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Subcutaneous insulin absorption is one of the key factors affecting glycemic control in patients with diabetes mellitus under insulin therapy. Insulin-induced subcutaneous lipohypertrophy has been reported to impair insulin regular absorption and hence glycemic control. So far, lipohypertrophy diagnosis has only been clinical. This study aims at evaluating the possible role of ultrasound scan in the assessment of subcutaneous lipohypertrophy in patients affected by type 1 diabetes mellitus. A pilot observational retrospective study was performed in 20 patients affected by type 1 diabetes mellitus. In these patients the areas with clinical evidence of lipohypertrophy dependent on the insulin injections were characterized by the presence of tissues that at the ultrasound scan resulted similar to fibrotic tissues (hyperechogenic) or to an interstitial edema or to fat tissues (hypoechogenic). It was utilized a multi frequency linear probe (6–18 MHz). The patients were advised to avoid insulin injections on the areas with lipohypertrophy scanned by the ultrasound and the HbA1c changes were evaluated 3 months later. The lipohypertrophic areas presented at least three different aspects upon ultrasound assessment: the iso-hyperechogenic one, with a predominant fibrotic component; the isoechogenic one, with “large tangles” fibrotic elements and the iso-hypoechogenic aspect with no fibrotic elements. When patients were advised to avoid insulin injections on areas with lipohypertrophy defined by ultrasound scan, 3 months after the first evaluation HbA1c had significantly improved (basal HbA1c 7.87 ± 0.56 versus 7.67 ± 0.52 3 months later, p = 0.029). No significant improvements of the HbA1c were found in the control matched group in which lipohypertrophy was only clinically valued through inspection and palpation. Ultrasound scan can help identify and characterize the lipohypertrophic areas and this might be useful to improve glycemic control.

The aim of this study was to evaluate the ability of ultrasound (US) to measure the parietal thickness of varicose veins.In a blind in vitro analysis, 28 great saphenous veins, obtained after stripping surgery from 28 patients with chronic venous insufficiency, were examined with a digital US scanner ATL-HDI5000, linear 5-1 to 2-MHz broadband probe, compound imaging technique and analogic-digital zooming. We obtained one to three progressive measurements for each vein wall (total 67 parietal thicknesses). The samples, fixed in formalin, were sent to the pathology laboratory: sections were obtained at the same level of the sonographic planes, and images were obtained by digital camera mounted on an optical microscope. Measurements obtained at histology were considered as the gold standard. K-statistic was applied to compare sonographic and histologic measurements.Considering only the hypoechoic wall portion, 29/29 (100%) diagnoses of hypotrophy (K=0.91), 19/22 (86%) diagnoses of normotrophy (K=0,47) and 12/16 (75%) diagnoses of hypertrophy (K=0.7) were obtained by sonography.In our preliminary experience, the in vitro study of varicose veins allows precise, at least morphological, detection of hypotrophic walls. If these preliminary data are confirmed in vivo, sonography could be used to discriminate patients eligible for conservative treatment instead of surgery.

