Effects of chronic treatment with statins and fenofibrate on rat skeletal muscle: a biochemical, histological and electrophysiological study.

BACKGROUND AND PURPOSE: Skeletal muscle injury by hypolipidemic drugs is not fully understood. An extensive analysis of the effect of chronic treatment with fluvastatin (5 mgkg(-1) and 20 mgkg(-1)), atorvastatin (10 mgkg(-1)) and fenofibrate (60 mgkg(-1)) on rat skeletal muscle was undertaken. EXPERIMENTAL APPROACH: Myoglobinemia as sign of muscle damage was measured by enzymatic assay. Histological and immunohistochemical techniques were used to estimate muscle integrity and the presence of aquaporin-4, a protein controlling water homeostasis. Electrophysiological evaluation of muscle Cl(-) conductance (gCl) and mechanical threshold (MT) for contraction, index of intracellular calcium homeostasis, was performed by the two-intracellular microelectrodes technique. KEY RESULTS: Fluvastatin (20 mgkg(-1)) increased myoglobinemia. The lower dose of fluvastatin did not modify myoglobinemia, but reduced urinary electrolytes, suggesting direct effects on renal function. Atorvastatin also increased myoglobinemia, with slight effects on urinary parameters. No treatment caused any histological damage to muscle or modification in the number of fibres expressing aquaporin-4. Either fluvastatin (at both doses) or atorvastatin reduced sarcolemma gCl and changed MT. Both statins produced slight effects on total cholesterol, suggesting that the observed modifications occur independently of HMGCoA-reductase inhibition. Fenofibrate increased myoglobinemia and decreased muscle gCl, whereas it did not change the MT, suggesting a different mechanism of action from the statins. CONCLUSIONS AND IMPLICATIONS: This study identifies muscle gCl and MT as early targets of drugs action that may contribute to milder symptoms of myotoxicity, such as muscle cramps, while the increase of myoglobinemia is a later phenomenon.

Progesterone supplement in pregnancy: an immunologic therapy?

One of the most interesting functions of the placenta is the regulation of the maternal immune response such that the fetal semi-allograft is tolerated during pregnancy. Trophoblasts are presumed to be essential to this phenomenon because they lie at the maternal-fetal interface, where they are in direct contact with cells of the maternal immune system. Trophoblasts do not express classic major histocompatibility complex (MHC) class II molecules. Surprisingly, cytotrophoblasts express more HLA-G, a MHC class Ib molecule, as they invade the uterus. Progesterone plays an important role in postovulatory regulation of the menstrual cycle. If fertilization occurs, progesterone supports implantation of the ovum and maintains the pregnancy. Progesterone has been named the 'hormone of pregnancy', because in preparing the endometrium for embryo implantation and facilitating endometrial development, it is critical to the very survival of a pregnancy. In addition, this key hormone inhibits the rejection of T cell-mediated tissue and also decreases myometrial activity and sensitivity throughout pregnancy. The cellular actions of progesterone are mediated through intracellular progesterone receptors (PRs), which are well studied gene regulators, not express classic major histocompatibility complex. The more used paradigm is relative to the alteration of relationship TH1/TH2, but the complexity of the respective distributions of cytokines at the materno-fetal interface, strongly suggest that, as useful as it certainly was for a while, the Th1/Th2 paradigm must now be considered as an oversimplification. Rather, the existing data point to sequential windows and are suggestive of a system where an extreme complexity is allied to very precise timing and tuning. They also suggest that the materno-fetal relationship is not simply maternal tolerance of a foreign tissue, but a series of intricate mutual cytokine interactions governing selective immune regulation and also control of the adhesion and vascularization processes during this dialogue. However, as shifting the immune response toward the Th2 pattern (IL-4, IL-5, IL-6) may benefit the fetus, whereas development of proinflammatory Th1 cells (secreting IL-2, IFN g, TNF a) may be harmful. Now we are working to open comprise the precise behaviour of NK populations, with the hope of obtaining a diagnostic test of the condition of abortion from 'immunological causes'.

DNA adducts, benzo(a)pyrene monooxygenase activity, and lysosomal membrane stability in Mytilus galloprovincialis from different areas in Taranto coastal waters (Italy).

