Limb-girdle myopathy and mild intellectual disability: The expanding spectrum of TANGO2-related disease.

TANGO2-related disease is an autosomal recessive multisystem disease associated with developmental delay and infancy-onset recurrent metabolic crises with early mortality. Several studies have reported dysfunction in endoplasmic reticulum-to-Golgi traffic and mitochondrial homoeostasis as the underlying pathophysiology. We report a 40-year-old woman affected by limb-girdle weakness and mild intellectual disability caused by the recurrent deletion of exons 3-9 in homozygosity in the TANGO2 gene. Physical examination revealed hyperlordosis, waddling gait, calf pseudohypertrophy, and Aquilian tendon retractions. Laboratory investigations revealed elevation of serum biomarkers suggestive of mitochondrial dysfunction together with hypothyroidism. At the age of 24, the patient suffered a metabolic crisis with severe rhabdomyolysis and malignant cardiac arrhythmia. After recovery, no metabolic or arrhythmic crisis has recurred. Muscle histology two years later revealed increased endomysial fibrosis and other myopathic changes. Our findings illustrate the mildest end of the phenotypic spectrum of TANGO2-related disease and reveal further aspects related to chronic muscle damage in this disorder.

Identification of two novel RRM2B variants associated with autosomal recessive progressive external ophthalmoplegia in a family with pseudodominant inheritance pattern.

RRM2B encodes the p53-inducible small subunit (p53R2) of ribonucleotide reductase, a key protein for mitochondrial DNA (mtDNA) synthesis. Pathogenic variants in this gene result in familial mitochondrial disease in adults and children, secondary to a maintenance disorder of mtDNA. This study describes two patients, mother and son, with early-onset chronic progressive external ophthalmoplegia (PEO). Skeletal muscle biopsy from the latter was examined: cytochrome c oxidase (COX)-negative fibres were shown, and molecular studies revealed multiple mtDNA deletions. A next-generation sequencing gene panel for nuclear-encoded mitochondrial maintenance genes identified two unreported heterozygous missense variants (c.514 G > A and c.682 G > A) in the clinically affected son. The clinically affected mother harboured the first variant in homozygous state, and the clinically unaffected father harboured the remaining variant in heterozygous state. In silico analyses predicted both variants as deleterious. Cell culture studies revealed that patients' skin fibroblasts, but not fibroblasts from healthy controls, responded to nucleoside supplementation with enhanced mtDNA repopulation, thus suggesting an in vitro functional difference in patients' cells. Our results support the pathogenicity of two novel RRM2B variants found in two patients with autosomal recessive PEO with multiple mtDNA deletions inherited with a pseudodominant pattern.

HLA-DQB1*05:02, *05:03, and *03:01 alleles as risk factors for myasthenia gravis in a Spanish cohort.

BACKGROUND: Myasthenia gravis (MG) is a very heterogenic chronic autoimmune disease caused by the failure of neuromuscular transmission. The HLA gene complex has conventionally been recognized as its main genetic risk and phenotype modifying factor. Our aim was to investigate the prevalence of HLA class I and II alleles and to identify possible risk factors for sporadic MG in a Spanish cohort. METHODS: We designed a clinical case-control study comparing HLA alleles and haplotype frequencies in a cohort of 234 patients with sporadic autoimmune MG with data from a group of 492 randomly selected healthy subjects. Using a high-resolution next-generation sequencing (NGS)-based HLA genotyping assay, we investigated the contribution of HLA genotypes and haplotypes in the resulting phenotype, especially, the age at onset, sex, onset MGFA class, thymic histopathology, and serological status. RESULTS: We found that the DQB1*05:02 and DQB1*05:03 alleles could be novel risk factors for Spanish MG cases. The HLA alleles A*01:01, B*08:01, DRB1*03:01, DRB1*14:54, and DQB1*02:01 were also risk factors for the disease. DQB1*03:01 acted as a risk factor for EOMG in women with AChR-positive antibodies and thymus hyperplasia. Additionally, several alleles were identified as potential phenotype-modifying factors that could exert a protective effect: HLA-B*35:08, DRB1*13:01, and DQB1*06:03 in MG; HLA-A*24:02 in women and DRB1*07:01 and DQB1*02:02 for early onset. HLA-C*07:01 and haplotype A1-B8-C7-DR3-DQ2 were associated with an early-onset phenotype.

