Sergio Generini

NGF, a useful tool in the treatment of chronic vasculitic ulcers in rheumatoid arthritis

Vasculitic necrosis and ulceration of the skin are frequent complications of connective tissue diseases and are very difficult to heal. We treated chronic vasculitic leg ulcers in rheumatoid arthritis and systemic sclerosis by topical application of nerve growth factor (NGF). In all patients with rheumatoid arthritis, NGF led to rapid healing, whereas less striking results were obtained in patients with systemic sclerosis. The efficacy of NGF could be due to its promoting activity on keratinocytes proliferation and vascular neoangiogenesis. We suggest that topical application of NGF could represent a powerful pharmacological tool for the treatment of vasculitic ulcers.

High prevalence of polymorphisms of angiotensin-converting enzyme (I/D) and endothelial nitric oxide synthase (Glu298Asp) in patients with systemic sclerosis

PURPOSE Systemic sclerosis is characterized by progressive microvascular occlusion and fibrosis and by an imbalance in the fibrinolytic system. In vivo and in vitro studies suggest that the renin-angiotensin system partly regulates vascular fibrinolytic balance. Angiotensin II increases the production and secretion of plasminogen activator inhibitor-1, while angiotensin-converting enzyme (ACE) contributes to reduced production of tissue plasminogen activator and endothelial nitric oxide synthesis by bradykinin degradation. The aim of our study was to investigate the effects of ACE insertion/deletion (I/D) and endothelial nitric oxide synthase (eNOS) Glu298Asp (G894-->T) and T-786-->C polymorphisms in patients with systemic sclerosis. SUBJECTS AND METHODS We studied 73 consecutive patients (47 with limited and 26 with diffuse cutaneous systemic sclerosis) and 112 control subjects. ACE I/D and eNOS polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS The ACE I/D and the eNOS G894-->T polymorphisms were more common in patients than in controls (for the ACE D allele: odds ratio [OR] = 3.4; 95% confidence interval [CI]: 1.5 to 7.9; P = 0.003; for the eNOS T allele: OR = 1.9; 95% CI: 1.0 to 3.4; P = 0.04). There was no association between the eNOS T-786-->C polymorphism and systemic sclerosis. CONCLUSIONS Our findings of an increased risk of systemic sclerosis in ACE D and eNOS 894T allele carriers suggest that these polymorphisms may contribute to the pathogenesis of the disease.

Activin, a Grape Seed-derived Proanthocyanidin Extract, Reduces Plasma Levels of Oxidative Stress and Adhesion Molecules (ICAM-1, VCAM-1 and E-selectin) in Systemic Sclerosis

This study evaluated whether a new generation antioxidant Activin derived from the grape seed proanthocyanidins, could reduce the induction of the adhesion molecules as a result of inflammatory response in the plasma of systemic sclerosis (SSc) patients. SSc patients were divided into two groups: one group was treated with Activin, a grape seed‐derived proanthocyanidins, while the other group served as control. Patients were given Activin 100 mg/day orally for one month after which the blood samples were withdrawn from both groups of the patients. Blood was also taken from normal human volunteers. Plasma was obtained in fasting state between 8 to 9 A.M. from two groups of SSc patients and controls. Soluble adhesion molecules including ICAM‐1, VCAM‐1, E‐selectin and P‐selectin as well as malonaldehyde, a marker for oxidative stress, were measured. The results of our study demonstrated up‐regulation of these soluble adhesion molecules except for P‐selectin, in the plasma of the SSc patients compared to those obtained from human volunteers. Activin significantly attenuated the increased expression of these adhesion molecules. In addition, there was a significant increase in the amount of malondialdehyde formation in the plasma of the SSc patients, which was also attenuated by Activin. The results of this study demonstrated that Activin could reduce the inflammatory response and the oxidative stress developed in SSc patients.

