Inhibition of mTOR prevents glucotoxicity-mediated increase of SA-beta-gal, p16INK4a, and insulin hypersecretion, without restoring electrical features of mouse pancreatic islets.

An over-activation of the mechanistic target of rapamycin (mTOR) pathway promotes senescence and age-related diseases like type 2 diabetes. Besides, the regenerative potential of pancreatic islets deteriorates with aging. Nevertheless, the role of mTOR on senescence promoted by metabolic stress in islet cells as well as its relevance for electrophysiological aspects is not yet known. Here, we investigated whether parameters suggested to be indicative for senescence are induced in vitro in mouse islet cells by glucotoxicity and if mTOR inhibition plays a protective role against this. Islet cells exhibit a significant increase (~ 76%) in senescence-associated beta-galactosidase (SA-beta-gal) activity after exposure to glucotoxicity for 72 h. Glucotoxicity does not markedly influence p16INK4a protein within 72 h, but p16INK4a levels increase significantly after a 7-days incubation period. mTOR inhibition with a low rapamycin concentration (1 nM) entirely prevents the glucotoxicity-mediated increase of SA-beta-gal and p16INK4a. At the functional level, reactive oxygen species, calcium homeostasis, and electrical activity are disturbed by glucotoxicity, and rapamycin fails to prevent this. In contrast, rapamycin significantly attenuates the insulin hypersecretion promoted by glucotoxicity by modifying the mRNA levels of Vamp2 and Snap25 genes, related to insulin exocytosis. Our data indicate an influence of glucotoxicity on pancreatic islet-cell senescence and a reduction of the senescence markers by mTOR inhibition, which is relevant to preserve the regenerative potential of the islets. Decreasing the influence of mTOR on islet cells exposed to glucotoxicity attenuates insulin hypersecretion, but is not sufficient to prevent electrophysiological disturbances, indicating the involvement of mTOR-independent mechanisms.

Molecular changes underlying pulmonary emphysema and chronic bronchitis in Chronic Obstructive Pulmonary Disease: An updated review.

The aim of this review is to update and synthesize the molecular mechanisms that lead to the heterogeneous effect on tissue remodeling observed in the two most important clinical phenotypes of chronic obstructive pulmonary disease (COPD), pulmonary emphysema (PE) and chronic bronchitis (CB). Clinical and experimental evidence suggests that this heterogeneous response to promote PE, CB, or both, is related to differentiated genetic, epigenetic, and molecular conditions. Specifically, a tendency toward PE could be related to a variant in the DSP gene, SIRT1 downregulation, macrophage polarization to M1, as well as the involvement of the noncanonical Wnt5A signaling pathway, among other alterations. Additionally, in advanced stages of COPD, PE development is potentiated by dysregulations in autophagy, which promotes senescence and subsequently cell apoptosis, through exacerbated inflammasome activation and release of caspases. On the other hand, CB or the pro-fibrotic phenotype could be potentiated by the downregulated activity of HDAC2, the activation of the TGF-ß/Smad or Wnt/ß-catenin signaling pathways, macrophage polarization to M2, upregulation of TIMP-1, and/or the presence of the epithelial-mesenchymal transition (EMT) mechanism. Interestingly, the upregulated activity of MMPs, especially MMP-9, is widely involved in the development of both phenotypes. Furthermore, MMP-9 and MMP-12 enhance the severity, perpetuation, and exacerbation of COPD, as well as the development of autoimmunity in this disease.

Maize Flavonoid Biosynthesis, Regulation, and Human Health Relevance: A Review.