Cardioprotective Properties of Phenolic Compounds: A Role for Biological Rhythms.

Cardiovascular diseases (CVD) are the leading cause of deaths worldwide and their prevalence is continuously increasing. Available treatments may present several side effects and therefore the development of new safer therapeutics is of interest. Phenolic compounds have shown several cardioprotective properties helpful in reducing different CVD risk factors such as inflammation, elevated blood pressure, hyperlipidemia, or endothelial dysfunction. These factors are significantly influenced by biological rhythms which are in fact emerging as key modulators of important metabolic and physiological processes. Thus, increased events of CVD have been observed under circadian rhythm disruption or in winter versus other seasons. These rhythms can also affect the functionality of phenolic compounds. Indeed, different effects have been observed depending on the administration time or under different photoperiods. Therefore, in this review the focus will be on the potential of phenolic compounds as therapeutics to prevent CVD via biological rhythm modulation.

Time-of-Day Circadian Modulation of Grape-Seed Procyanidin Extract (GSPE) in Hepatic Mitochondrial Dynamics in Cafeteria-Diet-Induced Obese Rats.

Major susceptibility to alterations in liver function (e.g., hepatic steatosis) in a prone environment due to circadian misalignments represents a common consequence of recent sociobiological behavior (i.e., food excess and sleep deprivation). Natural compounds and, more concisely, polyphenols have been shown as an interesting tool for fighting against metabolic syndrome and related consequences. Furthermore, mitochondria have been identified as an important target for mediation of the health effects of these compounds. Additionally, mitochondrial function and dynamics are strongly regulated in a circadian way. Thus, we wondered whether some of the beneficial effects of grape-seed procyanidin extract (GSPE) on metabolic syndrome could be mediated by a circadian modulation of mitochondrial homeostasis. For this purpose, rats were subjected to "standard", "cafeteria" and "cafeteria diet + GSPE" treatments (n = 4/group) for 9 weeks (the last 4 weeks, GSPE/vehicle) of treatment, administering the extract/vehicle at diurnal or nocturnal times (ZT0 or ZT12). For circadian assessment, one hour after turning the light on (ZT1), animals were sacrificed every 6 h (ZT1, ZT7, ZT13 and ZT19). Interestingly, GSPE was able to restore the rhythm on clock hepatic genes (Bmal1, Per2, Cry1, Rorα), as this correction was more evident in nocturnal treatment. Additionally, during nocturnal treatment, an increase in hepatic fusion genes and a decrease in fission genes were observed. Regarding mitochondrial complex activity, there was a strong effect of cafeteria diet at nearly all ZTs, and GSPE was able to restore activity at discrete ZTs, mainly in the diurnal treatment (ZT0). Furthermore, a differential behavior was observed in tricarboxylic acid (TCA) metabolites between GSPE diurnal and nocturnal administration times. Therefore, GSPE may serve as a nutritional preventive strategy in the recovery of hepatic-related metabolic disease by modulating mitochondrial dynamics, which is concomitant to the restoration of the hepatic circadian machinery.

Efficacy of therapeutic lifestyle changes on lipid profiles assessed by NMR in children with familial and non-familial hypercholesterolemia.