The aim of this study was to investigate the impact of environmental pollution at different stations along the Taranto coastline (Ionian Sea, Puglia, Italy) using several biomarkers of exposure and the effect on mussels, Mytilus galloprovincialis, collected in October 2001 and October 2002. Five sampling sites were compared with a "cleaner" reference site in the Aeronautics Area. In this study we also investigated the differences between adduct levels in gills and digestive gland. This Taranto area is the most significant industrial settlement on the Ionian Sea known to be contaminated by polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc. Exposure to PAHs was evaluated by measuring DNA adduct levels and benzo(a)pyrene monooxygenase activity (B(a)PMO); DNA adducts were analyzed by 32P-postlabeling with nuclease P1 enhancement in both gills and digestive glands to evaluate differences between DNA adduct levels in the two tissues. B(a)PMO was assayed in the microsomal fraction of the digestive glands as a result of the high expression of P450-metabolizing enzymes in this tissue. Lysosomal membrane stability, a potential biomarker of anthropogenic stress, was also evaluated in the digestive glands of mussels, by measuring the latent activity of beta-N-acetylhexosaminidase. Induction of DNA adducts was evident in both tissues, although the results revealed large tissue differences in DNA adduct formation. In fact, gills showed higher DNA adduct levels than did digestive gland. No significant differences were found in DNA adduct levels over time, with both tissues providing similar results in both years. DNA adduct levels were correlated with B(a)PMO activity in digestive gland in both years (r = 0.60 in 2001; r = 0.73 in 2002). Increases were observed in B(a)PMO activity and DNA adduct levels at different stations; no statistical difference was observed in B(a)PMO activity over the two monitoring campaigns. The membrane labilization period in mussels from some stations was decreased in both years. No statistical differences were established in the membrane labilization times from 2001 to 2002. Our results suggest the existence of different sources and amounts of environmental contaminants at the stations investigated. The formation of DNA adducts confirms the existence of activation pathways in mussels and shows the importance of DNA adduct analysis in the gill tissue in addition to the more commonly used digestive gland; these results confirm the utility of lysosomal membrane stability as a biomarker of general stress. Overall, the integrated use of biomarkers of exposure and the effects of environmental contaminants on living marine organisms may help to better interpret the impact of pollutants in a marine coastal environment.

Flexible treatment of gestational diabetes modulated on ultrasound evaluation of intrauterine growth: a controlled randomized clinical trial.

OBJECTIVES: In order to prevent abnormalities of fetal growth still characterizing pregnancies complicated by Gestational Diabetes (GDM), in the present study we evaluated a therapeutic strategy for GDM based on ultrasound (US) measurement of fetal insulin-sensitive tissues. METHODS: All GDM women diagnosed before 28th week immediately started diet and self-monitoring of blood glucose; after 2 weeks they were randomized to conventional (C) or modified (M) management. In C the glycemic target (GT) was fixed at 90 fasting/120 post-prandial mg/dl; in M GT varied, according to US measurement of the Abdominal Circumference (AC) centile performed every 2 weeks: 80/100 if AC > or =75th, 100/140 if AC<75th. Therapy was tailored to mean fasting (FG) and postprandial glycemia (PPG). RESULTS: Globally, 229 women completed the study, 78 in C, 151 in M. Use of insulin was 16.7% in C, 30.4% in M (total groups), significantly more frequent in M than in C (59.7% vs 15.4%) when considering only women with AC > or =75th c. Mean metabolic data were similar in the 2 groups, but in M a tightly-optimized subgroup, resulting from the lowering of GT due to AC > or =75th, coexisted with a less-controlled one, whose higher GT was justified by AC<75th. Pregnancy outcome was better in M, with lower (p<0.05*) rate of LGA* (7.9% vs 17.9%), SGA (6.0% vs 9.0%) and Macrosomia* (3.3% vs 11.5%). CONCLUSIONS: Our data show the value of a flexible US-based approach to the treatment of GDM. This model does not necessarily involve a generalized aggressive treatment, allowing to concentrate therapeutical efforts on a small subgroup of women showing indirect US evidence of fetal hyperinsulinization. Such a selective approach allowed to obtain a near-normalization of fetal growth, with clear advantages on global pregnancy outcome.

Pregnancy outcome in 100 women with autoimmune diseases and anti-Ro/SSA antibodies: a prospective controlled study.

