*68, -B*15, -B*49, and -C*03. Platelet HLA class I expression did not change over time or during freezing-thawing cycles. The analysis of platelet subpopulations showed a statistically significant higher expression of HLA class I molecules on large platelets than on normal-sized platelets. Moreover, donors with high HLA class I expression showed a higher frequency of large platelets (p<0.0001). The analysis of P-LCR in both groups of donors showed a statistically significant difference (p<0.05) within high HLA-expressing donors. DISCUSSION: Our data suggest an allele-dependent expression of HLA class I molecules on human platelets with distinct HLA allele group frequencies and different platelet subpopulation frequencies among blood donors.

Screening of Organ-Specific Autoantibodies in a Large Cohort of Patients with Autoimmune Thyroid Diseases.

Background: Autoimmune diseases tend to cluster in the same individual or in families. Four types of autoimmune polyglandular syndromes (APS) have been described based on the combination of endocrine and/or non-endocrine autoimmune diseases. In particular, type-3 APS is defined by the association of an autoimmune thyroid disease (ATD) and other autoimmune diseases and has a multifactorial etiology. The natural history of autoimmune diseases is characterized by three stages: potential, subclinical, and clinical. Methods: To determine the prevalence of organ-specific autoantibodies (anti-adrenal, anti-ovary [StCA], anti-pituitary [APA], anti-parietal cells [PCA], anti-tissue transglutaminase [tTGAb], anti-mitochondrial [AMA], anti-glutamic acid decarboxylase [GADA], anti-nicotinic acetylcholine receptor) in patients with ATD and to define the stage of the disease in patients with positive autoantibodies. From January 2016 to November 2018, 1502 patients (1302 female; age 52.7 ± 14.7 [mean ± standard deviation] years, range 18-86 years) with ATD (1285/1502 [85.6%] with chronic autoimmune thyroiditis and 217/1502 [14.4%] with Graves' disease) were prospectively enrolled. Results: The most common organ-specific autoantibodies were PCA (6.99%) and GADA (2.83%), while the prevalence of the remaining autoantibodies was ≤1%. All autoimmune diseases, but celiac disease, were predominant at the potential stage. Sex, ATD type, smoking habit, and coexistence of other autoimmune diseases correlated with the susceptibility to develop chronic atrophic gastritis (CAG) or autoimmune diabetes mellitus. Conclusions: The association between ATD and CAG was the most common manifestation of type-3 APS, mainly at the potential stage, that could lead to appropriate follow-up for early detection and timely treatment of the disease.

Differentially activated Src kinase in chemo-naïve human primary osteosarcoma cells and effects of a Src kinase inhibitor.

The therapeutic treatment of osteosarcoma (OS), a rare malignant teenage cancer of the skeletal system, still represents a great challenge as patient survival after conventional protocol chemotherapy treatment has not improved in the last four decades leaving poor patient prognoses. Therefore, many efforts have been done to find increasingly reliable OS cell models and to identify "druggable" targets in OS, in order to identify novel effective therapeutic approaches and treatment strategies. In this contest, the more successful use of patient-derived cell cultures in respect to human commercial lines and findings of Src kinase deregulation in cancer, prompted us to study for the first time the activation state of Src and the potential activity of our Src inhibitor SI-83 in a number of chemo-naïve patient-derived primary OS cells. We here demonstrate that Src is hyperactivated in OS cells in respect to the nonmalignant counterpart and that SI-83 is able to strongly decrease cell viability, proliferation, Src416 phosphorylation, and cell migration. © 2017 BioFactors, 43(6):801-811, 2017.

Histological and Ultrastructural Characterization of Alkaptonuric Tissues.

Alkaptonuria (AKU) is a hereditary disorder that results from altered structure and function of homogentisate 1,2 dioxygenase (HGD). This enzyme, predominantly produced by liver and kidney, is responsible for the breakdown of homogentisic acid (HGA), an intermediate in the tyrosine degradation pathway. A deficient HGD activity causes HGA levels to rise systemically. The disease is clinically characterized by homogentisic aciduria, bluish-black discoloration of connective tissues (ochronosis) and joint arthropathy. Additional manifestations are cardiovascular abnormalities, renal, urethral and prostate calculi and scleral and ear involvement. While the radiological aspect of ochronotic spondyloarthropathy is known, there are only few data regarding an exhaustive ultrastructural and histologic study of different tissues in AKU. Moreover, an in-depth analysis of tissues from patients of different ages, having varied symptoms, is currently lacking. A complete microscopic and ultrastructural analysis of different AKU tissues, coming from six differently aged patients, is here presented thus significantly contributing to a more comprehensive knowledge of this ultra-rare pathology.