BACKGROUND AND AIMS: The first line of therapy in children with hypercholesterolaemia is therapeutic lifestyle changes (TLSC). The efficacy of lifestyle intervention in children with familial hypercholesterolaemia (FH), where LDL-C levels are genetically driven, deserves a focused study. AIMS: To evaluate the impact of a lifestyle education program, focused on food patterns and physical activity, on lipid profiles assessed by nuclear magnetic resonance (NMR) in children with FH vs. non-FH. METHODS: Phase 1 was a cross-sectional study of baseline characteristics, and phase 2 was a prospective TLSC intervention study. In total, the study included 238 children (4 to 18 years old; 47% girls) attending the lipid unit of our hospital due to high cholesterol levels. Eighty-five were diagnosed with FH (72% genetic positive), and 153 were diagnosed with non-Familial hypercholesterolaemia. A quantitative food frequency questionnaire (FFQ) including 137 items was used. Physical activity (PA) was assessed by the Minnesota questionnaire. The lipid profile was assessed using the 2D-1H-NMR (Liposcale test). A total of 127 children (81 in the FH group) participated in the prospective phase and were re-assessed after 1 year of the TLSC intervention, consisting of education on lifestyle changes delivered by a specialized nutritionist. RESULTS: The FH and non-FH groups were similar in anthropometry and clinical data, except that those in the FH were slightly younger than those in the non-FH group. Both the FH and non-FH groups showed a similar diet composition characterized by a high absolute calorie intake and a high percentage of fat, mainly saturated fat. The PA was below the recommended level in both groups. After one year of TLSC, the percentage of total and saturated fats was reduced, and the amount of fiber increased significantly in both groups. The percentage of protein increased slightly. The number of children engaged in at least 1 hour/day of PA increased by 56% in the FH group and by 53% in the non-FH group, and both these increases were significant. The total and small-LDL particle numbers were reduced in both groups, although the absolute change was greater in the FH group than in the non-FH group. CONCLUSIONS: Educational strategies to implement TLSC in children lead to empowerment, increased adherence, and overall metabolic improvement in children with high blood cholesterol, including those with FH.

The Immune Response in Nonmetastatic Axillary Lymph Nodes Is Associated with the Presence of Axillary Metastasis and Breast Cancer Patient Outcome.

Tumor cells can modify the immune response in primary tumors and in the axillary lymph nodes with metastasis (ALN+) in breast cancer (BC), influencing patient outcome. We investigated whether patterns of immune cells in the primary tumor and in the axillary lymph nodes without metastasis (ALN-) differed between patients diagnosed without ALN+ (diagnosed-ALN-) and with ALN+ (diagnosed-ALN+) and the implications for clinical outcome. Eleven immune markers were studied using immunohistochemistry, tissue microarray, and digital image analysis in 141 BC patient samples (75 diagnosed-ALN+ and 66 diagnosed-ALN-). Two logistic regression models were derived to identify the clinical, pathologic, and immunologic variables associated with the presence of ALN+ at diagnosis. There are immune patterns in the ALN- associated with the presence of ALN+ at diagnosis. The regression models revealed a small subgroup of diagnosed-ALN+ with ALN- immune patterns that were more similar to those of the ALN- of the diagnosed-ALN-. This small subgroup also showed similar clinical behavior to that of the diagnosed-ALN-. Another small subgroup of diagnosed-ALN- with ALN- immune patterns was found whose members were more similar to those of the ALN- of the diagnosed-ALN+. This small subgroup had similar clinical behavior to the diagnosed-ALN+. These data suggest that the immune response present in ALN- at diagnosis could influence the clinical outcome of BC patients.

Intravenous immunoglobulin to prevent myasthenic crisis after thymectomy and other procedures can be omitted in patients with well-controlled myasthenia gravis.

