Sauro Lorenzini

Diagnosis of calcium pyrophosphate dihydrate crystal deposition disease: ultrasonographic criteria proposed

Objective: To investigate by high frequency ultrasonography the appearance of calcium pyrophosphate dihydrate (CPPD) calcifications, in the most commonly affected sites in CPPD disease, and the relationship between ultrasonographic CPPD deposits and the presence of CPPD crystals in synovial fluid. Methods: Three ultrasonographic patterns of CPPD calcification were identified and 11 patients enrolled. A control group comprised 13 patients with no evidence of CPPD deposits. Synovial fluid was aspirated from all patients and controls and examined for identification of crystals. All patients underwent a standard radiography examination at the same sites investigated by ultrasound. Results: In all patients with ultrasonographically defined CPPD deposits, CPPD crystals were found in the synovial fluid. In two cases, standard radiographic examination did not show evidence of the calcific deposits that were identified by ultrasonography. CPPD crystals were not found in the synovial fluid of controls. In four control group patients, ultrasonography identified calcifications defined as deposits of another nature. Conclusions: The ultrasonographic pattern used in this study for the diagnosis of CPPD disease demonstrated a very high correlation with the presence of CPPD crystals in synovial fluid. Ultrasonography demonstrated a sensitivity and specificity at least equal to that of radiography in identifying CPPD crystal calcifications.

Alkaptonuria, ochronosis, and ochronotic arthropathy.

OBJECTIVES To describe the clinical presentation and course of a relatively large group of Italian adult patients screened for mutation of the homogentisate dioxygenase gene causing alkaptonuria (AKU) and ochronosis, and to review typical and atypical facets of this condition. METHODS We reviewed the medical records of 9 patients affected by ochronotic arthropathy who were observed in our institutions between 1979 and 2001. All patients were diagnosed as having AKU through a rapid urine test with alkali. Mutation screening was performed by single-strand conformation analysis of all homogentisate dioxygenase exons, followed by sequencing of altered conformers. RESULTS Our 9 cases had similar clinical features and they reflected those described in the literature: a progressive degenerative arthropathy mainly affecting axial and weight-bearing joints associated with extraarticular manifestation. Musculoskeletal symptoms began in most of our patients around the age of 30 years with back pain and stiffness: involvement of the large peripheral joints usually occurred several years after spinal changes. Ochronotic peripheral arthropathy generally was degenerative, but joint inflammation was observed in some cases; this could be attributed to an inflammatory reaction of the ochronotic shard in the synovial membrane. CONCLUSIONS Ochronosis is a model of arthropathy with known etiologic factors. Over time, AKU, the genetically determined metabolic defect, leads to the accumulation of pigment and the development of this crippling condition. Most of the clinical findings may be explained by inhibition of collagen crosslinks, but some require additional interpretation. For example, inflammatory features of the ochronotic joint only occur in a minority of cases, and may be attributable to ochronotic shards. Further studies are needed to establish the genotype-phenotype correlation to identify mutations that are predictive of severe disease. For this purpose, the Italian Study Group on Alkaptonuria (www.dfc.unifi.it/aku) is enrolling affected patients in an on-line database to characterize the molecular defects and their relationship to clinical data.

Age-Related Influence on Thiol, Disulfide, and Protein-Mixed Disulfide Levels in Human Plasma

In this study, plasma levels of both low-molecular-mass sulfhydryls/disulfides and mixed disulfides with proteins in 41 healthy humans aged 21-92 years were measured, with the aim of assessing whether there is a shift of the thiol/disulfide balance during aging and verifying some of the possible effects of the thiol imbalance. Our data suggest that aging is strictly correlated to a decrease in plasma glutathione and cysteinylglycine with the concomitant increase of most oxidized forms of thiols and a parallel increase in total cysteine and total homocysteine, probably due to an augmented efflux of these amino acids from various organs. The occurrence of two distinct regulatory systems for plasmatic pools of glutathione/cysteinylglycine on the one hand and cysteine/homocysteine on the other hand is hypothesized.

