A risk prediction model for head and neck cancers incorporating lifestyle factors, HPV serology and genetic markers.

Head and neck cancer is often diagnosed late and prognosis for most head and neck cancer patients remains poor. To aid early detection, we developed a risk prediction model based on demographic and lifestyle risk factors, human papillomavirus (HPV) serological markers and genetic markers. A total of 10 126 head and neck cancer cases and 5254 controls from five North American and European studies were included. HPV serostatus was determined by antibodies for HPV16 early oncoproteins (E6, E7) and regulatory early proteins (E1, E2, E4). The data were split into a training set (70%) for model development and a hold-out testing set (30%) for model performance evaluation, including discriminative ability and calibration. The risk models including demographic, lifestyle risk factors and polygenic risk score showed a reasonable predictive accuracy for head and neck cancer overall. A risk model that also included HPV serology showed substantially improved predictive accuracy for oropharyngeal cancer (AUC = 0.94, 95% CI = 0.92-0.95 in men and AUC = 0.92, 95% CI = 0.88-0.95 in women). The 5-year absolute risk estimates showed distinct trajectories by risk factor profiles. Based on the UK Biobank cohort, the risks of developing oropharyngeal cancer among 60 years old and HPV16 seropositive in the next 5 years ranged from 5.8% to 14.9% with an average of 8.1% for men, 1.3% to 4.4% with an average of 2.2% for women. Absolute risk was generally higher among individuals with heavy smoking, heavy drinking, HPV seropositivity and those with higher polygenic risk score. These risk models may be helpful for identifying people at high risk of developing head and neck cancer.

Germline determinants of humoral immune response to HPV-16 protect against oropharyngeal cancer.

Although several oropharyngeal cancer (OPC) susceptibility loci have been identified, most previous studies lacked detailed information on human papillomavirus (HPV) status. We conduct a genome-wide analysis by HPV16 serology status in 4,002 oral cancer cases (OPC and oral cavity cancer (OCC)) and 5,256 controls. We detect four susceptibility loci pointing to a distinct genetic predisposition by HPV status. Our most notable finding in the HLA region, that is now confirmed to be specific of HPV(+)OPC risk, reveal two independent loci with strong protective effects, one refining the previously reported HLA class II haplotype association. Antibody levels against HPV16 viral proteins strongly implicate the protective HLA variants as major determinants of humoral response against L1 capsid protein or E6 oncoprotein suggesting a natural immune response against HPV(+)OPC promoted by HLA variants. This indicates that therapeutic vaccines that target E6 and attenuate viral response after established HPV infections might protect against HPV(+)OPC.

Vorinostat increases carboplatin and paclitaxel activity in non-small-cell lung cancer cells.

We observed a 53% response rate in non-small cell lung cancer (NSCLC) patients treated with vorinostat plus paclitaxel/carboplatin in a Phase I trial. Studies were undertaken to investigate the mechanism (s) underlying this activity. Growth inhibition was assessed in NSCLC cells by MTT assay after 72 hr of continuous drug exposure. Vorinostat (1 microM) inhibited growth by: 17% +/- 7% in A549, 28% +/- 6% in 128-88T, 39% +/- 8% in Calu1 and 41% +/- 7% in 201T cells. Vorinostat addition to carboplatin or paclitaxel led to significantly greater growth inhibition than chemotherapy alone in all 4 cell lines. Vorinostat (1 microM) synergistically increased the growth inhibitory effects of carboplatin/paclitaxel in 128-88T cells. When colony formation was measured after drug withdrawal, vorinostat significantly increased the effects of carboplatin but not paclitaxel. The % colony formation was control 100%; 1 microM vorinostat, 83% +/- 10%; 5 microM carboplatin, 41% +/- 11%; carboplatin/vorinostat, 8% +/- 4%; 2 nM paclitaxel, 53% +/- 11%; paclitaxel/vorinostat, 46% +/- 21%. In A549 and 128-88T, vorinostat potentiated carboplatin induction of gamma-H2AX (a DNA damage marker) and increased alpha-tubulin acetylation (a marker for stabilized mictrotubules). In A549, combination of vorinostat with paclitaxel resulted in a synergistic increase in alpha-tubulin acetylation, which reversed upon drug washout. We conclude that vorinostat interacts favorably with carboplatin and paclitaxel in NSCLC cells, which may explain the provocative response observed in our clinical trial. This likely involves a vorinostat-mediated irreversible increase in DNA damage in the case of carboplatin and a reversible increase in microtubule stability in the case of paclitaxel.