BACKGROUND: Myasthenic crisis (MC) is a potentially life-threatening complication of myasthenia gravis. Its precipitating factors include surgical procedures, particularly thymectomy. The role of preoperative intravenous immunoglobulin (IVIg) in preventing MC in patients scheduled for thymectomy and other surgery with general anaesthesia is unknown. Our objective was to test the hypothesis that preoperative IVIg is effective in preventing myasthenic crisis in patients with myasthenia gravis scheduled for surgery under general anaesthesia, including thymectomy. METHODS: A prospective, randomized, double-blind, single-centre study was conducted over a 4-year period. The treatment group received IVIg, 0.4 g/kg/day preoperatively for 5 consecutive days, and the placebo group received saline solution under the same conditions. The two groups were age-matched, with similar functional status, and Myasthenia Gravis Foundation of America class. All patients had well-controlled myasthenia gravis with minimal manifestations before surgery. The primary outcome measured was MC. Intubation times, time in the recovery room, number of postoperative complications, and days of hospitalization were the secondary outcomes measured. RESULTS: A total of 47 patients were randomized, 25 to the IVIg group and 22 to placebo. There were 19 men and 28 women, with a mean age of 58.6 years, mean body mass index of 27.8 kg/m2, and mean acetylcholine receptor antibodies of 12.9 nmol/l. The mean forced vital capacity was 84.4%. The mean quantitative myasthenia gravis sum score was 6.3. Ten patients (five in each arm) had a history of MC. Thymectomy was performed in 16 patients. Only one patient in the placebo group presented with MC requiring non-invasive ventilation (but no reintubation) for 6 days. Neither differences between groups in the univariate analysis nor risk factors for MC in the multivariate analysis were found. CONCLUSIONS: Preoperative IVIg to prevent MC does not appear to be justified in well-controlled myasthenia gravis patients. This study provides class I evidence that preparation with IVIg to prevent MC is not necessary in well-controlled myasthenia gravis patients scheduled for surgery with general anaesthesia.

Transthyretin stabilization activity of the catechol-O-methyltransferase inhibitor tolcapone (SOM0226) in hereditary ATTR amyloidosis patients and asymptomatic carriers: proof-of-concept study.

Objective: To assess the transthyretin (TTR) stabilization activity of tolcapone (SOM0226) in patients with hereditary ATTR amyloidosis, asymptomatic carriers and healthy volunteers. Methods: A phase IIa proof-of-concept trial included two phases separated by a 6-week washout period. Phase A: single 200 mg dose of tolcapone; phase B: three 100 mg doses taken at 4 h intervals. The primary efficacy variable was TTR stabilization. Results: Seventeen subjects were included (wild type, n = 6; mutation TTR Val30Met, n = 11). TTR stabilization was observed in all participants. Two hours after dosing, 82% of participants in phase A and 93% of those in phase B reached a TTR stabilization value of at least 20%. In phase A, there was an increase of 52% in TTR stabilization vs baseline values 2 h after dosing, which decreased to 22.9% at 8 h. In phase B, there was a significant increase of 38.8% in TTR stabilization 2 h after the first 100 mg dose. This difference was maintained after 10 h and decreased after 24 h. No serious adverse events were observed. Conclusions: The ability of tolcapone for stabilizing TTR supports further development and repositioning of the drug for the treatment of ATTR amyloidosis. EudraCT trial number: 2014-001586-27 ClinicalTrials.gov Identifier: NCT02191826.

Intravenous immunoglobulin as monotherapy for myasthenia gravis during pregnancy.

INTRODUCTION: Pregnant women with myasthenia gravis (MG) are at increased risk of complications and adverse outcomes, including the teratogenic effects of many drugs used to treat MG women of childbearing age. The effectiveness of intravenous immunoglobulins (IVIg) on other autoimmune mediated diseases has been extensively reported in recent years, although little is known about the role of IVIg in the treatment of MG during pregnancy. We designed this study to determine the effectiveness of IVIg as monotherapy during pregnancy for women with MG. MATERIAL AND METHODS: Five pregnant MG patients (mean age at delivery 36.4years, SD 5.8, range 29.4-45.2) were studied in 2013-14. Their treatment was switched to monthly IVIg cycles 2months before the pregnancy. Follow-up included monthly neurological QMG throughout the pregnancy and postpartum, obstetrical monitoring during monthly visits in the first two trimesters of the pregnancy, fortnightly visits between week 32 and week 36, and weekly visits after 36weeks, and neonatal follow-up after delivery. RESULTS: We observed no exacerbations during pregnancy, delivery or post-partum. The mean QMG score at baseline (before pregnancy) was 7.4 points in five women with generalized forms of MG. The maximum mean value reached during pregnancy was 8.6 points. The mean pregnancy duration was 38 w+5 d. No infant with transient neonatal myasthenia gravis. CONCLUSIONS: These results suggest that monotherapy with IVIg during pregnancy in MG patients could be promising, although confirmation is required in studies with larger populations.