Anti-Ro/SSA antibodies are associated with neonatal lupus but are also considered a possible cause for unexplained pregnancy loss and adverse pregnancy outcome. In a large multicentres cohort study we have prospectively followed 100 anti-Ro/SSA positive women (53 systemic lupus erythematosus (SLE)) during their 122 pregnancies and 107 anti-Ro/SSA negative women (58 SLE) (140 pregnancies). Anti-Ro/SSA antibodies were tested by immunoblot and counterimunoelectrophoresis. Mean gestational age at delivery (38 vs 37.9 weeks), prevalence of pregnancy loss (9.9 vs 18.6%), preterm birth (21.3 vs 13.9%), cesarean sections (49.2 vs 53.4%), premature rupture of membranes (4.9 vs 8.1%), preeclampsia (6.6 vs 8%), intrauterine growth retardation (0 vs 2.3%)and newborns small for gestational age (11.5 vs 5.8%) were similar in anti-Ro/SSA positive and negative SLE mothers; findings were similar in non-SLE women. Two cases of congenital heart block were observed out of 100 anti-Ro/SSA positive women. In conclusion, anti-Ro/SSA antibodies are responsible for congenital heart block but do not affect other pregnancy outcomes, both in SLE and in non-SLE women. The general outcome of these pregnancies is now very good, ifprospectively followed by multidisciplinary teams with ample experience in this field.

Risk of congenital complete heart block in newborns of mothers with anti-Ro/SSA antibodies detected by counterimmunoelectrophoresis: a prospective study of 100 women.

OBJECTIVE: To assess the true prevalence of congenital complete heart block (CCHB) in infants of anti-Ro/SSA-positive women known to have connective tissue disease (CTD) and, secondarily, to evaluate the prevalence of other electrocardiographic abnormalities in these newborns at birth. METHODS: A prospective study was conducted in 4 referral hospitals. One hundred anti-Ro/SSAA-positive mothers were followed up before they became pregnant and during the index pregnancy. Counterimmunoelectrophoresis and immunoblotting were used to test for antibodies to extractable nuclear antigens. RESULTS: Of the 100 women with anti-Ro/SSA antibodies, 2 had infants who developed CCHB in utero (2%). The CCHB was detected at 22 weeks and 20 weeks, respectively. One of the 2 mothers had primary Sjögren's syndrome (SS), and the other had undifferentiated CTD (UCTD). No case of CCHB occurred among the infants of 53 mothers with systemic lupus erythematosus (SLE). No fetal death occurred due to CCHB. In 2 centers, electrocardiography was recorded in 24 unselected newborns, and 4 were found to have sinus bradycardia. CONCLUSION: The prevalence of CCHB in newborns of prospectively followed up women already known to be anti-Ro/SSA positive and with known CTD was 2%. This finding is useful with regard to preconception counseling of these women. The risk of delivering an infant with CCHB may be higher in mothers with primary SS or UCTD than in those with SLE. Additional electrocardiographic abnormalities such as sinus bradycardia and prolongation of the QT interval may be present in their children.

[Neonatal lupus: clinical features and risk of congenital cardiac heart block in newborns from mothers with anti Ro/SSA antibodies] / Lupus neonatale: manifestazioni cliniche e rischio di blocco cardiaco congenito in neonati da madri con anticorpi antiRo/SS-A.

OBJECTIVE: To assess the prevalence of Congenital Heart Block (CHB) in newborns from anti Ro/SS-A antibodies positive mothers affected by connective tissue diseases (CTD) and to evaluate the prevalence of other manifestations of Neonatal Lupus (NL) and the electrocardiographic abnormalities. METHODS: A prospective study was conducted on 100 anti Ro/SS-A positive mothers that were followed before and during their 118 pregnancies (4 twin pregnancies and 18 second pregnancies). Counterimmunoelectroforesis (CIE) and immunoblot (IB) were used to test antibodies to extractable nuclear antigens (ENA). RESULTS: Only 2 cases of CHB (1.8%) were found among the 112 living newborns. In one case the mother with primary Sjögren's Syndrome (pSS) was anti Ro 60 and 52kD positive while in the other case the mother affected by undifferentiated connective tissue disease (UCTD) was anti Ro 60kD and anti La positive. No fetal death was due to CHB. There were no cutaneous rashes at birth while mild hepatic enzyme alterations were observed in 21 (68%) of the 31 tested newborns. In 22 healthy newborns an ECG have been registered and in 4 cases (18.2%) sinus bradycardia was found. During the follow up 7 suckling showed Cutaneous Neonatal Lupus. Moreover a six month girl developed Kawasaki Syndrome. CONCLUSIONS: The risk of delivering a child with CHB is 1.8% in anti Ro/SS-A positive mothers with CTD. This finding is extremely important in the preconceptional counseling of anti-Ro/SS-A positive women. Furthermore mild electrocardiographic abnormalities may be found in their healthy newborns.

Effects of Aroclor 1254 on intercellular communication in human keratinocytes.