Background The role of rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP) in the response to treatment for rheumatoid arthritis (RA) is not fully understood. Studies investigating changes in the levels of RF in response to synthetic and biological DMARDs have not been able to confirm a definitive relationship between decreased RF subtypes and clinical response, while studies investigating changes in anti-CCP levels have yielded conflicting results. In addition, to our knowledge the correlation between autoantibodies and tocilizumab treatment has been poorly investigated. Objectives This study investigates the relationship between the presence and levels of RF and anti-CCP and clinical response to tocilizumab in patients with RA. Methods This was an observational longitudinal study in 27 patients with active, long-standing RA despite previous treatment with >2 DMARDs and/or steroids. Patients were treated with tocilizumab 8 mg/kg every 4 weeks. All patients were assessed using approved clinical scales (VES, PCR, HAQ, DAS28-VES, DAS28-PCR, CDAI, and SDAI). IgM-, IgA- and IgG-RFs and anti-CCP antibodies were assessed using ELISA at baseline, 3 months (T1), 6 months (T2), and 12 months (T3). Results All patients showed significant and sustained clinical response to tocilizmuab treatment. All clinical scales with the exception of HAQ significantly decreased. There was a significant correlation (p=0.03) between anti-CCP and SDAI changes from baseline at T1 and T2. Likewise there were no significant correlations between antibody count at T0 and changes in the DAS-28 VES at T1 and at T2. No significant relationship between clinical scales and antibody levels RF-IgG, IgA, IgM and anti-CCP levels were observed. Conclusions Tocilizumab is effective in treating the clinical symptoms of RA, and the efficacy of this molecule was not correlated with either RF or anti-CCP levels. There is now a growing body of evidence suggesting that markers associated with clinical response may not be the same biomarkers that predict risk of further joint damage References MIKULS TR, O’DELL JR, STONER JA, ET AL: Association of rheumatoid arthritis treatment response and disease duration with declines in serum levels of IgM rheumatoid factor and anti-cyclic citrullinated peptide antibody. Arthritis Rheum 2004; 50: 3776–82. KARSDAL MA, WOODWORTH T, HENRIKSEN K, ET AL: Biochemical markers of ongoing joint damage in rheumatoid arthritis--current and future applications, limitations and opportunities. Arthritis Res Ther 2011; 13: 215 TAKEUCHI T, TANAKA Y, AMANO K, ET AL: Clinical, radiographic and functional effectiveness of tocilizumab for rheumatoid arthritis patients-REACTION 52-week study. Rheumatology 2011; 50: 1908–15. Disclosure of Interest None Declared

An investigation was performed in order to evaluate the presence as polluting agents of chlorinated hydrocarbons in complex cosmetic matrices. Chlorinated pesticides, such as Dieldrin, alpha-Endosulfan, Heptachlor, alpha-Hexachlorocyclohexane (alpha-HCH), Lindane and polychlorinated biphenyls (PCBs) such as Aroclor 1254, were studied in two ingredients and in three finished commercial products. The analytical procedure consists of extraction, extract clean-up, separation of pesticides from PCBs, identification and measurement. Analysis was performed by gas chromatography with electron-capture detector, identity of pesticides and PCBs was assumed from retention times and quantification was based on peak-height measurements.

The control of vascular function is a central issue in hypertension, where total peripheral resistance is increased, implying a state of relative vasoconstriction.

The aortic muscle of spontaneously hypertensive (SHR) and normotensive control (WKY) rats was stimulated with phorbol esters and the contractile response was measured as isometric tension. Phorbol esters are known activators of protein kinase C. The aortas of SHR were characterized by the following distinct alterations in the response to phorbol myristate acetate: (1) increased sensitivity: half-maximal force was achieved at 62 +/- 6 nmol/l phorbol myristate acetate in SHR and 105 +/- 8 nmol/l in WKY; (2) increased contractility: the maximal force developed by phorbol myristate acetate was greater in SHR aortas (1.9 +/- 0.3 versus 1.6 +/- 0.2 g in WKY) compared with the decreased contractility generated with noradrenaline and high levels of potassium; (3) decreased dependency on extracellular calcium for half-maximal tension: in the presence of 3 mumol/l phorbol myristate acetate 50% of maximal force was attained at 21 +/- 8 mumol/l extracellular calcium compared with 49 +/- 9 mumol/l in WKY; (4) diminished relaxation in response to excess extracellular calcium: phorbol myristate acetate-precontracted WKY aortas began to relax when calcium was raised above 4 mmol/l in the bath and relaxation reached 51% at 8-10 mmol/l. Relaxation was almost absent in SHR (3-7%). Hence, there is an abnormality in the response to protein kinase C activation by phorbol esters in SHR vascular smooth muscle. Intracellular calcium appears to be involved. Studies of protein kinase C will prove important in understanding vascular smooth muscle function in normal and abnormal states.

