Agonistic anti-human Fas monoclonal antibody induces fibroblast-like synoviocyte apoptosis in haemophilic arthropathy: potential therapeutic implications.

Haemophilic arthropathy (HA) is characterized by chronic proliferative synovitis leading to cartilage destruction and shares some pathological features with rheumatoid arthritis (RA). Apoptosis has been implicated in RA pathogenesis, and an agonistic anti-Fas monoclonal antibody (mAb) was found to induce RA fibroblast-like synoviocyte (FLS) apoptosis and suppress synovial hyperplasia in animal models of RA. The aim of this study was to evaluate the effect of anti-Fas mAb on HA-FLS. FLS were isolated from knee synovial biopsies from six HA patients, six RA patients and six healthy subjects. The expression of Fas in synovial biopsies was investigated by immunohistochemistry. FLS were stimulated with anti-Fas mAb at different concentrations, alone or in combination with tumour necrosis factor-α (TNF-α) and basic fibroblast growth factor (bFGF). Fas expression in FLS was assessed by Western blot. Cell viability was studied with the WST-1 assay. Active caspase-3 levels were measured using ELISA and Western blot. A strong Fas-immunoreactivity was observed in different cells of HA synovium, including FLS, inflammatory cells and endothelial cells. Fas antigen was constitutively overexpressed in cultured HA-FLS. Anti-Fas mAb had a significant cytotoxicity on HA-FLS in a dose-dependent manner, either alone or in combination with TNF-α and bFGF. These cytotoxic effects were due to the ability of anti-Fas to induce HA-FLS apoptosis, as shown by the increased active caspase-3 levels. Anti-Fas mAb exhibited a more pronounced pro-apoptotic effect on HA-FLS than RA-FLS. Fas antigen is highly expressed on HA-FLS and its stimulation by anti-Fas mAb may be an effective strategy to induce HA-FLS apoptosis.

Cryptic chromosome rearrangements in five patients, with normal and/or abnormal karyotypes, associated with mental retardation, autism and/or epilepsy, detected by BAC genome array-CGH.

This report describes the usefulness of the BAC genome array-CGH platform in the detection of cryptic rearrangements. We examined ten patients with normal and/or abnormal karyotypes and dysmorphic features, associated with mental retardation, autism and/or epilepsy. This approach led us to discover further cryptic chromosomal rearrangements, not previously detected by conventional cytogenetic procedures, and allowed us to better delineate genotype/phenotype correlation. Our experience shows the validity of the BAC platform as a reliable method for genome-wide screening of chromosomal aberrations in patient with idiopathic mental retardation and/or in association with autism and epilepsy.

Severe fibrotic changes and altered expression of angiogenic factors in maternal scleroderma: placental findings.

OBJECTIVE: Pregnant women with systemic sclerosis (SSc; scleroderma) have an increased risk of premature delivery and small full-term infants. During placental development, angiogenesis and vascular remodelling are essential for a successful pregnancy outcome. An analysis was made of the pathological changes and expression of angiogenic factors in SSc placentas. METHODS: Placenta biopsies were obtained from three patients with SSc and four healthy uncomplicated pregnancies after delivery at 34-38 weeks of gestation. The sections were stained with Masson's trichrome and phosphotungstic-acid-haematoxylin and immunostained for connective tissue growth factor (CTGF), alpha-smooth muscle actin (alpha-SMA), vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and receptors VEGFR-1 and VEGFR-2. RESULTS: The pathological findings were signs of decidual vasculopathy, increased syncytiotrophoblast knotting, placental infarcts and villous hypoplasia. Severe and diffuse perivascular and stromal fibrosis of decidua and chorionic villi, and extensive deposition of fibrinoid material around decidual vessels and in intervillous spaces were observed. Strong CTGF expression in the vessel wall, decidual cells and fibroblasts and alpha-SMA+ myofibroblasts were found. VEGF and VEGFR-2 expression was stronger in SSc than in healthy placentas, while VEGFR-1 expression was similar to controls. PlGF immunopositivity was weaker in SSc. CONCLUSION: In SSc placentas, severe fibrosis and abnormal vascular remodelling were detected. This may result in reduced blood flow leading to deep sufferance of maternal placenta and possible premature delivery.