Oxidative stress in Systemic Sclerosis

In 63 patients affected by Systemic Sclerosis (SSc) (limited subset., 40; diffuse subset: 23; early: 30; advanced: 33) the peroxidation product diene-conjugates (DC) and antibodies against oxidised low density lipoproteins (Ab oxLDL) were tested in serum by a spectrophotometer (absorbance 234 mn) and by a standard ELISA respectively. The data were compared with those obtained by 21 healthy subjects. DC was significantly higher in patients (73.3 ± 37.2 μM/l; p < 0.0001) than in controls (48.4 ± 16.7) as well as in the limited (80 ± 48.8; p < 0.05) than in the diffuse subset (64.5 ± 36.4); and in early (84.1 ± 31.4; p < 0.05) than in advanced stage of the disease (67.9 ± 42.5). The levels Ab oxLDL were significantly higher in SSc patients (309.5 ± 367.2 mU/ml; p < 0.0001) in all its subsets (limited: 351.9 ± 351.1, p < 0.0001; diffuse: 207.7 ± 316. 1, p < 0.05; early: 428.9 ± 417.1, p < 0.001; advanced: 302.7 ± 89.9, p < 0.0001) than in controls (89.3 ± 29.1). These antibodies levels were higher in limited subset than in diffuse (p < 0.05) and in early SSc than in advanced SSc (p < 0.05). The highest values of parameters of oxidative stress are found in the early stages, when the episodes of reperfusion after ischemic episodes (Raynauds phenomenon) are very ferequent. Moreover, the damage is higher in the early stages of SSc, with intact microvessels, than in late stages, when microvessels are very reduced in number, destroyed by the worsening of the disease. These radicals products works as well in other diseases such as myocardial ischemia and pulmonary fibrosis.These data show that the respiratory burst deduced their lipoperoxidation is higher in SSe than in controls, may be an important pathogenetic factors involved in tissue changes in SSe.

Effect of N-acetyl-l-cysteine on peroxynitrite and superoxide anion production of lung alveolar macrophages in systemic sclerosis

Lung macrophages may play a relevant role in oxidative processes producing both superoxide anion (O(2)(-)) and NO. In this view, an antioxidant therapy can be useful in the treatment of systemic sclerosis (SSc) patients. N-Acetylcysteine (NAC) is able to expand natural antioxidant defenses by increasing intracellular gluthatione concentration and it has been proposed as an antioxidant therapy in respiratory distress syndromes. The aim of our study was to determine whether lung macrophages obtained from SSc patient bronchoalveolar lavage (BAL) express the inducible form of nitric oxide synthase (iNOS) and whether NAC can reduce the peroxynitrite (ONOO(-)) and O(2)(-) production of these cells. Alveolar macrophages were isolated from BAL of 32 patients and used for the immunocytochemical determination of iNOS, and the production of ONOO(-) and O(2)(-) was measured by fluorimetric or spectrophotometric methods, respectively. Lung macrophages obtained from SSc patients expressed a higher level of iNOS compared to healthy subject cells. NAC preincubation (5 x 10(-5)M, 24h) significantly reduced (-21%) the ONOO(-) production in formyl Met-Leu-Phe (fMLP)-activated cells and slightly reduced it under resting conditions, whereas NAC preincubation was unable to modify the release of O(2)(-) both in basal condition and in fMLP-stimulated cells. We conclude that since SSc lung macrophages express high levels of iNOS and produce a significant quantity of ONOO(-), NAC administration reduces ONOO(-) production and can be an useful treatment to alleviate SSc symptoms.

Increased circulating nerve growth factor is directly correlated with disease activity in juvenile chronic arthritis.

OBJECTIVE: To determine the circulating serum concentrations of nerve growth factor (NGF) and compare them with indices of disease activity in juvenile chronic arthritis. METHODS: NGF concentrations were evaluated with a two site immunoenzymatic assay (ELISA), in 17 children with systemic, 39 with polyarticular, and 24 with pauciarticular onset juvenile chronic arthritis. Each subset was divided according to different variables, appropriate to each subset, reflecting active and inactive disease. RESULTS: NGF concentrations were significantly higher in children with systemic [254 (SD 256.1) pg ml-1; P < 0.001], polyarticular [165.2 (300.8) pg ml-1; P < 0.05], and pauciarticular [106.8 (111.8) pg ml-1; P < 0.005] onset juvenile chronic arthritis than in controls. In all subsets, NGF concentrations were higher in the active than in the inactive phase of the disease. A significant direct correlation between NGF concentrations and erythrocyte sedimentation rate was found both in the systemic and in the polyarticular onset juvenile chronic arthritis. CONCLUSIONS: The increase in NGF concentrations in all juvenile chronic arthritis subsets and the correlation with disease activity suggest that NGF may take an active part in joint inflammation.

THE NERVOUS SYSTEM IN SYSTEMIC SCLEROSIS (SCLERODERMA): Clinical Features and Pathogenetic Mechanisms

The involvement of the nervous system in SSc is well recognized today. Different pathogenetic mechanisms are suggested that may alternatively explain the multiform appearance of the clinical spectrum (mononeuritis, mononeuritis multiplex, carpal tunnel syndrome, and so forth). It is now clear that the ANS is the earliest structure targeted by the disease in the gastrointestinal tract. The importance of this observation has not yet been adequately interpreted but may, together with the increasing evidence of the nervous system involvement in SSc, become a leading factor in understanding of the importance of the nervous system in the onset, development, and maintenance of the disease.