Spontaneously hypertensive rats have long been used as an animal counterpart of human essential hypertension. The validation of this strain as a model rests mainly on the "clinical" similarity of the two syndromes, but it has scarcely been founded on numerical comparison of measurable parameters. We investigated three hematological indexes previously recognized to be altered in spontaneously hypertensive rats: the single-cell volume of erythrocytes, the single-cell volume of platelets, and the erythrocyte number. Erythrocyte volume was lower by 7%, platelet volume was higher by 12%, and erythrocyte count was higher by 22% in spontaneously hypertensive rats in comparison with Wistar-Kyoto controls. More unexpectedly, it was found that erythrocyte volume is lower by 2%, platelet volume is higher by 3%, and erythrocyte number is higher by 6% in essential hypertensive subjects when compared with normotensive healthy subjects. These results, combined with previously reported blood cell alterations in subjects and rats, reinforce the evidence of a biological similarity between essential and spontaneous hypertension.

The intracellular Ca indicator fura-2 was used for simultaneous measurements of intracellular free Ca (Ca2+i) and force in arterial smooth muscle. Rat aortic medial rings were submitted to fluorometry in a geometrical arrangement resembling that of adherent cell layers. A rigid force-transducing system served to immobilize the tissue and record the developed force quasi-isometrically. Stimulation was performed with norepinephrine (NE), KCl depolarization (high K), and a nonfluorescent Ca ionophore (ionomycin) at varying extracellular Ca concentrations. The following facts were observed. NE, high K, and ionomycin increased tension along with fura-2-reported Ca2+i; under any circumstances tension was Ca2+i dependent and could be varied by manipulating Ca2+i. However, NE and high K determined a parallel increase in the effectiveness of Ca2+i in comparison with the simple ionophore, i.e., they increased the force-to-Ca2+i ratio. NE and high K produced half-maximal tension at fura-2 estimated Ca2+i of 0.10 and 0.13 microM, whereas ionomycin required 0.6 microM to achieve the same amount of force. It is inferred that Ca2+i is a determinant of vascular contraction, but some results suggest the existence of factors that sensitize the contractile machinery to Ca.

The effect of synthetic human atrial natriuretic factor [ANF-(99-126)] on human omental arteries was investigated. The compound produced concentration-dependent relaxation, 10, 50 and 90% of maximum effect being observed at about 1, 10 and 100 nmol/l, respectively. These concentrations are considerably higher than those measured by radio-immunoassay in human plasma, even under extreme conditions (0.001-0.3 nmol/l). Therefore, ANF can dilate human resistance-size arteries, but whether it does so under physiological conditions has not been established.

Diminished beta-adrenoceptor mediated relaxation and increased reactivity to norepinephrine have been observed in the arteries of spontaneously hypertensive rats (SHR). However, some reports are conflicting, while the complexity of endothelial, neural and purely morphological factors regulating the response of whole vessels is increasingly appreciated. Thus a dissection of the complex vascular contraction mechanism into single-step events is needed. Here characterization of adrenoceptors in isolated aortic smooth muscle cells of SHR by radioligand binding techniques is reported. The density of beta-adrenoceptors was decreased at 4 (-23%), 7(-20%) and 13(-39%) weeks in SHR compared with age-matched normotensive Wistar-Kyoto rats. In both strains beta-adrenoceptor density decreased significantly with age. alpha 1-Adrenoceptors exhibited complex, though slight, changes in SHR at different ages. The alpha/beta adrenoceptor density ratio was constantly increased in SHR (11.9 versus 7.6 at 7 weeks; 19.1 versus 15.8 at 13 weeks). These findings suggest that the abnormal contractility of SHR arteries may at least partially depend on changes at the adrenoceptor level. A loss of arterial beta-adrenoceptors early in the life of SHR can be an event leading to increased peripheral resistance and elevation of blood pressure.