Influence of early neurological complications on clinical outcome following lung transplant.

BACKGROUND: Neurological complications after lung transplantation are common. The full spectrum of neurological complications and their impact on clinical outcomes has not been extensively studied. METHODS: We investigated the neurological incidence of complications, categorized according to whether they affected the central, peripheral or autonomic nervous systems, in a series of 109 patients undergoing lung transplantation at our center between January 1 2013 and December 31 2014. RESULTS: Fifty-one patients (46.8%) presented at least one neurological complication. Critical illness polyneuropathy-myopathy (31 cases) and phrenic nerve injury (26 cases) were the two most prevalent complications. These two neuromuscular complications lengthened hospital stays by a median period of 35.5 and 32.5 days respectively. However, neurological complications did not affect patients' survival. CONCLUSIONS: The real incidence of neurological complications among lung transplant recipients is probably underestimated. They usually appear in the first two months after surgery. Despite not affecting mortality, they do affect the mean length of hospital stay, and especially the time spent in the Intensive Care Unit. We found no risk factor for neurological complications except for long operating times, ischemic time and need for transfusion. It is necessary to develop programs for the prevention and early recognition of these complications, and the prevention of their precipitant and risk factors.

Study of the prevalence of familial autoimmune myasthenia gravis in a Spanish cohort.

BACKGROUND: Myasthenia gravis (MG) is an autoimmune disease caused by a failure of neuromuscular transmission. Familial clustering has been reported despiteMG usually manifesting as a sporadic condition presumed not to be inherited. Our study investigated the prevalence of FAMG in a Spanish cohort, characterizing their phenotype,antibody titres and thymus findings. MATERIAL/METHODS: We investigated the presence of familial cases in 462 MG patients, characterizing by age and MGFA class at debut, quantitative MG score, antibody titres, MGFA post-intervention status and thymus pathology. RESULTS: Sixteen cases from8 unrelated pedigrees were identified. The prevalence of FAMG caseswas 3.46%.Mean age at onset was 57.8 ± 17.4 years (range=23­82). Distribution at debut was: 6 ocular, 4 IIa, 4IIb, 1 IIIa and 1 IIIb. Thymoma was identified in two of the 7 thymectomized individuals. CONCLUSIONS: The prevalence of FAMG in Spain is similar to other populations. Post-intervention status did not differ from sporadic autoimmune MG. As in other neuromuscular disorders, phenotype and inheritance heterogeneity are present in FAMG. In addition to the interfamilial heterogeneity observed, members of the same family affected with FAMG may even present different ages of onset, severity and thymus involvement. Further studies are necessary to clarify the role of genetic risk factors in this form of autoimmune MG.

Autoimmune-like hepatitis during masitinib therapy in an amyotrophic lateral sclerosis patient.

We report a case of acute severe hepatitis resulting from masitinib in a young amyotrophic lateral sclerosis patient. Hepatotoxicity induced by masitinib, a tyrosine kinase inhibitor, is usually transient with mild elevation of transaminases, although acute hepatitis has been not reported to date. The hepatitis was resolved after masitinib was discontinued and a combination of prednisone and azathioprine was started. The transaminases returned to baseline normal values five months later. This is the first case in the hepatitis literature associated with masitinib. The autoimmune role of this drug-induced liver injury is discussed. Physicians should be aware of this potential complication.

Differential modulation of apoptotic processes by proanthocyanidins as a dietary strategy for delaying chronic pathologies.