Establishment of Four New Human Primary Cell Cultures from Chemo-Naïve Italian Osteosarcoma Patients.

Osteosarcoma (OS) is a primary highly malignant tumor of bone, affecting predominately adolescents and young adults between 10 and 20 years of age. OS is characterized by an extremely aggressive clinical course, with a rapid development of metastasis to the lung and distant bones.

Human platelet releasates combined with polyglycolic acid scaffold promote chondrocyte differentiation and phenotypic maintenance.

In the present study, we aimed to demonstrate the differentiating properties of platelet-rich plasma releasates (PRPr) on human chondrocytes seeded on a polygtlycolic acid (PGA) 3D scaffold. Gene expression and biochemical analysis were carried out to assess the improved quality of our PGA-based cartilage constructs supplemented with PRPr. We observed that the use of PRPr as cell cultures supplementation to PGA-chondrocyte constructs may promote chondrocyte differentiation, and thus may contribute to maintaining the chondrogenic phenotype longer than conventional supplementation by increasing high levels of important chondrogenic markers (e.g. sox9, aggrecan and type II collagen), without induction of type I collagen. Moreover, our constructs were analysed for the secretion and deposition of important ECM molecules (sGAG, type II collagen, etc.). Our results indicate that PRPr supplementation may synergize with PGA-based scaffolds to stimulate human articular chondrocyte differentiation, maturation and phenotypic maintenance.

Amyloidosis, inflammation, and oxidative stress in the heart of an alkaptonuric patient.

BACKGROUND: Alkaptonuria, a rare autosomal recessive metabolic disorder caused by deficiency in homogentisate 1,2-dioxygenase activity, leads to accumulation of oxidised homogentisic acid in cartilage and collagenous structures present in all organs and tissues, especially joints and heart, causing a pigmentation called ochronosis. A secondary amyloidosis is associated with AKU. Here we report a study of an aortic valve from an AKU patient. RESULTS: Congo Red birefringence, Th-T fluorescence, and biochemical assays demonstrated the presence of SAA-amyloid deposits in AKU stenotic aortic valve. Light and electron microscopy assessed the colocalization of ochronotic pigment and SAA-amyloid, the presence of calcified areas in the valve. Immunofluorescence detected lipid peroxidation of the tissue and lymphocyte/macrophage infiltration causing inflammation. High SAA plasma levels and proinflammatory cytokines levels comparable to those from rheumatoid arthritis patients were found in AKU patient. CONCLUSIONS: SAA-amyloidosis was present in the aortic valve from an AKU patient and colocalized with ochronotic pigment as well as with tissue calcification, lipid oxidation, macrophages infiltration, cell death, and tissue degeneration. A local HGD expression in human cardiac tissue has also been ascertained suggesting a consequent local production of ochronotic pigment in AKU heart.

Proteomics and phosphoproteomics provide insights into the mechanism of action of a novel pyrazolo[3,4-d]pyrimidine Src inhibitor in human osteosarcoma.

Osteosarcoma (OS) is a highly malignant bone tumour, affecting mainly children and young adults between 10 and 20 years of age. It represents the most frequent primitive malignant tumour of the skeletal system and is characterized by an extremely aggressive clinical course, with rapid development of lung metastases. In the last few years, targeting Src in the treatment of OS has become one of the major challenges in the development of new drugs, since an elevated Src kinase activity has been associated with the development and the maintenance of the OS malignant phenotype. Recently, SI-83, a novel pyrazolo[3,4-d]pyrimidine derivate Src inhibitor, was selected as a promising OS therapeutic drug because of its elevated anti-tumour effects toward human OS. In the present study, gel-based proteomics and phosphoproteomics revealed significant changes in proteins involved in many cancer related processes. We got insight into SI-83 proapoptotic and antiproliferative properties (overrepresentation of GRIA1, GRP78, and CALR and underrepresentation of NPM1, RCN, and P4HB). Nevertheless, the most significant findings of our work are the SI-83 induced dephosphorylation of ARPC5L, a subunit of the actin related Arp2/3 complex, and the decrease of other cytoskeleton proteins. These data, together with a dramatic impairment of SaOS-2 cell migration and adhesion, suggest that SI-83 may have antimetastatic features that enhance its use as a potent OS chemotherapeutic drug.