It has been previously described that Aroclor 1254 can inhibit GJIC in rodent liver cells where it is known to be a tumor promoter, while the possibility that Aroclor 1254 exerts its inhibitory effects on GJIC in human keratinocytes and acts as a human skin tumor promoter, deserves further attention. In the present study the effects of Aroclor 1254 were examined on gap junction channel permeability, on connexin 43 (Cx 43) expression at mRNA and protein level and on ultrastructural modification to add further experimental evidence to its inhibitory effect on GJIC. The results were compared to those induced by 12-O-tetradecanoylphorbol-13 acetate (TPA), a tumor promoter known to be a potent inhibitor of GJIC in human skin cells and to those induced by benzo[a]pyrene (B[a]P) known for its genotoxic activity. Our data show increased Cx 43 protein expression in Aroclor 1254 and TPA-treated cultures compared to controls, decreased Cx 43 protein level in those exposed to B[a]P, while Cx 43 gene expression (Cx 43 mRNA) was unaffected by the treatments. In Aroclor and TPA-treated keratinocytes, the ultrastructural examination showed residues of junctional systems expressed by specular, short tracts of the faced plasma membranes. In contrast, the contacts between plasma membranes of adjacent B[a]P treated keratinocytes were more extended. A clear inhibition of gap junction channel permeability due to Aroclor 1254 and TPA was also manifest by Lucifer yellow dye test compared to B[a]P-treated cultures where dye spreading to the neighbouring cells and to the extracellular space occurred. The present data, in addition to confirming inhibition of GJIC mediated by Aroclor 1254 in human keratinocytes, which were found to be comparable to those induced by TPA, suggest that GJIC inhibition is associated with increased Cx 43 protein expression without significant modification of its gene expression.

The influence of cytochrome P450 1A1 and glutathione S-transferase M1 genotypes on biomarker levels in coke-oven workers.

The present study has the aim of evaluating gene-environment interaction on the levels of different biomarkers in coke-oven workers exposed to PAH. In order to assess whether the levels of some biomarkers (PAH-DNA adducts, nitro-PAH adducts to Hb and MN frequency) could be modulated by the genetic metabolic polymorphisms for CYP1A1 and GSTM1, we analysed in 76 coke-oven workers and 18 controls the CYP1A1 (MspI and Ile/Val sites) and the GSTM1 genotypes by a PCR assay. In individuals with shared setup of CYP1A1 or GSTM1 genotypes, we analysed how the specified biomarkers correlated with total PAH exposure (urinary levels of 1-hydroxypyrene) both by a stratified analysis and logistic regression modelling. Statistically significant (P = 0.03 and P = 0.01) higher percentages of the more susceptible GSTM1- subjects compared to the GSTM1+ subjects and of the more susceptible CYP1A1 Ile/Val individuals compared to the CYP1A1 Ile/Ile individuals were detected for high levels of PAH-DNA adducts in the high exposure group (namely high levels of 1-OHP). A statistically significant association was observed between increased PAH-DNA adduct levels and the more susceptible GSTM1- genotype (P.O.R. = 4.18, P = 0.03) in a logistic regression modelling and a significant interaction between PAH exposure and GSTM1-genotype was found for PAH-DNA adducts. No effect of these metabolic genotypes was observed for MN frequency and nitro-PAH adducts to Hb. In conclusion, a gene-environment interaction between PAH exposure and two metabolic genotypes involved in activation (CYP1A1) and detoxification (GSTM1) of PAHs, respectively, has been identified.

[Clinical course of pre- and post-natal isolated congenital atrioventricular block diagnosed in utero]. / Evoluzione pre e postnatale del blocco atrioventricolare congenito isolato diagnosticato in utero.

We evaluated the pre- and postnatal outcome of isolated atrioventricular (AV) block detected during fetal life in order to identify factors that may affect the natural history of this lesion and to assess prenatal therapy. Over the past eight years, we consecutively evaluated 10 fetuses with complete AV block. The mean gestational age at diagnosis was 25.3 weeks and the mean heart rate was 57 bpm; two fetuses were hydropic. During pregnancy, one fetus suddenly died, while 6 out of 9 fetuses had a mean reduction in heart rate of 17.8 bpm; 4 patients had heart rate < 50 bpm. Five fetuses developed heart failure, which was severe in 2 cases and mild in 3. The mean gestational age at delivery was 31 weeks. Dexamethasone was administered to the mothers during pregnancy in 4 cases without modification of AV block and/or of heart rate, but in 3 out of 4 fetuses the general condition remained stable in spite of the reduction in heart rate in two of them. Sympathomimetic drugs were employed in 3 cases with an increase in fetal heart rate, but maternal discomfort appeared in two cases. Three newborns died during the first week of life, two of hydrops and one of persistent pulmonary hypertension. Cardiac pacing was performed in 6/9 patients within the first 8 months of life and in 3 within the first 2 days. In conclusion, morbidity and mortality are high when AV block is detected during fetal life. Negative prognostic factors are hydrops and a heart rate < 50 bpm. Pre-term delivery to enable cardiac pacing is probably the therapy of choice if gestational age is > 27-28 weeks. Sympathomimetic drugs are effective but are poorly tolerated by the mothers. Dexamethasone has no effect on AV block and/or heart rate, but may improve clinical tolerance of conduction disturbance.

