Sarcoglycanopathies: a multiplex molecular analysis for the most common mutations.

Sarcoglycanopathies (SGpathies) are highly frequent among severely affected limb-girdle muscular dystrophy patients. On the basis of the findings of 5 common mutations in the 4 sarcoglycan (SG) genes in the Brazilian population, we standardized a multiplex polymerase chain reaction-single-strand conformation polymorphism methodology for their concomitant analysis in DNA samples. The test was able to confirm the diagnosis in about 63% of new patients with a suspected SGpathy and was particularly important in patients in advanced stages of the disease, when obtaining a muscle biopsy for analysis may be very difficult. As common mutations have been described in several countries, this multiplex analysis could be useful for the diagnosis of SGpathies if established according to the most prevalent mutations in each population. Besides, even though the disorder studied is rare, the technique could be broadly applicable to other genes and disorders.     

Challenge tests of monoaminergic systems: neurophysiological aspects.

Monoaminergic challenge tests allow investigating central nervous changes in humans under acute depletion of specific neurotransmitters (5-HT, DA, NE). Along with studies using alpha-methyl-para-tyrosine test (AMPT) and phenylalanine/tyrosine depletion test (APTD), the tryptophan depletion test (ATDT) represents the currently most established human challenge tool for the assessment of brain serotonin functioning. Neurophysiological studies in healthy and clinical samples may contribute to the search for a non-invasive and reliable biological marker of monoaminergic vulnerability or dysfunction. In the design of these studies, various biochemical and methodological aspects have to be taken into account. This article focuses on electrophysiological methodology and results of monoamine depletion studies (i.e., electroencephalography, magnetoencephalography, polysomnography, auditory evoked potentials and startle response).     

The angiotensin II type 1-receptor blocker candesartan increases cerebral blood flow, reduces infarct size, and improves neurologic outcome after transient cerebral ischemia in rats.

The goal of the present study was to test the impact of administration time of the angiotensin II type 1-receptor blocker candesartan on cerebral blood flow (CBF), infarct size, and neuroscore in transient cerebral ischemia. Therefore, 1-hour middle cerebral artery occlusion (MCAO) was followed by reperfusion. Rats received 0.5-mg/kg candesartan intravenously 2 hours before MCAO (pretreatment), 24 hours after MCAO, every 24 hours after MCAO, or 2 hours before and every 24 hours after MCAO. Infarct size (mm3) and a neuroscore at day 7 were compared with controls. CBF was quantified by radiolabeled microspheres and laser-Doppler flowmetry. Compared with controls (95 +/- 8), infarct size in candesartan-treated groups was smaller (59 +/- 5, 68 +/- 10, 28 +/- 3, and 15 +/- 3, respectively; P<0.05). Although there was no difference in neuroscore between pretreatment and controls (1.55 +/- 0.18, 1.80 +/- 0.13), other treatment regimens resulted in improved neuroscores (1.33 +/- 0.16, 1.11 +/- 0.11, 0.73 +/- 0.15; P<0.05). CBF in pretreated animals at 0.5 hours after MCAO was significantly higher than in controls (0.58 +/- 0.09 mL x g(-1) x min(-1) and 44% +/- 7% of baseline compared with 0.49 +/- 0.06 mL x g(-1) x min(-1) and 37% +/- 6%, microspheres and laser-Doppler flowmetry; P<0.05). Thus, candesartan reduces infarct size even if administered only during reperfusion. Apart from pretreatment, other treatment regimens result in significantly improved neuroscores. In the acute phase of cerebral ischemia, candesartan increases CBF.     