H3-dihydroalprenolol (H3-DHA) binding in isolated smooth muscle cells has been studied. Two different binding sites have been detected: a high affinity (highly specific) and a low affinity (nonspecific) binding site. Local anaesthetics are able to displace H3-DHA from nonspecific sites with a rank order of potency that mirrors their local anaesthetic activity. This finding correlates, at the molecular level, with the local anaesthetic properties of beta-blockers, as concluded from classical pharmacological experiments.

1. The cytoplasmic free calcium concentration ([Ca2+]i) was assessed with the fluorescent dye Quin 2 in platelets and lymphocytes of spontaneously hypertensive rats (SHR), normotensive Wistar—Kyoto rats (WKY), essential hypertensive patients (EHP) and normotensive human control subjects (NCS). 2. [Ca2+]i was significantly higher in the platelets of 8- and 20-week-old SHR in comparison with WKY. However, no difference was evident after weaning. Changes of cellular calcium in hypertensive rats apparently evolved simultaneously with the development of high arterial pressure. 3. [Ca2+]i was significantly higher in platelets of EHP than in NCS. 4. In lymphocytes of SHR, [Ca2+]i was not different from WKY at 4 and 8 weeks, but was increased at 14 weeks and at older ages. In EHP, intralymphocytic [Ca2+] was only modestly higher than in controls. On the whole, the results suggest that control of cytoplasmic calcium in these blood cells is similarly affected in human and animal models of primary hypertension.

Cytoplasmic free calcium (Ca2+i) is increased in platelets and lymphocytes of spontaneously hypertensive rats (SHR) and, to a lesser extent, in essential hypertensive patients. In this study, a method was devised to evaluate cellular Ca fluxes and the cellular Ca buffering power by means of the intracellular Ca indicator Quin-2. Ca influx was measured under conditions of Ca-pump inhibition, either by vanadate or ATP depletion. Lymphocytes of 5-month-old SHR were compared with those of normotensive Wistar-Kyoto rats (WKY). In both strains, the Ca entry rate and the cellular buffering capacity were higher in vanadate-treated than in ATP-depleted cells. SH rats exhibited a higher Ca influx and a greater intracellular Ca buffering power when vanadate was used to stop the Ca pump; these differences, however, were abolished in ATP-depleted lymphocytes. It is suggested that Ca entry in lymphocytes is ATP-dependent. Accelerated Ca entry in SHR can account for the previously reported higher levels of intracellular free Ca.

It is scarcely necessary to stress the importance of adrenoceptor regulation in arterial hypertension. In this disease an altered response to adrenergic agonists has been described in different organs, and particularly in the blood vessels [1–6]. Moreover, antiadrenergic drugs, and particularly alpha and beta blockers, are effective antihypertensive agents.

In order to explore a previously observed association between raised blood cell cytosolic calcium [Ca2+]i and primary hypertension, resting levels of [Ca2+]i in the Milan hypertensive rat strain (MHS) were measured. The [Ca2+]i was increased significantly at 7 weeks in MHS (116 +/- 8 versus 93 +/- 6 mumol/l, P0.005, n = 7) and non-significantly at 5 weeks (109 +/- 7 versus 85 +/- 9 mumol/l, n = 5). In Okamoto-Aoki spontaneous hypertensive rats (SHR), calcium fluxes were measured with Quin 2 in platelets and lymphocytes. Calcium influx was measured during calcium-pump inhibition either with vanadate or ATP-depletion. Influx was increased in vanadate-treated (but not in ATP-depleted) cells of SHR. This suggests an ATP dependence of calcium influx in blood cells which is more pronounced in SHR. Calcium efflux was determined when the influx was stopped by external EGTA. Calcium efflux was [Ca2+]i-dependent and was moderately increased in SHR. The faster efflux in SHR was due to higher [Ca2+]i. The [Ca2+]i-dependency of calcium efflux was comparable in SHR and Wistar-Kyoto (WKY) rats. These results suggest that a slight but significant rise of cytosolic calcium occurs in blood cells in primary hypertension. In Okamoto-Aoki SHR this rise was due to a higher calcium entry rate.