Antioxidants inhibit SAA formation and pro-inflammatory cytokine release in a human cell model of alkaptonuria.

OBJECTIVE: Alkaptonuria (AKU) is an ultra-rare autosomal recessive disease that currently lacks an appropriate therapy. Recently we provided experimental evidence that AKU is a secondary serum amyloid A (SAA)-based amyloidosis. The aim of the present work was to evaluate the use of antioxidants to inhibit SAA amyloid and pro-inflammatory cytokine release in AKU. METHODS: We adopted a human chondrocytic cell AKU model to evaluate the anti-amyloid capacity of a set of antioxidants that had previously been shown to counteract ochronosis in a serum AKU model. Amyloid presence was evaluated by Congo red staining. Homogentisic acid-induced SAA production and pro-inflammatory cytokine release (overexpressed in AKU patients) were evaluated by ELISA and multiplex systems, respectively. Lipid peroxidation was evaluated by means of a fluorescence-based assay. RESULTS: Our AKU model allowed us to prove the efficacy of ascorbic acid combined with N-acetylcysteine, taurine, phytic acid and lipoic acid in significantly inhibiting SAA production, pro-inflammatory cytokine release and membrane lipid peroxidation. CONCLUSION: All the tested antioxidant compounds were able to reduce the production of amyloid and may be the basis for establishing new therapies for AKU amyloidosis.

Rosuvastatin inhibits spontaneous and IL-1ß-induced interleukin-6 production from human cultured osteoblastic cells.

OBJECTIVE: Experimental and clinical data suggest that statins may protect bone by inhibiting bone resorption and/or stimulating bone formation. Interleukin-6 (IL-6) is produced by osteoblasts, and potently stimulates osteoclast activation playing a key role in normal bone resorption as well as in post-menopausal and inflammation-driven osteoporosis. Although statins inhibit IL-6 production from different cell types, currently no data exist on osteoblasts. The aim of the study was to evaluate the effect of rosuvastatin on IL-6 production by human osteoblasts. METHODS: Osteoblasts from osteoarthritic patients were incubated with rosuvastatin (0.1-10 µmol/L)±IL-1ß, and IL-6 production was evaluated as cytokine concentration in the culture medium (ELISA), as well as mRNA expression in the cells (qPCR). Putative intracellular mechanisms of the drug, such as blocking HMG-CoA-reductase, and interference in the prenylation process were investigated by the addition of mevalonate and isoprenoids. The effect of rosuvastatin±IL-1ß on the anti-resorptive molecule osteoprotegerin (OPG) was also assessed (ELISA). RESULTS: Rosuvastatin significantly reduced IL-6 levels in the osteoblast culture medium, both in unstimulated and IL-1ß-stimulated cells. This effect was reversed by mevalonate or geranylgeraniol, but not farnesol. Moreover, the drug decreased both spontaneous and IL-1ß-induced IL-6 mRNA expression in osteoblasts. Conversely, rosuvastatin did not affect OPG levels in the culture medium. CONCLUSION: Our results show that rosuvastatin decreases IL-6 production by osteoblasts, thereby suggesting a possible inhibiting activity on osteoclast function in an indirect way. These data may provide further rationale for employing rosuvastatin to beneficially affect bone metabolism in post-menopausal women and possibly in inflammation-driven osteoporosis.

Polysorbate 80 and Helicobacter pylori: a microbiological and ultrastructural study.