Anterior Cruciate Ligament Reconstruction: State of the Art

Abstract The rupture of the Anterior cruciate ligament (ACL) belongs to the most common ligament injuries of the human knee joint. ACL rupture results in an increased anterior translation and internal rotation of the tibia. Untreated knee instability causes a disintegration of the roll and sliding movement and a high incidence of secondary meniscus and chondral damages with consecutive or advanced arthritic changes. For deciding on a conservative or operative therapy, it is necessary to develop a high-risk profile. Elderly, inactive patients without instability symptoms can be treated conservatively; younger, active people and complex ligament injuries should receive an ACL replacement. The goal is to eliminate instability by maintaining the physiological kinematics of the knee. Anterior cruciate ligament may be reconstructed arthroscopically assisted by autologous tendons. Predominantly, hamstring- and bone-patellar-tendon grafts are used. No significant differences in knee laxity, clinically and functionally, were observed between both grafts. Various reconstruction techniques, single- or double-bundle techniques, were described. Successful replacement depends on a correct tunnel placement and reconstruction of the physiological band tension, a sufficient mechanical stability of fixation, an impingement-free range of motion and an adequate rehabilitation. A high degree of patient satisfaction in clinical and functional outcome could be evaluated.     

In vivo anti-melanoma activities of the Melan-A/MART-1101–115 T CD4+ cell peptide

Malignant melanoma causes significant health problems. The identification of tumour-associated antigens has led to novel approaches to increase T cell mediated anti-tumour immune response. Melan-A/MART-1 has been use as target antigen for several T cell based immunotherapeutic treatments. More recently, the critical role of CD4+ T cells in inducing and maintaining anti-tumour immunity has been increasingly recognized. In order to optimize tumour immunotherapy, greater efforts have been concentrated on the identification of tumour antigens presented by MHC class II molecules to CD4+ T cells. In a publication, Tiwari et al. (2004) [1] have identified by a computational approach the 15-mer amino-acid sequence 101–115 (PPAYEKLSAEQSPPP) of the Melan-A/MART-1 as a good target for a vigorous and safe immunotherapy. Therefore, we have investigated the in vivo anti-tumour activity of this peptide in a murine melanoma model. For the prophylactic treatment, 20 μg or 50 μg peptide was subcutaneously injected in mice once a week during 3 weeks before tumour induction. Treatment with 50 μg peptide significantly affected tumour development. Thus, our preliminary data demonstrate potential in vivo prophylactic activity of the 101–115 peptide-based vaccine to control melanoma growth.     

Actin-free Gc globulin: a rapidly assessed biomarker of organ dysfunction in acute liver failure and cirrhosis.

Reductions in serum levels of Gc globulin, a hepatically synthesized component of the extracellular actin scavenger system responsible for complexing circulating actin and attenuating intravascular microthrombus formation, are associated with poor outcome in acute liver failure. Clinically applicable assays of the important actin-free fraction (Af-Gc) have not been available until now. We measured actin-free Gc globulin levels with a novel, rapid assay in 61 cases of acute liver failure (ALF) and in 91 patients with cirrhosis (40 of whom were clinically unstable with extrahepatic organ dysfunction), and studied associations with liver dysfunction, extrahepatic organ dysfunction, indices of disseminated coagulation, and outcome. Reductions in Af-Gc levels mirrored hepatic dysfunction and organ dysfunction in both groups, and discriminated patients with poor prognosis from those with good prognosis in the ALF cohort. Levels were lowest in patients with ALF (10% of control values), but levels were also markedly reduced in both unstable (28%) and stable (44%) patients with cirrhosis. Associations with markers of disseminated intravascular coagulation were seen in both groups, most notably in the cirrhosis cohort, supporting a pathophysiological role for reduced Af-Gc in the evolution of organ dysfunction. In acetaminophen-induced ALF, Af-Gc identified patients with poor prognosis as well as did the Acute Physiology and Chronic Health Evaluation (APACHE II) score (area under the receiver operating characteristic curve, 0.7), and in cirrhosis, Af-Gc was an independent predictor of mortality by multifactorial analysis. In conclusion, the importance of Af-Gc reductions in the development of multiple organ dysfunction in ALF and cirrhosis is highlighted, probably resulting from reduced hepatic production and peripheral exhaustion of this arm of the extracellular actin scavenger system.     