Apoptosis is a biological process necessary for maintaining cellular homeostasis. Several diseases can result if it is deregulated. For example, inhibition of apoptotic signaling pathways is linked to the survival of pathological cells, which contributes to cancer, whereas excessive apoptosis is linked to neurodegenerative diseases, partially via oxidative stress. The activation or restoration of apoptosis via extrinsic or intrinsic pathways combined with cell signaling pathways triggered by reactive oxygen specises (ROS) formation is considered a key strategy by which bioactive foods can exert their health effects. Proanthocyanidins, a class of flavonoids naturally found in fruits, vegetables, and beverages, have attracted a great deal of attention not only because they are strong antioxidants but also because they appear to exert a different modulation of apoptosis, stimulating apoptosis in damaged cells, thus preventing cancer or reducing apoptosis in healthy cells, and as a result, preserving the integrity of normal cells and protecting against neurodegenerative diseases. Therefore, proanthocyanidins could provide a defense against apoptosis induced by oxidative stress or directly inhibit apoptosis, and they could also provide a promising treatment for a variety of diseases. Emerging data suggest that proanthocyanidins, especially those that humans can be persuaded to consume, may be used to prevent and manage cancer and mental disorders.

Grape seed proanthocyanidin extract improves the hepatic glutathione metabolism in obese Zucker rats.

SCOPE: Increased oxidative stress may play an important role in metabolic syndrome and related manifestations, including obesity, atherosclerosis, hypertension, and insulin resistance. Its relation to obesity is due to increased reactive oxygen species and/or decreased glutathione (GSH) antioxidant metabolism. Consequently, the activation of glutathione metabolism appears to be a central defense response to prevent oxidative stress. In this sense, dietary supplements with natural antioxidant molecules, including proanthocyanidins, may present a useful strategy of controlling and reducing complications of obesity, including hepatic steatosis. MATERIALS AND RESULTS: We assessed the grape seed proanthocyanidin extract (GSPE) effect on oxidative alterations related to genetically obese rats (Zucker rats) and, more specifically, to hepatic GSH metabolism. We demonstrate that the administration of GSPE reduced the oxidized glutathione accumulation increasing the total GSH/oxidized glutathione hepatic ratio and consequently decreasing the activation of antioxidant enzymes, including glutathione peroxidase, glutathione reductase, and glutathione S-transferase, and increasing the total antioxidant capacity of the cell. CONCLUSION: In Zucker rats, the obesity-induced oxidative stress related to liver glutathione alteration was mitigated by GSPE administration.

Grape seed procyanidin extract reduces the endotoxic effects induced by lipopolysaccharide in rats.

Acute inflammation is a response to injury, infection, tissue damage, or shock. Bacterial lipopolysaccharide (LPS) is an endotoxin implicated in triggering sepsis and septic shock, and LPS promotes the inflammatory response, resulting in the secretion of proinflammatory and anti-inflammatory cytokines such as the interleukins (IL-6, IL-1ß, and IL-10) and tumor necrosis factor-α by the immune cells. Furthermore, nitric oxide and reactive oxygen species levels increase rapidly, which is partially due to the activation of inducible nitric oxide synthase in several tissues in response to inflammatory stimuli. Previous studies have shown that procyanidins, polyphenols present in foods such as apples, grapes, cocoa, and berries, have several beneficial properties against inflammation and oxidative stress using several in vitro and in vivo models. In this study, the anti-inflammatory and antioxidant effects of two physiological doses and two pharmaceutical doses of grape seed procyanidin extract (GSPE) were analyzed using a rat model of septic shock by the intraperitoneal injection of LPS derived from Escherichia coli. The high nutritional (75mg/kg/day) and the high pharmacological doses (200mg/kg/day) of GSPE showed anti-inflammatory effects by decreasing the proinflammatory marker NOx in the plasma, red blood cells, spleen, and liver. Moreover, the high pharmacological dose also downregulated the genes Il-6 and iNos; and the high nutritional dose decreased the glutathione ratio (GSSG/total glutathione), further illustrating the antioxidant capability of GSPE. In conclusion, several doses of GSPE can alleviate acute inflammation triggered by LPS in rats at the systemic and local levels when administered for as few as 15 days before the injection of endotoxin.