Breast cancer-derived M543V mutation in helix 12 of estrogen receptor alpha inverts response to estrogen and SERMs.

We have isolated from human breast cancers several mutations in the Helix 12 component of activation function 2 (AF-2) in the estrogen receptor alpha (ERalpha). We used a novel approach to detect changes in the hormone-binding domain of ERalpha, based on the evidence that antiestrogens, such as 4-hydroxytamoxifen (ZOHT) and ICI 182,780, block the function of ERalpha by binding and folding the AF-2 transcriptional domain in a way that inhibits its association with coactivator proteins. We have identified a Helix 12 mutation, M543V, which leads to greater ERalpha transcription with ZOHT and other antiestrogens (including 1,1-dichloro-2,2,3-triarylcyclopropanes, DTACs) than with 17-beta estradiol (E2). We also found an independent mutation at the same position, M543I, which did not show this inverted ligand phenotype. In comparison to further Helix 12 mutations made in vitro, it appears that relative hydrophobicity of the amino acid side chains on the inner face of Helix 12 is key to maintaining the transcriptionally active, agonist conformation with bound E2. This active conformation can be induced, resulting in increased transcription, by adding excess p160 coactivator AIB1 in transcriptional assays with E2-bound receptors, while the ZOHT-bound receptors were not further activated by AIB1. Other experiments show that the cross talk between ERalpha and AP-1 protein from AP-1-binding sites is not dependent on Helix 12 integrity. We show that two alleles containing a proline substitution in Helix 12 that inactivate AF-2 function of ERalpha at EREs have little negative effect on function through AP-1 elements, supporting a prominent role for the N-terminal AF-1 of ERalpha in AP-1/ERalpha transcriptional cross talk.

Preclinical Evidence for Combined Use of Aromatase Inhibitors and NSAIDs as Preventive Agents of Tobacco-Induced Lung Cancer.

INTRODUCTION: A hormonal role in NSCLC development is well documented. We previously showed that the aromatase inhibitor (AI) anastrozole decreased development of tobacco carcinogen-induced lung tumors in a murine lung cancer prevention model and that aromatase and estrogen receptor were expressed in pulmonary inflammatory cells. METHODS: We utilized a tobacco carcinogen-induced lung tumor mouse model by treatment with 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), to determine whether an AI combined with nonsteroidal anti-inflammatory drugs results in greater lung tumor prevention effects compared to single-agent treatment. RESULTS: Combination of anastrozole (0.1 mg/kg/d) with aspirin (25 mg/kg/d) after NNK exposure resulted in significantly fewer and smaller lung tumors than did single-agent treatments and was accompanied by maximum decreases in circulating ß-estradiol (E2) and interleukin-6, tumor-infiltrating macrophages, and tumoral Ki67, phospho-mitogen-activated protein kinase, phospho-signal transducer and activator of transcription 3, and interleukin-17A expression. Preneoplasia arising after combination treatment showed the lowest Sox-2 expression, suggesting an inhibitory effect on proliferative capacity in the airways by blocking both E2 and inflammation. Anastrozole combined with ibuprofen instead of aspirin also showed enhanced antitumor effects. Moreover, male mice treated with NNK that received E2 in their drinking water showed greater levels of pulmonary macrophages and inflammatory markers than did the control, confirming an E2 effect on inflammation in the microenvironment. CONCLUSIONS: Our results suggest a benefit to joint targeting of the estrogen and inflammatory pathways for NSCLC prevention. Combining AIs with nonsteroidal anti-inflammatory drugs reduces circulating E2, proinflammatory cytokines, and macrophage recruitment in the lung microenvironment after tobacco exposure. This strategy could be particularly effective in women who have underlying pulmonary inflammatory diseases.