Uncommon case of symmetrical fibrous hyperplasia of the hard palate.

The fibrous hyperplasia of the palate (fibroma) is a benign tumor which has its origin in the soft tissue and whose frequency is estimated to be 1.2% on adult subjects. Rarely the size exceeds the cm, but in these cases a complaint of increasing difficulty of mastication and swallowing appears. We describe the case of a young white woman affected with two peculiar symmetric lesions of the secondary hard palate, that could be the consequence of an abnormal answer to a chronic inflammatory stimulation.

Differential expression of tissue kallikrein in the skin of systemic sclerosis.

Systemic sclerosis (SSc) is characterised by ischemic damage, impaired angiogenesis and skin fibrosis. Tissue kallikrein (t-kallikrein) is involved through kinins in inflammation, vasorelaxation and angiogenesis. T-kallikrein is synthetised by endothelial, smooth muscle, and inflammatory cells and, in skin, also by dark cells of the sweat glands, where it is involved in sweat formation. Our aim was to analyse, by immunohistochemistry and RT-PCR, the expression of t-kallikrein in the skin of patients with different SSc subsets, limited (lSSc) and diffuse (dSSc), and phases, early and advanced. Skin biopsies were taken from 18 SSc patients and 10 controls. Immunohistochemistry was performed on paraffin sections with an antibody against human urinary t-kallikrein. For RT-PCR, cDNA from skin biopsies was amplified using primers specific for human t-kallikrein. In the control skin, dark cells of the secretory units of sweat glands showed immunopositivity for t-kallikrein as well as blood vessels. In the lSSc skin, immunoreactivity was observed only in some glands, with weak staining in the advanced phase. In early lSSc skin, immunoreactivity was observed in microvessel walls and in the inflammatory infiltrate. In dSSc skin, dark cells of the glandular fundus units, and the few remaining vessels showed scarcity (early phase) or lack (advanced phase) of immunoreactivity for t-kallikrein. RT-PCR confirmed a decrease of t-kallikrein mRNA levels from early to advanced phase in SSc subsets, reaching its lowest level in advanced dSSc. In conclusion, immunohistochemical and biomolecular results indicate that t-kallikrein is decreased in the skin of SSc patients and decreases progressively from the early to advanced phase of lSSc and dSSc. The decreased expression of t-kallikrein may be involved in the impairment of the sweating process, vessel functionality and angiogenesis.

Cardiovascular effects of nociceptin in unanesthetized mice.

We evaluated the systemic hemodynamic effects induced by nociceptin (NC) and NC-related peptides, including the NC receptor antagonist [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 ([F/G]NC(1-13)NH2) in unanesthetized normotensive Swiss Morini mice. Bolus intravenous injection of NC decreased mean blood pressure and heart rate. The hypotensive response to 10 nmol/kg NC lasted <10 minutes, whereas a more prolonged hypotension was evoked by 100 nmol/kg (from 114+/-3 to 97+/-2 mm Hg at 10 minutes, P<0.01). The latter dose reduced heart rate from 542+/-43 to 479+/-31 beats/min (P<0.05) and increased aortic blood flow by 41+/-5% (P<0.05). Hypotension and bradycardia were also evoked by NC(1-17)NH2 and NC(1-13)NH2 fragments, whereas NC(1-13)OH and NC(1-9)NH2 were ineffective. Thiorphan, an inhibitor of neutral endopeptidase 24.11, enhanced the hypotension induced by NC(1-13)NH2 and revealed the ability of NC(1-13)OH to decrease mean blood pressure. [F/G]NC(1-13)NH2, a recently synthesized antagonist of the NC receptor, did not alter basal mean blood pressure or heart rate, but it prevented the hypotension, bradycardia, and increase in aortic blood flow evoked by NC. In contrast, [F/G]NC(1-13)NH2 did not alter the hypotension induced by bradykinin or endomorphin-1 (a micro-receptor agonist), and the bradycardia induced by leu-enkephalin (a delta-receptor agonist) or U504885 (a synthetic kappa-receptor agonist). In conclusion, NC and some of its fragments cause hypotension and bradycardia and increase aortic blood flow in mice, with the NC(1-13) sequence being critical for these biological effects. Our results also demonstrate that the compound [F/G]NC(1-13)NH2 is a potent and selective antagonist of the NC receptor in vivo.