BACKGROUND: The frequent occurrence of chemoresistant strains reduces the chances of eradication of H. pylori infection and prompted the investigation of non-antibiotic substances active against this organism. Some surfactants enhance the effectiveness of antibiotics for their permeabilizing properties towards bacteria. We examined the antimicrobial activity to H. pylori of the surfactant polysorbate 80, used alone and in association with amoxicillin, clarithromycin, metronidazole, levofloxacin and tetracycline. We also aimed to study the ultrastructural alterations caused upon H. pylori by polysorbate 80, alone and in combination with antibiotics. Twenty-two H. pylori strains were tested using the broth dilution method. After incubation, broth from each dilution was subcultured onto agar enriched with foetal bovine serum to determine the minimum bactericidal concentration (MBC). Synergistic effect of polysorbate 80 with antibiotics was investigated by the broth dilution and disc diffusion techniques. Ultrastructural alterations of organisms treated with polysorbate 80, alone and in association with antibiotics were analyzed by transmission electron microscopy. RESULTS: MBCs of polysorbate 80 ranged from 2.6 (1.1) µg/ml to 32 (0) µg/ml. Polysorbate 80 exerted a synergistic effect when associated with metronidazole and clarithromycin: polysorbate 80 and metronidazole MBCs decreased by ≥ 4 fold; clarithromycin MBCs for two resistant strains decreased by 20 and 1000 times. The principal alteration caused by polysorbate 80 consisted in the detachment of the outer membrane of bacteria. CONCLUSIONS: The bactericidal activity of polysorbate 80 and the synergistic effect of the association with metronidazole and clarithromycin could be useful in the treatment of H. pylori infection.

Serum amyloid A circulating levels and disease activity in patients with juvenile idiopathic arthritis.

The aim of our study was to evaluate the association between circulating levels of serum amyloid A protein (SAA) and disease activity in patients with juvenile idiopathic arthritis (JIA). Our study group included 41 JIA patients (9 male, 32 female), classified according to the International League of Associations for Rheumatology (ILAR) criteria (5); 16 had polyarticular onset disease and 25 had oligoarticular onset disease. Among 25 patients with oligoarticular disease, three had extended oligoarthritis. Serum amyloid A (SAA), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were measured in both patients and 26 healthy controls. SAA levels were higher in JIA patients versus healthy controls (p<0.001). Significant positive correlations were found between SAA and the presence of active joints (rho=0.363, p<0.05), the number of active joints (rho=0.418, p<0.05), ESR (R=0.702, p<0.05) and CRP (R=0.827, p<0.05). No significant correlations between ESR and the presence of active joints (rho=0.221, p=0.225) or between ESR and the number of active joints (rho=0.118, p=0.520) were demonstrated in JIA patients. No significant correlations were obtained between CRP and the presence of active joints (rho=0.034, p=0.855) or between CRP and the number of active joints (rho=0.033, p=0.859). We discovered a significant increase in SAA levels in JIA patients, compared to controls, and a strong positive correlation between SAA level and JIA disease activity. We also discerned SAA to be a more sensitive laboratory marker than ESR and CRP for evaluating the presence and number of active joints. We suggest that SAA can be used as an additional indicator of disease activity in JIA.

Alkaptonuria is a novel human secondary amyloidogenic disease.

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates.

Post-genomics of bone metabolic dysfunctions and neoplasias.

Post-genomic research on osteoblastic and osteoclastic cells, in contrast to that on many other cell types, has only been undertaken recently. Nevertheless, important information has been gained from these investigations on the mechanisms involved in osteoblast differentiation and on markers relevant for tissue regeneration and therapeutic validation of drugs, hormones and growth factors. These protein indicators may also have a diagnostic and prognostic value for bone dysfunctions and tumors. Some reviews have already focused on the application of transcriptomics and/or proteomics for exploring skeletal biology and related disorders. The main goal of the present review is to systematically summarize the most relevant post-genomic studies on various metabolic bone diseases (osteoporosis, Paget's disease and osteonecrosis), neoplasias (osteosarcoma) and metabolic conditions that indirectly affect bone tissue, such as alkaptonuria.

Biochemical and proteomic characterization of alkaptonuric chondrocytes.

Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as "black" AKU chondrocytes, while those coming from the white portion were referred to as "white" AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both "white" and "black" AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in "black" AKU chondrocytes.

Homogentisate 1,2 dioxygenase is expressed in human osteoarticular cells: implications in alkaptonuria.

Alkaptonuria (AKU) results from defective homogentisate1,2-dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT-PCR, mono- and 2D-Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy.