Relationship between oxidative stress and essential hypertension.

This study investigated the association of blood pressure with blood oxidative stress-related parameters in normotensive and hypertensive subjects. A cross-sectional design was applied to 31 hypertensive patients and 35 healthy normotensive subjects. All subjects were men between the ages of 35 and 60 years. Exclusion criteria were obesity, dyslipidemia, diabetes mellitus, smoking and current use of any medication. All patients underwent 24-h ambulatory blood pressure monitoring and sampling of blood and urine. Antioxidant enzymes activity, reduced/oxidized glutathione ratio (GSH/GSSG), and lipid peroxidation (malondialdehyde) were determined in erythrocytes. Parameters measured in the plasma of test subjects were plasma antioxidant status, lipid peroxidation (8-isoprostane), plasma vitamin C and E, and the blood pressure modulators renin, aldosterone, endothelin-1 and homocysteine. Daytime systolic and diastolic blood pressures of hypertensives were negatively correlated with plasma antioxidant capacity (r=-0.46, p<0.009 and r=-0.48, p<0.007), plasma vitamin C levels (r=-0.53, p<0.003 and r=-0.44, p<0.02), erythrocyte activity of antioxidant enzymes, and erythrocyte GSH/GSSG ratio, with hypertensives showing higher levels of oxidative stress. Blood pressures showed a positive correlation with both plasma and urine 8-isoprostane. Neither plasma vitamin E nor the assessed blood pressure modulator levels showed significant differences between the groups or correlation with blood pressures. These findings demonstrate a strong association between blood pressure and some oxidative stress-related parameters and suggest a possible role of oxidative stress in the pathophysiology of essential hypertension.     

Human antibody against C domain of tenascin-C visualizes murine atherosclerotic plaques ex vivo.

Targeting proteins that are overexpressed in atherosclerotic plaques may open novel diagnostic applications. The C domain of tenascin-C is absent from normal adult tissues but can be inserted during tumor progression or tissue repair into the molecule by alternative splicing. We tested the ability of the human antibody G11, specific to this antigen, to reveal murine atherosclerotic plaques ex vivo. The antibody directed against the extra domain B of fibronectin (L19) was used as a reference. METHODS: We intravenously injected (125)I-labeled G11 or L19 antibodies into apolipoprotein E-deficient (ApoE(-/-)) mice and harvested the aortae 4 or 24 h later. En face analyses of distal aortae and longitudinal sections of the aortic arch were performed to compare antibody uptake using autoradiography with plaque staining using oil red O. Plaque macrophages were detected by immunohistochemistry (anti-CD68 staining). Biodistribution of injected antibodies was investigated in aortae and blood at 4 and 24 h. RESULTS: En face analyses revealed a significant correlation between radiolabeled G11 and fat-stained areas, increasing from 4 to 24 h, with a correlation coefficient of 0.92 (P < 0.0001) and an average signal-to-noise ratio of 104:1 at 24 h. Plaque imaging using L19 showed similar results (r = 0.86; P < 0.0001; signal-to-noise ratio, 72:1 at 24 h). Uptake of radiolabeled antibodies in histologic sections colocalized with fat staining and activated macrophages in aortic plaques. Biodistribution analyses confirmed specific accumulation in aortic plaques as well as rapid blood pool clearance of the antibodies 24 h after injection. Immunofluorescence analyses revealed increased expression of tenascin and fibronectin isoforms in macrophage-rich plaques. CONCLUSION: The antibody G11, specific to the C domain of tenascin-C, visualizes murine atherosclerotic plaques ex vivo. In conjunction with the increased expression of the C domain of tenascin-C in macrophage-rich plaques, the colocalization of G11 uptake with activated macrophages, and the favorable target-to-blood ratio at 24 h, this antibody may be useful for molecular imaging of advanced atherosclerotic plaques in the intact organism.