A “new” technique for the diagnosis of chondrocalcinosis of the knee: sensitivity and specificity of high-frequency ultrasonography

According to the criteria proposed by Ryan and McCarty,1 the diagnosis of calcium pyrophosphate dihydrate (CPPD) deposition disease has been based on radiological evidence of the characteristic calcifications and on verification of the synovial liquid of CPPD crystals. Joint ultrasonography is an innocuous diagnostic technique that is well tolerated by patients, and is the elected method for observing calcified deposits in soft tissues.2 We carried out a longitudinal study, enrolling patients affected with ultrasonographic chondrocalcinosis according to previously proposed criteria3 from a sample of consecutive patients that came to our joint ultrasonography department for gonalgia (fig. 1). A total of 47 patients were identified, of which 14 had joint effusion. Figure 1  Hyperechoic deposits. Deposits (arrows) are shown that are compatible with calcium pyrophosphate dihydrate (CPPD) calcifications in the context of the synovial membrane (1), hyaline cartilage of the …

Synthetic cannabinoid ajulemic acid exerts potent antifibrotic effects in experimental models of systemic sclerosis

Background Cannabinoids modulate fibrogenesis in scleroderma. Ajulemic acid (AjA) is a non-psychoactive synthetic analogue of tetrahydrocannabinol that can bind the peroxisome proliferator-activated receptor-γ (PPAR-γ). Recent evidence suggests a key role for PPAR-γ in fibrogenesis. Objective To determine whether AjA can modulate fibrogenesis in murine models of scleroderma. Material and methods Bleomycin-induced experimental fibrosis was used to assess the antifibrotic effects of AjA in vivo. In addition, the efficacy of AjA in pre-established fibrosis was analysed in a modified model of bleomycin-induced dermal fibrosis and in mice overexpressing a constitutively active transforming growth factor β (TGFβ) receptor I. Skin fibrosis was evaluated by quantification of skin thickness and hydroxyproline content. As a marker of fibroblast activation, α-smooth muscle actin was examined. To study the direct effect of AjA in collagen neosynthesis, skin fibroblasts from patients with scleroderma were treated with increasing concentrations of AjA. Protein expression of PPAR-γ, and its endogenous ligand 15d-PGJ2, and TGFβ were assessed before and after AjA treatment. Results AjA significantly prevented experimental bleomycin-induced dermal fibrosis and modestly reduced its progression when started 3 weeks into the disease. AjA strongly reduced collagen neosynthesis by scleroderma fibroblasts in vitro, an action which was reversed completely by co-treatment with a selective PPAR-γ antagonist. Conclusions AjA prevents progression of fibrosis in vivo and inhibits fibrogenesis in vitro by stimulating PPAR-γ signalling. Since therapeutic doses of AjA are well tolerated in humans, it is suggested that AjA as an interesting molecule targeting fibrosis in patients with scleroderma.

Human rheumatoid synoviocytes express functional P2X7 receptors

Human type B synoviocytes are involved in joint injury during rheumatic diseases by producing inflammatory mediators such as interleukin-6 (IL-6). The increased level of purine and pirimidine nucleotides in the synovial fluid of rheumatoid arthritis (RA) patients could activate the large family of P2 receptors. Thus, we investigated the presence of P2 receptors in human type B synoviocytes from rheumatoid joints, also evaluating whether the P2X7 receptor is involved in IL-6 release. Reverse transcriptase polymerase chain reaction analysis revealed messenger ribonucleic acid (mRNA) expression for the P2X1, P2X2, P2X4, P2X5, P2X6, P2X7, P2Y1, P2Y4, P2Y11, P2Y12, P2Y13, and P2Y14 but not the P2X3, P2Y2, and P2Y6 receptors. The expression of the P2X7 receptor was confirmed by Western blot analysis. Adenosine triphosphate (ATP) and the P2X7 receptor agonist 2′-3′-O-(4-benzoylbenzoyl)ATP (BzATP) triggered an increase in intracellular calcium, thereby suggesting the expression of functional P2 receptors, including the P2X7 receptor. Moreover, BzATP treatment upregulated both IL-6 mRNA and protein expression. Synoviocytes spontaneously released low quantities of IL-6; the incubation with BzATP induced the release of larger amounts of the cytokine, and such a release was blunted by the P2X7 antagonist oxidized ATP. The selective P2X1 and P2X3 receptor agonist α,β-methylene ATP did not affect IL-6 release. Finally, BzATP failed to induce a significant uptake of the large-molecule YO-PRO, thus suggesting the lack of pore formation after P2X7 receptor stimulation. In conclusion, among the different P2 receptors expressed on human RA type B synoviocytes, the P2X7 receptor may modulate IL-6 release but not inducing changes in cell membrane permeability.