Fetal blood sampling immediately before and within 24 hours of death in monochorionic twin pregnancies complicated by single intrauterine death.

OBJECTIVE: Our goal was to investigate the mechanisms that play a role in intrauterine death in monochorionic twins and that contribute to the high perinatal mortality and morbidity in the survivors. STUDY DESIGN: In 8 monochorionic twin pregnancies complicated by the intrauterine death of a single twin, we took samples from 5 twin fetuses immediately before death and from 4 of their cotwins and also from 4 surviving fetuses within 24 hours after death of the cotwin. RESULTS: Four of the 5 fetuses sampled who subsequently died were acidemic and 3 were hypoxemic. None of these fetuses or their cotwins were anemic at that time. All 4 survivors sampled within 24 hours of the death of each cotwin had low hematocrits. CONCLUSION: Fetal anemia, probably the consequence of acute blood loss just before the time of death of the cotwin, may play a role in the high mortality and morbidity found in the surviving twin. It is unlikely that immediate delivery of the surviving twin after death could affect the outcome.

Should we treat minor degrees of glucose intolerance in pregnancy?

We examined the pregnancy outcome of 112 women classified as minor degrees of glucose intolerance (MDGI) in pregnancy in a screening program based on Carpenter and Coustan's criteria. The MDGI group comprised 49 women with abnormal oral glucose challenge test (OGCT) followed by normal OGTT (group A), and 63 with "borderline" OGTT (1 abnormal value, group B). No treatment was offered to 88 MDGI women, while 26 received dietary advice and metabolic monitoring. A control group was constituted from 112 age- and BMI-matched negative screenees. Similar rates of cesarean sections and macrosomia, but higher rate of large for gestational age (LGA) babies (25.9% vs 14.3%) were found in MDGI, without difference between groups A and B. When comparing treated and untreated MDGI, lower LGA incidence (11.5% vs 30.2%) and no macrosomia were found in the former. In conclusion, untreated MDGI may present excessive fetal growth, which can be normalized by dietary treatment and metabolic monitoring.

[Prenatal diagnosis of anatomo-pathologic aspects of 2 cases of sirenomelia]. / Diagnosi prenatale e quadro anatomopatologico di 2 casi di sirenomelia.

The antenatal echographic diagnostic aspects and anatomopathological features of two foetuses suffering from sirenomelia, or caudal pole regression syndrome, are examined. Conduction of the two cases is compared. In the first case, observed at the 22nd week, antenatal diagnosis of sirenomelia led to the therapeutic interruption of pregnancy. The second case, seen close to term, closed with the birth of an already dead sirenomelic foetus.

Pharmacokinetics of VP16-213 given by different administration methods.

Plasma pharmacokinetics of VP16-213 were investigated after a 30-60 min infusion in 14 adult patients and six children. In adult the elimination half-life (T1/2 beta), plasma clearance (Clp) and volume of distribution (Vd) were respectively 7.05 +/- 0.67 h, 26.8 +1- 2.4 ml/min/m2, and 15.7 +1- 1.8 l/m2; in children 3.37 +/- 0.5 h, 39.34 +1- 6.6 ml/min m2, and 9.97 +/- 3.7 l/m2. After repeated daily doses no accumulation of VP16-213 was found in plasma. The unchanged drug found in the 24 h urine after administration amounted to 20-30% of the dose. In eight choriocarcinoma patients plasma levels of VP16-213 were measured after oral capsules and drinkable ampoules. The bioavailability compared to the i.v. route was variable, mean values being 57% for capsules and 91% for ampoules. In one further patient, with abnormal d-Xylose absorption results, VP16-213 was not detectable in plasma after the oral ampoule dose. Steady state levels investigated in three patients after 72 h continuous VP16-213 infusion (100 mg/m2/24h) were around 2-5 micrograms/ml. Levels of VP16-213 were undetectable in CSF after i.v. or oral administration.