Maize is one of the most important crops for human and animal consumption and contains a chemical arsenal essential for survival: flavonoids. Moreover, flavonoids are well known for their beneficial effects on human health. In this review, we decided to organize the information about maize flavonoids into three sections. In the first section, we include updated information about the enzymatic pathway of maize flavonoids. We describe a total of twenty-one genes for the flavonoid pathway of maize. The first three genes participate in the general phenylpropanoid pathway. Four genes are common biosynthetic early genes for flavonoids, and fourteen are specific genes for the flavonoid subgroups, the anthocyanins, and flavone C-glycosides. The second section explains the tissue accumulation and regulation of flavonoids by environmental factors affecting the expression of the MYB-bHLH-WD40 (MBW) transcriptional complex. The study of transcription factors of the MBW complex is fundamental for understanding how the flavonoid profiles generate a palette of colors in the plant tissues. Finally, we also include an update of the biological activities of C3G, the major maize anthocyanin, including anticancer, antidiabetic, and antioxidant effects, among others. This review intends to disclose and integrate the existing knowledge regarding maize flavonoid pigmentation and its relevance in the human health sector.

In Vivo Healthy Benefits of Galacto-Oligosaccharides from Lupinus albus (LA-GOS) in Butyrate Production through Intestinal Microbiota.

Animal digestive systems host microorganism ecosystems, including integrated bacteria, viruses, fungi, and others, that produce a variety of compounds from different substrates with healthy properties. Among these substrates, α-galacto-oligosaccharides (GOS) are considered prebiotics that promote the grow of gut microbiota with a metabolic output of Short Chain Fatty Acids (SCFAs). In this regard, we evaluated Lupinus albus GOS (LA-GOS) as a natural prebiotic using different animal models. Therefore, the aim of this work was to evaluate the effect of LA-GOS on the gut microbiota, SCFA production, and intestinal health in healthy and induced dysbiosis conditions (an ulcerative colitis (UC) model). Twenty C57BL/6 mice were randomly allocated in four groups (n = 5/group): untreated and treated non-induced animals, and two groups induced with 2% dextran sulfate sodium to UC with and without LA-GOS administration (2.5 g/kg bw). We found that the UC treated group showed a higher goblet cell number, lower disease activity index, and reduced histopathological damage in comparison to the UC untreated group. In addition, the abundance of positive bacteria to butyryl-CoA transferase in gut microbiota was significantly increased by LA-GOS treatment, in healthy conditions. We measured the SCFA production with significant differences in the butyrate concentration between treated and untreated healthy groups. Finally, the pH level in cecum feces was reduced after LA-GOS treatment. Overall, we point out the in vivo health benefits of LA-GOS administration on the preservation of the intestinal ecosystem and the promotion of SCFA production.

Lupin γ-conglutin protects against cell death induced by oxidative stress and lipotoxicity, but transiently inhibits in vitro insulin secretion by increasing KATP channel currents.

Lupin γ-conglutin beneficially modulates glycemia, but whether it protects against oxidative and lipotoxic damage remains unknown. Here, we studied the effects of γ-conglutin on cell death provoked by hydrogen peroxide and palmitate in HepG2 hepatocytes and insulin-producing MIN6 cells, and if a modulation of mitochondrial potential and reactive oxygen species (ROS) levels was involved. We also investigated how γ-conglutin influences insulin secretion and electrical activity of ß-cells. The increased apoptosis of HepG2 cells exposed to hydrogen peroxide was prevented by γ-conglutin, and the viability and ROS content in γ-conglutin-treated cells was similar to that of non-exposed cells. Additionally, γ-conglutin partially protected MIN6 cells against hydrogen peroxide-induced death. This was associated with a marked reduction in ROS. No significant changes were found in the mitochondrial potential of γ-conglutin-treated cells. Besides, we observed a partial protection against lipotoxicity only in hepatocytes. Unexpectedly, we found a transient inhibition of insulin secretion, plasma membrane hyperpolarization, and higher KATP channel currents in ß-cells treated with γ-conglutin. Our data show that γ-conglutin protects against cell death induced by oxidative stress or lipotoxicity by decreasing ROS and might also indicate that γ-conglutin promotes a ß-cell rest, which could be useful for preventing ß-cell exhaustion in chronic hyperglycemia.

Glucokinase activation as antidiabetic therapy: effect of nutraceuticals and phytochemicals on glucokinase gene expression and enzymatic activity.