Renovascular hypertension in bradykinin B2-receptor knockout mice.

We evaluated whether kinins exert a protective action against the development of two-kidney, one clip (2K1C) hypertension, a model characterized by an activated renin-angiotensin system in the ischemic kidney and increased expression of the bradykinin (BK) B2 receptor in the contralateral kidney. BK B2-receptor knockout (B2-/-), wild-type (B2+/+), and heterozygous (B2+/-) mice underwent clipping of the left renal artery, with the other kidney remaining untouched. Basal systolic blood pressure (SBP, via tail-cuff plethysmography) was higher in B2-/- mice than in B2+/- or B2+/+ mice (121+/-2 versus 113+/-2 and 109+/-1 mm Hg; P<0.05 for both comparisons). SBP did not change from basal values after sham operation, but it increased in mice that underwent clipping. The increase in SBP was greater in 2K1C B2-/- mice than in B2+/- or B2+/+ mice (28+/-2 versus 14+/-2 and 14+/-2 mm Hg, respectively, at 2 weeks; P<0.05 for both comparisons). Blockade of the BK B2 receptor by Icatibant enhanced the pressure response to clipping in B2+/+ mice (29+/-2 mm Hg at 2 weeks). Intra-arterial mean blood pressure (MBP) was higher in 2K1C than in respective sham-operated mice, with the MBP difference being higher in B2-/- mice (32 and 38 mm Hg, at 2 and 4 weeks, respectively), and higher in B2+/+ mice given Icatibant (30 and 32 mm Hg) than in B2+/+ mice without Icatibant (17 and 18 mm Hg). At 4 weeks, acute injection of an angiotensin type 1 receptor antagonist normalized the MBP of 2K1C hypertensive mice. A tachycardic response was observed 1 week after clipping in B2-/- and B2+/- mice, but this effect was delayed in B2+/+ mice. However, the HR response to clipping in B2+/+ mice was enhanced by Icatibant. Within each strain, heart weight to body weight ratio was greater in 2K1C hypertensive mice than in sham-operated control animals (B2-/-: 5.7+/-0.1 versus 5.2+/-0.1; B2+/+: 5.1+/-0.1 versus 4.5+/-0.1; P<0.01 for both comparisons). The clipped kidney weight to nonclipped kidney weight ratio was consistently reduced in mice with 2K1C hypertension. Our results indicate that kinins acting on the BK B2 receptor exert a protective action against excessive blood pressure elevation during early phases of 2K1C hypertension.

Endocrine functioning in multitransfused prepubertal patients with homozygous beta-thalassemia.

Endocrine function was evaluated in 20 prepubertal patients with homozygous beta-thalassemia treated with frequent transfusions and long term iron chelation therapy. FSH, LH, PRL, and TSH secretion were evaluated by LRH and TRH testing and L-dopa and ACTH were used to assess GH and adrenocortical reserve. No statistically significant differences were found between FSH, LH, PRL, GH, and cortisol secretion in the patients and in normal subjects. There was a relatively high incidence (35%) of primary thyroid impairment since 1 patient had primary hypothyroidism and 6 others had evidence of subclinical hypothyroidism as manifested by increased TSH responses to TRH. However, no statistically significant correlations were found between either serum ferritin levels, total blood transfusions received, and thyroid function.

Enhanced blood pressure sensitivity to deoxycorticosterone in mice with disruption of bradykinin B2 receptor gene.