Is it necessary to evaluate nuclei in HER2 FISH evaluation?

A new method that simplifies the evaluation of the traditional HER2 fluorescence in situ hybridization (FISH) evaluation in breast cancer was proposed. HER2 status was evaluated in digital images (DIs) captured from 423 invasive breast cancer stained sections. All centromeric/CEP17 and HER2 gene signals obtained from separated stacked DIs were manually counted on the screen. The global ratios were compared with the traditional FISH evaluation and the immunohistochemical status. The 2 FISH scores were convergent in 96.93% of cases, showing an "almost perfect" agreement with a weighted k of 0.956 (95% confidence interval, 0.928-0.985). The new method evaluates at least 3 times more nuclei than traditional methods and also has an almost perfect agreement with the immunohistochemical scores. The proposed enhanced method substantially improves HER2 FISH assessment in breast cancer biopsy specimens because the evaluation of HER2/CEP17 copy numbers is more representative, easier, and faster than the conventional method.

miRNAs, polyphenols, and chronic disease.

MicroRNAs (miRNAs) are small noncoding RNAs, approximately 18-25 nucleotides in length, that modulate gene expression at the posttranscriptional level. Thousands of miRNAs have been described, and it is thought that they regulate some aspects of more than 60% of all human cell transcripts. Several polyphenols have been shown to modulate miRNAs related to metabolic homeostasis and chronic diseases. Polyphenolic modulation of miRNAs is very attractive as a strategy to target numerous cell processes and potentially reduce the risk of chronic disease. Evidence is building that polyphenols can target specific miRNAs, such as miR-122, but more studies are necessary to discover and validate additional miRNA targets.

Digital image analysis in breast cancer: an example of an automated methodology and the effects of image compression.

In the current practice of pathology, the evaluation of immunohistochemical (IHC) markers represents an essential tool. The manual quantification of these markers is still laborious and subjective, and the use of computerized systems for digital image (DI) analysis has not yet resolved the problems of nuclear aggregates (clusters). Furthermore, the volume of DI storage continues to be an important problem in computer-assisted pathology. In the present study we have developed an automated procedure to quantify IHC nuclear markers in DI with a high level of clusters. Furthermore the effects of JPEG compression in the image analysis were evaluated. The results indicated that there was an agreement with the results of both methods (automated vs. manual) in almost 90% of the analyzed images. On the other hand, automated count differences increase as the compression level increase, but only in images with a high number of stained nuclei (>nuclei/image) or with high area cluster (>25µm2). Some corrector factors were developed in order to correct this count differences. In conclusion, the proposed automated procedure is an objective, faster than manual counting and reproducible method that has more than 90% of similarity with manual count. Moreover, the results demonstrate that with correction factors, it is possible to carry out unbiased automated quantifications on IHC nuclear markers in compressed DIs.

Hypolipidemic effects of proanthocyanidins and their underlying biochemical and molecular mechanisms.

Proanthocyanidins are the most abundant polyphenols in human diets. Epidemiological studies strongly suggest that proanthocyanidins protect against cardiovascular diseases. Despite the antioxidant and anti-inflammatory properties of these flavonoids, one of the mechanisms by which proanthocyanidins exert their cardiovascular protection is improving lipid homeostasis. Animal studies demonstrate that proanthocyanidins reduce the plasma levels of atherogenic apolipoprotein B-triglyceride-rich lipoproteins and LDL-cholesterol but increase antiatherogenic HDL-cholesterol. The results in humans, however, are less clear. This review summarizes the results that have been published on plasma triglyceride, apolipoprotein B, HDL-cholesterol and LDL-cholesterol levels in humans and animal models in response to proanthocyanidin extracts and proanthocyanidin-rich foods. The physiological processes and biochemical pathways that are related to lipid homeostasis and affected by proanthocyanidin consumption are also discussed. Intestinal lipid absorption, chylomicron secretion by the intestine and VLDL secretion by the liver are the processes that are most repressed by proanthocyanidins, which, therefore, induce hypolipidemic effects.