Subcutaneous panniculitis-like T-cell lymphoma misdiagnosed as lupus erythematosus panniculitis

We report a case of subcutaneous panniculitis-like T-cell lymphoma (SPTCL), associated with macrophage activation syndrome, mimicking a lupus erythematosus panniculitis (LEP). A 29-year-old woman presented with high fever, general malaise, nausea, vomiting, and subcutaneous nodules and ulcerating lesions located on the lower extremities. The histopathology showed an infiltration of the panniculus, mostly involving fat, and periadnexial and perivascular structures consistent with lymphocytic lobular panniculitis (LLP). LLP is a shared feature of LEP and SPTCL. The immunophenotyping of the cell infiltrate was crucial for a correct diagnosis.

Cannabinoids inhibit fibrogenesis in diffuse systemic sclerosis fibroblasts

OBJECTIVE It has been demonstrated that the endocannabinoid system is up-regulated in pathologic fibrosis and that modulation of the cannabinoid receptors might limit the progression of uncontrolled fibrogenesis. The aim of this study was to investigate whether the synthetic cannabinoid receptor agonist WIN55,212-2 could modulate fibrogenesis in an in vitro model of dcSSc. METHODS The expression of cannabinoid receptors CB1 and CB2 was assessed in dcSSc fibroblasts and healthy control fibroblasts. To investigate the effect of WIN55,212-2 on dcSSc fibrogenesis, we studied type I collagen, profibrotic cytokines, fibroblast transdifferentiation into myofibroblasts, apoptotic processes and activation of the extracellular signal-related kinase 1/2 pathway prior to and after the treatment with the synthetic cannabinoid at increasing concentrations. RESULTS Both CB1 and CB2 receptors were over-expressed in dcSSc fibroblasts compared with healthy controls. WIN55,212-2 caused a reduction in extracellular matrix deposition and counteracted several behavioural abnormalities of scleroderma fibroblasts including transdifferentiation into myofibroblasts and resistance to apoptosis. The anti-fibrogenic effect of WIN55,212-2 was not reverted by selective cannabinoid antagonists. CONCLUSIONS Our preliminary findings suggest that cannabinoids are provided with an anti-fibrotic activity, thereby possibly representing a new class of agents targeting fibrosis diseases.

Oxygen Radical Scavenger Activity, EPR, NMR, Molecular Mechanics and Extended-Hückel Molecular Orbital Investigation of the Bis(Piroxicam)Copper(II) Complex.