Diabetes represents an important public health problem. Recently, new molecular targets have been identified and exploited to treat this disease. Due to its pivotal role in glucose homeostasis, glucokinase (GCK) is a promising target for the development of novel antidiabetic drugs; however, pharmacological agents that modulate GCK activity have been linked to undesirable side-effects, limiting its use. Interestingly, plants might be a valuable source of new therapeutic compounds with GCK-activating properties and presumably no adverse effects. In this review, we describe biochemical characteristics related to the physiological and pathological importance of GCK, as well as the mechanisms involved in its regulation at different molecular levels. Posteriorly, we present a compendium of findings supporting the potential use of nutraceuticals and phytochemicals in the management of diabetes through modulation of GCK expression and activity. Finally, we propose critical aspects to keep in mind when designing experiments to evaluate GCK modulation properly.

Effect of the acute and chronic administration of Lupinus albus ß-conglutin on glycaemia, circulating cholesterol, and genes potentially involved.

Constituents of lupin seeds, like γ-conglutin and lupanine, have gained attention as potential complementary treatments for dysglycaemia management. Notwithstanding, the effect of other lupin components on carbohydrate metabolism, including ß-conglutin protein, has received little attention. Here, we investigated the influence of the acute and chronic administration of ß-conglutin on glycaemia modulation in normal and streptozotocin induced-to-diabetes rats. We analysed the liver transcriptome modulation exerted by ß-conglutin in diabetes-induced rats using DNA microarrays to scout for potential molecular targets and pathways involved in this biological response. The acute administration of ß-conglutin reduced the incremental area under the curve of glycaemia in normal and diabetes-induced animals. In a seven-day study with diabetic animals, glycaemia increased significantly in non-treated animals but remained unchanged in animals treated with a daily dose of ß-conglutin. Total cholesterol was significantly lower at the end of the experimental period (-21.8 %, p = 0.039). The microarray and gene ontology analyses revealed several targets and pathways potentially modulated by ß-conglutin treatment, including a possible down-regulation of Jun kinase activity. Moreover, our data indicate that targets related to oxidative stress, inflammation, and estrogenic activity might orchestrate these metabolic effects. In conclusion, our findings show that ß-conglutin may help manage postprandial glycaemia and reduce cholesterol levels under the dysglycaemia stage. We identified and proposed new potential molecular targets for further research related to the mechanism of action of ß-conglutin.

Lupin gamma conglutin protein: effect on Slc2a2, Gck and Pdx-1 gene expression and GLUT2 levels in diabetic rats

ABSTRACT Recently, lupin seed (Lupinus albus L., Fabaceae) products have emerged as a functional food due to their nutritional and health benefits. Numerous reports have demonstrated the hypoglycemic effects of lupin's gamma conglutin protein; nonetheless, its mechanism of action remains elusive. To understand the role of this protein on glucose metabolism, we evaluated the effect of administering L. albus' gamma conglutin on Slc2a2, Gck, and Pdx-1 gene expression as well as GLUT2 protein tissue levels in streptozotocin-induced diabetic rats. While consuming their regular diet, animals received a daily gamma conglutin dose (120 mg/kg per body weight) for seven consecutive days. Serum glucose levels were measured at the beginning and at the end of the experimental period. At the end of the trial, we quantified gene expression in pancreatic and hepatic tissues as well as GLUT2 immunopositivity in Langerhans islets. Gamma conglutin administration lowered serum glucose concentration by 17.7%, slightly increased Slc2a2 and Pdx-1 mRNA levels in pancreas, up-regulated Slc2a2 expression in the liver, but it had no effect on hepatic Gck expression. After gamma conglutin administration, GLUT2 immunopositivity in Langerhans islets of diabetic animals resembled that of healthy rats. In conclusion, our results indicate that gamma conglutin up-regulates Slc2a2 gene expression in liver and normalizes GLUT2 protein content in pancreas of streptozotocin-induced rats.

Insulin glargine affects the expression of Igf-1r, Insr, and Igf-1 genes in colon and liver of diabetic rats.