Regulation of endogenous gene expression in human non-small cell lung cancer cells by estrogen receptor ligands.

Estrogen receptor (ER) agonists and antagonists elicit distinct responses in non-small cell lung cancer (NSCLC) cells. To determine how such responses are generated, the expression of ERalpha, ERbeta, and ER coregulators in human lung fibroblasts and human NSCLC cell lines was evaluated by immunoblot. Ligand-dependent estrogenic responses in NSCLC cells are probably generated via ERbeta and the p160 coactivator GRIP1/TIF2, because expression of these proteins was detected, but not full-length ERalpha or the p160 coactivator SRC-1. ERbeta and GRIP1/TIF2 are shown to interact in vitro in a ligand-dependent manner and thus may form functional transcription complexes in NSCLC cells. Furthermore, the capacity of ER ligands to regulate gene expression in NSCLC cells was explored using gene miniarrays. Expression profiles were examined after treatment with ER agonist 17-beta-estradiol (E2), the pure ER antagonist ICI 182,780 (fulvestrant, Faslodex), or epidermal growth factor, which served as a positive control for an alternative growth stimulus. E-cadherin and inhibitor of differentiation 2 were differentially regulated by E2 versus ICI 182,780 in 201T and 273T NSCLC cell lines. Epidermal growth factor also stimulated proliferation of these cells but had no effect on expression of E-cadherin and inhibitor of differentiation 2, suggesting they are specific targets of ER signaling. These data show that NSCLC cells respond to estrogens/antiestrogens by altering endogenous gene expression and support a model in which ICI 182,780 reduces proliferation of NSCLC cells via its ability to disrupt ER signaling. ICI 182,780 may therefore have therapeutic benefit in NSCLC.

Long-term depression in the adult hippocampus in vivo involves activation of extracellular signal-regulated kinase and phosphorylation of Elk-1.

Protein kinase cascades likely play a critical role in the signaling events that underlie synaptic plasticity and memory. The extracellular signal-regulated kinase (ERK) cascade is suited well for such a role because its targets include regulators of gene expression. Here we report that the ERK cascade is recruited during long-term depression (LTD) of synaptic strength in area CA1 of the adult hippocampus in vivo and selectively impacts on phosphorylation of the nuclear transcription factor Elk-1. Using a combination of in vivo electrophysiology, biochemistry, pharmacology, and immunohistochemistry, we found the following: (1) ERK phosphorylation, including phosphorylation of nuclear ERK, and ERK phosphotransferase activity are increased markedly, albeit transiently, after the induction of NMDA receptor-dependent LTD at the commissural input to area CA1 pyramidal cells in the hippocampus of anesthetized adult rats; (2) LTD-inducing paired-pulse stimulation fails to produce lasting LTD in the presence of the ERK kinase inhibitor SL327, which suggests that ERK activation is necessary for the persistence of LTD; and (3) ERK activation during LTD results in increased phosphorylation of Elk-1 but not of the transcription factor cAMP response element-binding protein. Our findings indicate that the ERK cascade transduces signals from the synapse to the nucleus during LTD in hippocampal area CA1 in vivo, as it does during long-term potentiation in area CA1, but that the pattern of coupling of the ERK cascade to transcriptional regulators differs between the two forms of synaptic plasticity.

Differential response to 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in non-small cell lung cancer cells with distinct oncogene mutations.