The renal kallikrein-kinin system is activated under conditions of mineralocorticoid excess. To evaluate whether endogenous kinins exert a protective role against the development of mineralocorticoid-induced hypertension, we studied the cardiovascular effects induced by long-term administration of deoxycorticosterone (DOC; 0.3 micromol/g body wt s.c. once per week for 6 weeks) or vehicle in transgenic mice (Bk2r-/-) lacking the bradykinin B2 receptor gene and in wild-type controls (Bk2r+/+). Under basal conditions, Bk2r-/- mice showed higher systolic blood pressure (tail-cuff plethysmography) than wild-type Bk2r+/+ and heterozygous Bk2r+/- mice (121+/-2 versus 114+/-2 and 115+/-2 mm Hg, respectively; P<0.05 for both comparisons). Heart rate was higher in Bk2r-/- and Bk2r+/- than in Bk2r+/+ (459+/-12 and 418+/-7 versus 390+/-7 bpm; P<0.05 for both comparisons). Systolic blood pressure was increased by DOC in transgenic as well as in wild-type mice, whereas no change was induced by the vehicle. The pressor response to DOC was more rapid and pronounced in Bk2r-/- than in Bk2r+/+ and Bk2r+/- (30+/-5 versus 15+/-4 and 6+/-3 mm Hg, respectively, at 3 weeks; P<0.01 for both comparisons). The difference in systolic blood pressure was consistent with that detected by direct intra-arterial measurements of mean blood pressure. Neither DOC nor its vehicle altered heart rate or gain in body weight over time. Under basal conditions, urinary sodium excretion did not differ between strains. During DOC administration, cumulative urinary sodium excretion was lower in Bk2r-/- than in Bk2r+/+ (2.59+/-0.15 versus 3.31+/-0.22 mmol, respectively, during the first week; P<0.05). Urinary kinin excretion was increased by DOC in both Bk2r-/- (from 0.65+/-0.17 to 4.27+/-0.80 pmol/24 h; P<0.01) and Bk2r+/+ (from 0.55+/-0.09 to 6.27+/-1.48 pmol/24 h; P<0.05). The increase in urinary kinin excretion was similar between strains. These results show that integrity of the bradykinin B2 receptor is essential for regulation of blood pressure and heart rate under basal conditions. In addition, they indicate that activation of the kallikrein-kinin system represents a compensatory response against the development of hypertension induced by mineralocorticoid excess.

Lipid peroxidation in regenerating rat liver.

Rats entrained to a strictly regulated lighting and feeding schedule have been subjected to partial hepatectomy or a sham operation. In the partially hepatectomised animals the period of liver regeneration is characterised by regular bursts of thymidine kinase activity. Liver microsomes from rats, at times corresponding to maximum thymidine kinase activity, have much reduced rates of lipid peroxidation compared to control preparations: this is due in part to increased levels of lipid-soluble antioxidant at times of maximal DNA synthesis. This temporal relationship between thymidine kinase and lipid peroxidation is consistent with the view that lipid peroxidation is decreased prior to cell division.

Low rate of NADPH/ADP-iron dependent lipid peroxidation in hepatic microsomes of DBA/2 mice.

Hepatic microsomal lipid peroxidation has been studied in 4 inbred strains of mice: C57BL/6, BALB/c, AKR and DBA/2. The rates of lipid peroxidation stimulated in vitro by carbon tetrachloride, ascorbate-iron and cumene hydroperoxide were similar in all 4 strains. Lipid peroxidation induced by NADPH/ADP-iron, however, proceeded at a substantially lower rate in the hepatic microsomes of DBA/2 mice. It is suggested that this low rate of enzymic iron-induced lipid peroxidation is a factor that may be involved in the resistance of this strain of mice to experimental hepatic porphyria induced by polyhalogenated aromatic hydrocarbons.

Left ventricular function in mice lacking the AT2 receptor.