OBJECTIVES: The mitogenic effect of the analogous insulin glargine is currently under debate since several clinical studies have raised the possibility that insulin glargine treatment has a carcinogenic potential in different tissues. This study aimed to evaluate the Igf-1r, Insr, and Igf-1 gene expression in colon and liver of streptozotocin-induced diabetic rats in response to insulin glargine, neutral protamine Hagedorn (NPH) insulin, and metformin treatments. MATERIALS AND METHODS: Male Wistar rats were induced during one week with streptozotocin to develop Type 2 Diabetes (T2D) and then randomly distributed into four groups. T2D rats included in the first group received insulin glargine, the second group received NPH insulin, the third group received metformin; finally, untreated T2D rats were included as the control group. All groups were treated for seven days; after the treatment, tissue samples of liver and colon were obtained. Quantitative PCR (qPCR) was performed to analyze the Igf-1r, Insr and Igf-1 gene expression in each tissue sample. RESULTS: The liver tissue showed overexpression of the Insr and Igf-1r genes (P>0.001) in rats treated with insulin glargine in comparison with the control group. Similar results were observed for the Insr gene (P>0.011) in colonic tissue of rats treated with insulin glargine. CONCLUSION: These observations demonstrate that insulin glargine promote an excess of insulin and IGF-1 receptors in STZ-induced diabetic rats, which could overstimulate the mitogenic signaling pathways.

Lupinus albus Conglutin Gamma Modifies the Gene Expressions of Enzymes Involved in Glucose Hepatic Production In Vivo.

Lupinus albus seeds contain conglutin gamma (Cγ) protein, which exerts a hypoglycemic effect and positively modifies proteins involved in glucose homeostasis. Cγ could potentially be used to manage patients with impaired glucose metabolism, but there remains a need to evaluate its effects on hepatic glucose production. The present study aimed to analyze G6pc, Fbp1, and Pck1 gene expressions in two experimental animal models of impaired glucose metabolism. We also evaluated hepatic and renal tissue integrity following Cγ treatment. To generate an insulin resistance model, male Wistar rats were provided 30% sucrose solution ad libitum for 20 weeks. To generate a type 2 diabetes model (STZ), five-day-old rats were intraperitoneally injected with streptozotocin (150 mg/kg). Each animal model was randomized into three subgroups that received the following oral treatments daily for one week: 0.9% w/v NaCl (vehicle; IR-Ctrl and STZ-Ctrl); metformin 300 mg/kg (IR-Met and STZ-Met); and Cγ 150 mg/kg (IR-Cγ and STZ-Cγ). Biochemical parameters were assessed pre- and post-treatment using colorimetric or enzymatic methods. We also performed histological analysis of hepatic and renal tissue. G6pc, Fbp1, and Pck1 gene expressions were quantified using real-time PCR. No histological changes were observed in any group. Post-treatment G6pc gene expression was decreased in the IR-Cγ and STZ-Cγ groups. Post-treatment Fbp1 and Pck1 gene expressions were reduced in the IR-Cγ group but increased in STZ-Cγ animals. Overall, these findings suggest that Cγ is involved in reducing hepatic glucose production, mainly through G6pc inhibition in impaired glucose metabolism disorders.

Lupanine Improves Glucose Homeostasis by Influencing KATP Channels and Insulin Gene Expression.

The glucose-lowering effects of lupin seeds involve the combined action of several components. The present study investigates the influence of one of the main quinolizidine alkaloids, lupanine, on pancreatic beta cells and in an animal model of type-2 diabetes mellitus. In vitro studies were performed with insulin-secreting INS-1E cells or islets of C57BL/6 mice. In the in vivo experiments, hyperglycemia was induced in rats by injecting streptozotocin (65 mg/kg body weight). In the presence of 15 mmol/L glucose, insulin secretion was significantly elevated by 0.5 mmol/L lupanine, whereas the alkaloid did not stimulate insulin release with lower glucose concentrations. In islets treated with l-arginine, the potentiating effect of lupanine already occurred at 8 mmol/L glucose. Lupanine increased the expression of the Ins-1 gene. The potentiating effect on secretion was correlated to membrane depolarization and an increase in the frequency of Ca(2+) action potentials. Determination of the current through ATP-dependent K⁺ channels (KATP channels) revealed that lupanine directly inhibited the channel. The effect was dose-dependent but, even with a high lupanine concentration of 1 mmol/L or after a prolonged exposure time (12 h), the KATP channel block was incomplete. Oral administration of lupanine did not induce hypoglycemia. By contrast, lupanine improved glycemic control in response to an oral glucose tolerance test in streptozotocin-diabetic rats. In summary, lupanine acts as a positive modulator of insulin release obviously without a risk for hypoglycemic episodes.