Raynaud’s Phenomenon and Vascular Disease in Systemic Sclerosis

Raynauds phenomenon (RP) is very often the first manifestation of SSc preceding the onset of all the other signs and symptoms of the disease. Two structures are involved in the pathogenesis of RP: the endothelium and the peripheral nervous system (PNS). The hypothesis is that SSc modifies consistently the activity of both these systems leading eventually to RP. The disease, through the injury to the endothelium, jeopardizes the basilar endothelial-dependent vascular tone control. An increase of endothelin, a potent endothelial-derived vasoconstrictor, and the reduction of nitric oxide, one of the main endothelial vasodilators, are two key events involved in the genesis of RP. The PNS is also targeted by the disease as demonstrated by the high incidence of neuropathy in SSc patients. A marked reduction of sensory fibres has been detected in SSc skin. Thus, the involvement of nerve terminals reduces the vasodilatory, endothelial dependent or independent, potential of the neuropeptides released by sensory nerve endings. Indeed, an increased sensitivity of alpha 2 adrenoceptors mediated vasoconstriction has been shown in SSc skin. The complex vasodilatory network formed by the interaction between the endothelium and the PNS seems greatly damaged by SSc leading inesorably toward vascular tone dysfunction clinically evident as RP.

Nerve growth factor and neuropeptides circulating levels in systemic sclerosis (scleroderma).

OBJECTIVE To determine the circulating levels of nerve growth factor (NGF), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP) in systemic sclerosis (SSc), and to correlate these levels with clinical and laboratory features. METHODS Forty four patients with SSc were evaluated for circulating NGF (immunoenzymatic assay), NPY and VIP (radioimmunoassay), anticentromere and antitopoisomerase I autoantibodies, lung disease (pulmonary function tests with carbon monoxide transfer factor (Tlco), ventilation scintiscan with 99mTc DTPA radioaerosol, high resolution computed tomography (HRCT), pulmonary pressure (echo colour Doppler)), heart disease (standard and 24 ECG, echocardiography), cutaneous involvement (skin score), joint involvement (evidence of tender or swollen joints, or both), peripheral nervous system (PNS) involvement (electromyography), rheumatoid factor, angiotensin converting enzyme (fluorimetric method), von Willebrand factor (ELISA), and erythrocyte sedimentation rate (ESR) (Westergren). RESULTS Circulating NGF levels in SSc were significantly increased compared with controls (p<0.00001) and significantly higher in the diffuse than in the limited subset of patients (p<0.01). Patients with articular disease had significantly higher levels of NGF. A significant indirect correlation between NGF levels and Tlco was detected (p<0.01), but no correlation was found between NGF and HRCT, DTPA, skin score, PNS involvement and angiotensin converting enzyme and von Willebrand factor levels, antitopoisomerase or anticentromere antibodies, and ESR. NGF levels increased progressively as the disease worsened. Similarly, VIP circulating levels were significantly increased in patients with SSc (p<0.001), whereas the increase of NPY levels did not reach statistical significance. However, both neuropeptides, following the same trend as NGF, increased as the disease worsened (skin score and lung disease). CONCLUSIONS The increase of NGF and VIP in patients with SSc, the former in the diffuse subset of the disease, and in patients with prominent articular disease, may suggest a link between neurotransmitters and the disease pathogenesis. Neuropeptide circulating levels seem to increase only in patients with the most severe disease.

The pathogenesis of inflammatory muscle diseases:: On the cutting edge among the environment, the genetic background, the immune response and the dysregulation of apoptosis

Inflammatory muscle diseases (IMD), including dermatomyositis (DM) and polymyositis (PM), affect skeletal muscle, leading to profound tissue modification. The etiology of IMD is unknown, but multiple steps of the disease pathogenesis have been identified. The main alterations involve the immune response. Cellular infiltrates found in the muscle provide strong evidence for the involvement of a preferential immune mechanism of muscle damage. The pathologic differences found between PM and DM indicate a different role played by cell-mediated and humoral immune alterations. It is well accepted that in the pathogenetic pathway both host genes and environmental factors are involved. Apoptosis, or programmed cell death, is a complex process that plays a key role in many physiological events. It regulates the turnover of immune cells and is one of the mechanisms involved in ensuring a competent, non-autoreactive repertoire of lymphocytes. Apoptosis as a mechanism of muscle fibre death has been described in several neuromuscular disorders and muscular dystrophies, and evidence of a lack of apoptosis in IMD suggests a failure of apoptotic clearance of inflammatory cells playing a role in the maintenance of chronic cytotoxic muscle fibre damage. Most likely, the failure of apoptosis seems to be the main hallmark of the pathogenesis of IMD.