OBJECTIVES: The role of the AT2 receptor in the heart is incompletely understood. We investigated left ventricular performance in AT2 receptor knockout mice, with and without deoxycorticosterone acetate (DOCA)-salt treatment. Given the putative opposing functions of the AT1 and AT2 receptor, we also analysed AT1 receptor expression in the left ventricle. METHODS: We used a miniaturized conductance-manometer system to measure pressure-volume loops for analysing left ventricular performance under baseline conditions and after increasing peripheral vascular resistance. We determined left ventricular AT1-receptor expression by RNase-protection assays. RESULTS: In AT2 receptor knockout mice, end-systolic and end-diastolic volumes were lower than in wild-type mice, so that pressure-volume loops were shifted leftward. Left ventricular systolic and diastolic kinetics were not different between the groups. AT2 receptor knockout mice and wild-type mice both stabilized their reduced stroke volume after laparatomy as peripheral resistance was increased. DOCA-salt treatment increased elastance in AT2 receptor knockout mice, compared to controls. Furthermore, AT2 receptor knockout mice had a steeper increase in dP/dtmax. Left ventricular AT1 receptor gene expression was increased in AT2 receptor knockout mice and was not down-regulated in response to DOCA-salt treatment. Finally, the hearts of AT2 receptor knockout mice were smaller than controls, but increased in size in response to DOCA-salt treatment. CONCLUSIONS: AT2 receptor knockout mice displayed no major changes in left ventricular function at baseline or in response to DOCA-salt treatment, compared to wild-type mice. The AT2 receptor may be important to AT1 receptor expression in response to DOCA-salt challenge and may have some influence on cardiac growth responses.

Long-term blood pressure telemetry in AT2 receptor-disrupted mice.

OBJECTIVES: The hypertension in AT2 receptor knockout mice is imperfectly defined. Therefore, we investigated the influence of dietary salt loading and deoxycorticosterone (DOCA)-salt treatment on blood pressure and diurnal patterns of blood pressure in these mice by radiotelemetry. METHODS: We used telemetry in AT2 receptor knockout and wild-type mice to measure blood pressure, heart rate, aortic pressure dp/dt, locomotor activity, and circadian rhythms. Salt-related effects were studied by increasing the salt in chow to 4%, adding 1% saline in drinking water, and by DOCA-salt treatment RESULTS: Baseline blood pressures were higher in AT2 receptor knockout than in wild-type mice and were not affected by increasing the salt intake. The blood pressure increase was steeper and greater in AT2 receptor knockout than in wild-type mice after DOCA-salt treatment A circadian rhythm of blood pressure and heart rate, with higher values during the night, was seen in wild-type, but not in AT2 receptor knockout mice. In AT2 receptor knockout mice, this rhythm was only significant when daily salt intake was increased or when DOCA-salt hypertension was induced. The acrophase of blood pressure and heart rate was found between 2000 and 2400 h and was in accordance with the maximum physical activity. CONCLUSION: These data suggest that AT2 knockout mice display slight hypertension which is not salt-sensitive. On the other hand, the susceptibility to develop DOCA-salt hypertension is increased. The study also illustrates the power of telemetry in monitoring long-term cardiovascular changes and circadian blood pressure and heart rate rhythms in genetically engineered mice.

Studies of the cardiovascular effects of nociceptin and related peptides.

Nociceptin, a novel opioid peptide, and its ORL(1) receptor share structural similarities with other opioid ligands and receptors. Although NC exerts evident cardiovascular effects at a central and peripheral level, its role in homeostatic mechanisms and disease states are just beginning to be understood, as only recently selective receptor antagonists became available. In this review, some of the new observations regarding the cardiovascular actions of NC, related peptides and newly synthesized receptor antagonists are discussed.

Role of calcitonin gene-related peptide and kinins in post-ischemic intestinal reperfusion.