Administration of Lupinus albus gamma conglutin (Cγ) to n5 STZ rats augmented Ins-1 gene expression and pancreatic insulin content.

Several studies support the health-promoting benefits of lupins, particularly lupin proteins. It has been demonstrated that Lupinus albus gamma conglutin (Cγ) protein lowered blood glucose levels; thus, Cγ showed promise as a new anti-diabetic compound for type 2 diabetes (T2D) treatment. The aim of this study was to evaluate the effect of Cγ on Ins-1 gene expression and on pancreatic insulin content in streptozotocin-mediated diabetic rats. Cγ was isolated from Lupinus albus seeds. Its identification was confirmed with polyacrylamide gel electrophoresis under native and denaturing conditions. We used streptozotocin (STZ) to induce T2D on the 5th day of life of newborn male Wistar rats (n5-STZ). After 20 weeks post-induction, these animals (glycemia > 200 mg/dL) were randomly assigned to three groups that received the following one-week treatments: vehicle, 0.90% w/v NaCl (n5 STZ-Ctrl); glibenclamide, 10 mg/kg (n5 STZ-Glib); or Cγ, 120 mg/kg (n5 STZ-Cγ). Glucose and insulin levels were measured before and after treatment. Ins-1 gene expression was quantified using real time polymerase chain reaction and the pancreatic insulin content was evaluated with immunohistochemistry. Post-treatment, the n5 STZ-Cγ and n5 STZ-Glib groups showed reductions in glucose, increments in serum insulin, and increases in Ins-1 gene expression and beta cell insulin content compared to the n5 STZ-Ctrl group. The results showed that Cγ had beneficial effects on Ins-1 gene expression and pancreatic insulin content. These biological effects of Cγ strengthen its promising potential as a nutraceutical and/or new agent for controlling hyperglycemia.

Reduction of lns-1 gene expression and tissue insulin levels in n5-STZ rats.

OBJECTIVE: The high global incidence of type 2 diabetes has challenged researchers to establish animal models that resemble the chronic stage observed in type 2 diabetes patients. One such model is induced by neonatal streptozotocin (n-STZ) administration to rat pups at 0, 2, or 5 days after birth. In this study, we assessed lns-1 gene expression and tissue insulin levels as well as serum concentration of glucose and insulin, insulin resistance, and histological changes of the islets of Langerhans in n5-STZ rats after 20-weeks post-induction. METHODS: Wistar rat pups were randomly distributed into a control group and a streptozotocin-induced group. Experimental induction involved a single intraperitoneal injection of streptozotocin (150 mg/kg) into neonates at five days after birth. RESULTS: At 20 weeks post-induction, streptozotocin-induced rats exhibited increased serum glucose levels, reduced serum insulin levels, impaired glucose metabolism and insulin resistance compared to control rats. Histologically, streptozotocin-induced rats exhibited atrophic islets, vacuolization, and significantly fewer insulin-positive cells. lns-1 gene expression was significantly decreased in n5-STZ rats in comparison to the control group. CONCLUSION: Our findings support that the n5-STZ model 20 weeks post-induction represents an appropriate experimental tool to study T2D and to evaluate novel therapeutic agents and targets that involve insulin gene expression and secretion, as well as complications caused by chronic diabetes.