Sex-based differences in susceptibility to respiratory and systemic pneumococcal disease in mice.

Systemic infection with Streptococcus pneumoniae was investigated in male and female mice in models of invasive pneumonia and sepsis. Male mice were found to be more susceptible to infection, exhibiting greater weight loss, marked decrease in body temperature, and a significantly higher mortality rate compared with female mice. For pneumonia, there were significant differences in survival rates. Female mice cleared their lung infections over time, whereas male mice, compared with female mice, had significantly increased numbers of colony-forming units in early stages of infection accompanied by higher levels of neutrophil recruitment in the first 24 hours after infection. Importantly, there were significant increases in proinflammatory cytokine levels during both sepsis and pneumonia in male compared with female mice. These cytokines were indicative of T-helper 1-type responses. The data presented here describe surprising differences in survival rates, neutrophil recruitment, and proinflammatory cytokine levels, indicating a sex-based difference in susceptibility to respiratory and systemic pneumococcal disease.

Different factors affecting human ANP amyloid aggregation and their implications in congestive heart failure.

AIMS: Atrial Natriuretic Peptide (ANP)-containing amyloid is frequently found in the elderly heart. No data exist regarding ANP aggregation process and its link to pathologies. Our aims were: i) to experimentally prove the presumptive association of Congestive Heart Failure (CHF) and Isolated Atrial Amyloidosis (IAA); ii) to characterize ANP aggregation, thereby elucidating IAA implication in the CHF pathogenesis. METHODS AND RESULTS: A significant prevalence (85%) of IAA was immunohistochemically proven ex vivo in biopsies from CHF patients. We investigated in vitro (using Congo Red, Thioflavin T, SDS-PAGE, transmission electron microscopy, infrared spectroscopy) ANP fibrillogenesis, starting from α-ANP as well as the ability of dimeric ß-ANP to promote amyloid formation. Different conditions were adopted, including those reproducing ß-ANP prevalence in CHF. Our results defined the uncommon rapidity of α-ANP self-assembly at acidic pH supporting the hypothesis that such aggregates constitute the onset of a fibrillization process subsequently proceeding at physiological pH. Interestingly, CHF-like conditions induced the production of the most stable and time-resistant ANP fibrils suggesting that CHF affected people may be prone to develop IAA. CONCLUSIONS: We established a link between IAA and CHF by ex vivo examination and assessed that ß-ANP is, in vitro, the seed of ANP fibrils. Our results indicate that ß-ANP plays a crucial role in ANP amyloid deposition under physiopathological CHF conditions. Overall, our findings indicate that early IAA-related ANP deposition may occur in CHF and suggest that these latter patients should be monitored for the development of cardiac amyloidosis.

Redox-proteomics of the effects of homogentisic acid in an in vitro human serum model of alkaptonuric ochronosis.

Alkaptonuria (AKU) is a rare inborn error of metabolism associated with a deficient activity of homogentisate 1,2-dioxygenase (HGO), an enzyme involved in tyrosine and phenylalanine metabolism. Such a deficiency leads to the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products in connective tissues, where melanin-like pigments accumulate (ochronosis). Ochronosis involves especially joints, where an ochronotic arthropathy develops. Little is known on the molecular mechanisms leading to ochronosis and ochronotic arthropathy in AKU. Previous works of ours showed that HGA in vitro propagates oxidative stress through its conversion into benzoquinone acetate (BQA). We hence used an in vitro model consisting of human serum treated with HGA and evaluated the activities of glutathione related anti-oxidant enzymes and levels of compounds indexes of oxidative stress. Proteomics and redox-proteomics were used to identify oxidized proteins and proteins more likely able to bind BQA. Overall, we found that the production of ochronotic pigment in HGA-treated serum is accompanied by lipid peroxidation, decreased activity of the enzyme glutathione peroxidase and massive depletion of thiol groups, together with increased protein carbonylation and thiol oxidation. We also found that BQA was likely to bind carrier proteins and naturally abundant serum proteins, eventually altering their chemico-physical properties. Concluding, our work points towards a critical importance of thiol compounds in counteracting HGA- and BQA- mediated stress in AKU, so that future research for disease biomarkers and pharmacological treatments for AKU and ochronosis will be more easily addressed.