The involvement of kinins, calcitonin gene-related peptide (CGRP), and tachykinins during mesenteric post-ischemic reperfusion was studied in anesthetized rats by using antagonists for bradykinin (BK) B1, BK B2, CGRP1, or tachykinin NK1 receptor, or by capsaicin-induced desensitization. B1, B2, or CGRP1 receptor antagonists or desensitization attenuated the transient hypotension and plasma protein and leukocyte infiltration of intestinal wall observed during post-ischemic reperfusion. These effects were abolished by the combination of B2 and CGRP1 blockade as well as by B2 antagonism in capsaicinized rats, while NK1 blockade was ineffective. Our results suggest that kinins and CGRP contribute to systemic vasodilatation and microvascular leakage during mesenteric reperfusion. Pharmacological blockade of these systems could help preventing hypotension and intestinal injury consequent to reperfusion.

An improved and simple method for determining diene conjugation in autoxidized polyunsaturated fatty acids.

The autoxidation of linolenic acid was produced at room temperature in a rotating apparatus by passing a stream of dry air over a thin layer of polyunsaturated fatty acid (PUFA). During these experiments, volatile thiobarbituric acid positive product(s), were recovered. The extent of fatty acid autoxidation was measured either (a) by the production of malonaldehyde (MDA) or (b) by the height of the peak at 233 nm of the second derivative spectrum, corresponding to the hidden maximum at 235 nm in the normal absorbance spectra of autoxidized PUFA. The identification of the conjugated double bond structure, arising from the autoxidation of linolenic acid was further confirmed by 1H-NMR spectrometric determinations.

Biochemical changes in the rat cerebellar cortex elicited by chronic treatment with methyl mercury.

Long-term (20 days) treatment with methyl mercury (MeHg) increases the total number of benzodiazepine binding sites and decreases essentially the content of cyclic GMP in the cerebellar cortex. In contrast, this treatment fails to modify the content of GABA and cyclic AMP, GAD activity and GABA binding sites in the same brain area. The changes in cyclic GMP and benzodiazepine binding sites in the cerebellar cortex are discussed in relation to the motor disturbances associated with MeHg intoxication.

Cytogenetic study on 282 patients with suspected chromosomal aberration.

This report shows the results of a karyotype analysis carried out on 282 patients clinically selected for some suspicion of chromosome abnormalities. This population showed a significantly higher incidence of chromosome anomalies (21.6%) than an unselected population (0.5-0.6%). The aim of this study is to determine the incidence of chromosome abnormalities in a selected Sardinian population and to compare these data with those of other authors.

Female genital cancer incidence in the Province of Sassari from 1974 to 1983.

The incidence of gynaecologic tumours in the Province of Sassari has been studied in order to estimate their value and to make a comparison with the ones industrialised countries. The global incidence of uterine cancer (corpus uteri plus cervix) is of 18.16/100,000 women; 11.99/100,000 for cancers of corpus uteri and 6.17/100,000 for cervical carcinoma including in situ forms. The incidence of gynaecologic tumors for the District of Sassari is equivalent to that reported in Western countries and it does confirm indeed a constant and progressive increase in tumours of the corpus uteri and a reduction in cervical carcinoma especially if in situ forms are excluded.

[Peripheral nervous system in limited systemic sclerosis]. / Il sistema nervoso periferico nella sclerosi sistemica limitata.

OBJECTIVES: PNS is involved in Systemic Sclerosis (SSc) since the earliest phases. Our aim is to perform an ultrastructural study on skin PNS fibers in SSc. METHODS: Skin biopsies were taken from forearms of 8 patients affected by limited SSc (lSSc) and 3 controls and processed for transmission electron microscopy (TEM). The semithin sections (2 mm) were observed at light microscope and optical fields were chosen for ultrathin sections (1 mm) preparation and TEM examination. RESULTS: In lSSc skin, in the semithin sections, damaged areas are close to apparently spared areas. At TEM, in early lSSc patients, signs of inflammation and damaged microvessels are visible in derma. PNS fibers are no damaged. In advanced lSSc, fibrosis prevails on inflammation, and slight ultrastructural alterations of PNS fibers are evident in papillar derma. CONCLUSIONS: PNS lesions are different in severity in lSSc according to the disease duration, resulting more severe in advanced than in early phase.