Reduction of lns-1 gene expression and tissue insulin levels in n5-STZ rats

Objective: The high global incidence of type 2 diabetes has challenged researchers to establish animal models that resemble the chronic stage observed in type 2 diabetes patients. One such model is induced by neonatal streptozotocin (n-STZ) administration to rat pups at 0, 2, or 5 days after birth. In this study, we assessed lns-1 gene expression and tissue insulin levels as well as serum concentration of glucose and insulin, insulin resistance, and histological changes of the islets of Langerhans in n5-STZ rats after 20-weeks post-induction. Methods: Wistar rat pups were randomly distributed into a control group and a streptozotocin-induced group. Experimental induction involved a single intraperitoneal injection of streptozotocin (150 mg/kg) into neonates at five days after birth. Results: At 20 weeks post-induction, streptozotocin-induced rats exhibited increased serum glucose levels, reduced serum insulin levels, impaired glucose metabolism and insulin resistance compared to control rats. Histologically, streptozotocin-induced rats exhibited atrophic islets, vacuolization, and significantly fewer insulin-positive cells. lns-1 gene expression was significantly decreased in n5-STZ rats in comparison to the control group. Conclusion: Our findings support that the n5-STZ model 20 weeks post-induction represents an appropriate experimental tool to study T2D and to evaluate novel therapeutic agents and targets that involve insulin gene expression and secretion, as well as complications caused by chronic diabetes.

Genetic polymorphisms of genes coding to alcohol-metabolizing enzymes in western Mexicans: association of CYP2E1*c2/CYP2E1*5B allele with cirrhosis and liver function.

BACKGROUND: Alcoholic cirrhosis constitutes a major public health problem in the world where ADH1B, ALDH2, and CYP2E1 polymorphisms could be playing an important role. We determined ADH1B*2, ALDH2*2, and CYP2E1*c2 allele frequencies in healthy control individuals (C) and patients with alcoholic cirrhosis (AC) from western Mexico. METHODS: Ninety C and 41 patients with AC were studied. Genotype and allele frequency were determined through polymerase chain reaction-restriction fragment length polymorphisms. RESULTS: Polymorphic allele distribution in AC was 1.6%ADH1B*2, 0.0%ALDH2*2, and 19.5%CYP2E1*c2; in C: 6.1%ADH1B*2, 0%ALDH2*2, and 10.6%CYP2E1*c2. CYP2E1*c2 polymorphic allele and c1/c2 genotype frequency were significantly higher (p < 0.05 and p < 0.01, respectively) in patients with AC when compared to C. Patients with AC, carrying the CYP2E1*c2 allele, exhibited more decompensated liver functioning evaluated by total bilirubin and prothrombin time, than c1 allele carrying patients (p < 0.05). Cirrhosis severity, assessed by Child's Pugh score and mortality, was higher in patients carrying the c2 allele, although not statistically significant. CONCLUSIONS: In this study, CYP2E1*c2 allele was associated with susceptibility to AC; meanwhile, ADH1B*2 and ALDH2*2 alleles were not. CYP2E1*c2 allele was associated with AC severity, which could probably be attributed to the oxidative stress promoted by this polymorphic form. Further studies to clearly establish CYP2E1*c2 clinical relevance in the development of alcohol-induced liver damage and its usefulness as a probable prognostic marker, should be performed. Also, increasing the number of patients and including a control group conformed by alcoholic patients free of liver damage may render more conclusive results. These findings contribute to the understanding of the influence of gene variations in AC development among populations, alcohol metabolism, and pharmacogenetics.

TGF-ß1 serum concentration as a complementary diagnostic biomarker of lung cancer: establishment of a cut-point value.