The oxygen radical scavenger activity (ORSA) of [CuII(Pir)2] (HPir = Piroxicam = 4-hydroxy -2- methyl -N-2- pyridyl -2H- 1,2-benzothiazine -3- carboxamide 1,1-dioxide) was determined by chemiluminescence of samples obtained by mixing human neutrophils (from healthy subjects) and [CuII(Pir)2(DMF)2] (DMF = N,N -dimethylformammide) in DMSO/GLY/PBS (2:1:2, v/v) solution (DMSO = dimethylsulfoxide, GLY = 1,2,3-propantriol, PBS = Dulbecco’s buffer salt solution). The ratio of the residual radicals, for the HPir (1.02·10−4M) and [CuII(Pir)2(DMF)2] (1.08·10−5M)/HPir (8.01·10−−5M) systems was higher than 12 (not stimulated) [excess of piroxicam was added (Cu/Pir molar ratio ≈1:10) in order to have most of the metal complexed as bischelate]. In contrast, the ratio of residual radicals for the CuCl2 (1.00·10−5M) and [CuII(Pir)2(DMF)2] (1.08·10−5M)/Hpir (8.01·10−5M)system was 5. The [CuII(Pir)2] compound is therefore a stronger radical scavenger than either HPir or CuCl2. A molecular mechanics (MM) analysis of the gas phase structures of neutral HPir, its zwitterionic (HPir+-) and anionic (Pir-) forms, and some CuII-piroxicam complexes based on X-ray structures allowed calculation of force constants. The most stable structure for HPir has a ZZZ conformation similar to that found in the CuII (and CdII complexes) in the solid state as well as in the gas phase. The structure is stabilized by a strong H bond which involves the N(amide)-H and O(enolic) groups. The MM simulation for the [CuII(Pir)2(DMF)2] complex showed that two high repulsive intramolecular contacts exist between a pyridyl hydrogen atom of one Pir- molecule with the O donor of the other ligand. These interactions activate a transition toward a pseudo-tetrahedral geometry, in the case the apical ligands are removed. On refluxing a suspension of [CuII(Pir)2(DMF)2] in acetone a brown microcystalline solid with the Cu(Pir)2·0.5DMF stoichiometry was in fact prepared. 13C spin-lattice relaxation rates of neutral, zwitterionic and anionic piroxicam, in DMSO solution are explained by the thermal equilibrium between the three most stable structures of the three forms, thus confirming the high quality of the force field. The EPR spectrum of [CuII(Pir)2(DMF)2] (DMSO/GLY, 2:1, v/v, 298 and 110 K) agrees with a N2O2+O2 pseudo-octahedral coordination geometry. The EPR spectrum of [CuII(Pir)2·0.5DMF agrees with a pseudo-tetrahedral coordination geometry. The parameters extracted from the room temperature spectra of the solution phases are in agreement with the data reported for powder and frozen solutions. The extended-Hückel calculations on minimum energy structures of [CuII(Pir)2(DMF)2] and [CuII(Pir)2] (square planar) revealed that the HOMOs have a relevant character of dx2−y2. On the other hand the HOMO of a computer generated structure for [CuII(Pir)2] (pseudo-tetrahedral) has a relevant character of dxy atomic orbital. A dxy orbital is better suited to allow a dπ-pπ interaction to the O2- anion. Therefore this work shows that the anti-inflammatory activity of piroxicam could be due in part to the formation of [CuII(Pir)2] chelates, which can exert a SOD-like activity.

Statins and the Joint: Multiple Targets for a Global Protection?

OBJECTIVES Evidence exists that the pleiotropic properties of the hydroxy-methyl-glutaryl Coenzyme A reductase inhibitors (statins) are not restricted to the cardiovascular system, as they can also favorably affect the joints, with intriguing implications for the treatment of many rheumatic diseases. In the view of the increasing interest on this topic, we here review the current state of the art. METHODS The PubMed database was searched for articles published between 1966 and 2010 for key words referring to statins and joint diseases. All relevant English-written articles were reviewed. RESULTS Many pivotal studies clearly demonstrated that HMG-CoA reductase inhibitors exert a wide spectrum of beneficial effects on the 3 main compartments of the joint, ie, the synovium, the cartilage, and the subchondral bone. Such (1) anti-inflammatory, (2) immunomodulating, and (3) anabolic effects strongly support a potential role of these drugs in the treatment and/or the prevention of the most important chronic joint diseases. However, although the majority of the in vivo studies with statins on animal models of inflammatory and degenerative joint diseases showed a marked protective activity substantially confirming the in vitro experiments, data arising from clinical trials are less probative and more conflicting. CONCLUSIONS Statins display multiple joint-protective effects. Since oral administration of statins could result in a relatively low drug bioavailability to the joints, alternative routes of administration of the drug (transdermal, intra-articular) and/or specific delivery systems should be developed to establish the entire therapeutic potential of statins in this clinical setting.