UNLABELLED: Lung cancer is a malignant disease with increasing mortality rates. Cytokines play a role in normal cell growth regulation and differentiation and are also implicated in malignant disease. Among these cytokines, Transforming Growth Factor ß type 1 (TGF-ß1) acts as a tumor promoter in malignant cells. Several clinical studies have found high levels of TGF-ß1 in various cancer types. The aim of this study was to establish a TGF-ß1 cut-off point as a complementary diagnostic tool in lung cancer detection. Therefore, 72 clinically well-characterized individuals were studied, 41 lung cancer patients and 31 healthy subjects. Serum TGF-ß1 concentration was measured by an enzyme-linked immunosorbent assay (ELISA). We compared statistically the serum TGF-ß1 concentration between both groups with analysis of variance, linear regression and receiver operating curve analysis. We observed that lung cancer patients produced higher TGF-ß1 levels than healthy individuals (37,225±9,436 vs. 28,416±9,324 pg/ml, P<0.001). The cut-point diagnostic value was 30,500 pg/ml with 80.5% sensitivity, 64.5% specificity and odds ratio: 7.5, 95% CI: 2.6-21.8. CONCLUSIONS: We found significantly higher TGF-ß1 levels in lung cancer patients than in healthy individuals. We propose the measurement of serum TGF-ß1 levels as a complementary diagnostic test in lung cancer detection.

Establishment of a cut-point value of serum TNF-alpha levels in the metabolic syndrome.

Cardiovascular diseases and type 2 diabetes are the major causes of mortality in Mexico. Metabolic syndrome (MS) is a cluster of factors that increase the risk to develop such diseases. Previous studies have shown that MS is associated with high tumor necrosis factor (TNF-alpha) levels. In fact, TNF-alpha has been proposed to be a useful marker for clinical diagnosis of inflammation at an early stage. Therefore, we analyzed TNF-alpha concentrations in Mexican individuals with or without MS and related these levels to the associated MS components. Clinical, anthropometric, and biochemical data were analyzed in 41 healthy and 39 MS individuals. Individuals were similarly grouped by age and gender.The serum TNF-alpha levels measured by a highly sensitive enzyme-linked immunosorbent assay (ELISA) kit were increased significantly in MS subjects compared with healthy individuals (P<0.001). The assay showed 78.1% sensitivity and 61.5% specificity with a cut-point level of 1.36 pg/mL. TNF-alpha levels higher than the cut-point value were correlated with insulin resistance indices. These findings support the hypothesis that serum TNF-alpha concentration could be a useful marker for early MS diagnosis. Nevertheless, we suggest the establishment of specific cut-point values in each studied population to evaluate potential clinical applications.

P16INK4a immunohistochemistry improves the reproducibility of the histological diagnosis of cervical intraepithelial neoplasia in cone biopsies.

OBJECTIVE: Cervical cancer is currently the most frequently occurring cancer among women in Mexico. Mexican cervical cancer prevention programs have been unsatisfactory in part because the tests used to diagnose precursor lesions have poor reproducibility. The implementation of specific biomarkers may overcome these limitations. Here, we analyzed whether immunohistochemistry for p16(INK4a) could improve the reproducibility of histopathological diagnoses of cervical precancerous lesions. METHODS: Serial sections of 78 specimens were stained for H&E and p16(INK4a) and independently interpreted by three Mexican pathologists. Specimens were interpreted and categorized in two ways: 1) four diagnostic categories including negative lesions, CIN1, CIN2, and CIN3, or 2) two diagnostic categories; either lesions that do not require therapy (negative, CIN1), or lesions that require therapy (>or=CIN2). The agreement in diagnoses between pairs of observers was evaluated by kappa statistics. RESULTS: The best concordance in diagnosing was observed with two categories and p16(INK4a) staining. Interestingly, the overall diagnostic discordances of higher than one CIN grade were 26.1% for H&E and 9.20% for p16(INK4a) (P<0.001). Using four diagnostic categories, weighted kappa values for each pair of observers were 0.28, 0.15, and 0.36 for H&E and 0.34, 0.35, and 0.60 for p16(INK4a) stains. Using two diagnostic categories, kappa values were 0.36, 0.12, and 0.18 for H&E and 0.59, 0.70, and 0.59, p16(INK4a) stains. CONCLUSION: These data show that p16(INK4a) immunohistochemistry substantially improved the reproducibility of interpreting histological slides. This approach may result in more accurate diagnoses and improved clinical management of patients with cervical precancerous lesions in Mexico and elsewhere.