We previously demonstrated that non-small cell lung cancer (NSCLC) cells and primary human lung tumors aberrantly express the vitamin D3-catabolizing enzyme, CYP24, and that CYP24 restricts transcriptional regulation and growth control by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in NSCLC cells. To ascertain the basis for CYP24 dysregulation, we assembled a panel of cell lines that represent distinct molecular classes of lung cancer: cell lines were selected which harbored mutually exclusive mutations in either the K-ras or the Epidermal Growth Factor Receptor (EGFR) genes. We observed that K-ras mutant lines displayed a basal vitamin D receptor (VDR)(low)CYP24(high) phenotype, whereas EGFR mutant lines had a VDR(high)CYP24(low) phenotype. A mutation-associated difference in CYP24 expression was also observed in clinical specimens. Specifically, K-ras mutation was associated with a median 4.2-fold increase in CYP24 mRNA expression (p=4.8×10(-7)) compared to EGFR mutation in a series of 147 primary lung adenocarcinoma cases. Because of their differential basal expression of VDR and CYP24, we hypothesized that NSCLC cells with an EGFR mutation would be more responsive to 1,25(OH)2D3 treatment than those with a K-ras mutation. To test this, we measured the ability of 1,25(OH)2D3 to increase reporter gene activity, induce transcription of endogenous target genes, and suppress colony formation. In each assay, the extent of 1,25(OH)2D3 response was greater in EGFR mutation-positive HCC827 and H1975 cells than in K-ras mutation-positive A549 and 128.88T cells. We subsequently examined the effect of combining 1,25(OH)2D3 with erlotinib, which is used clinically in the treatment of EGFR mutation-positive NSCLC. 1,25(OH)2D3/erlotinib combination resulted in significantly greater growth inhibition than either single agent in both the erlotinib-sensitive HCC827 cell line and the erlotinib-resistant H1975 cell line. These data are the first to suggest that EGFR mutations may identify a lung cancer subset which remains responsive to and is likely to benefit from 1,25(OH)2D3 administration. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

A local effect of CYP24 inhibition on lung tumor xenograft exposure to 1,25-dihydroxyvitamin D(3) is revealed using a novel LC-MS/MS assay.

The vitamin D(3) catabolizing enzyme, CYP24, is frequently over-expressed in tumors, where it may support proliferation by eliminating the growth suppressive effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). However, the impact of CYP24 expression in tumors or consequence of CYP24 inhibition on tumor levels of 1,25(OH)(2)D(3)in vivo has not been studied due to the lack of a suitable quantitative method. To address this need, an LC-MS/MS assay that permits absolute quantitation of 1,25(OH)(2)D(3) in plasma and tumor was developed. We applied this assay to the H292 lung tumor xenograft model: H292 cells eliminate 1,25(OH)(2)D(3) by a CYP24-dependent process in vitro, and 1,25(OH)(2)D(3) rapidly induces CYP24 expression in H292 cells in vivo. In tumor-bearing mice, plasma and tumor concentrations of 1,25(OH)(2)D(3) reached a maximum of 21.6 and 1.70ng/mL, respectively, following intraperitoneal dosing (20µg/kg 1,25(OH)(2)D(3)). When co-administered with the CYP24 selective inhibitor CTA091 (250µg/kg), 1,25(OH)(2)D(3) plasma levels increased 1.6-fold, and tumor levels increased 2.6-fold. The tumor/plasma ratio of 1,25(OH)(2)D(3) AUC was increased 1.7-fold by CTA091, suggesting that the inhibitor increased the tumor concentrations of 1,25(OH)(2)D(3) independent of its effects on plasma disposition. Compartmental modeling of 1,25(OH)(2)D(3) concentration versus time data confirmed that: 1,25(OH)(2)D(3) was eliminated from plasma and tumor; CTA091 reduced the elimination from both compartments; and that the effect of CTA091 on tumor exposure was greater than its effect on plasma. These results provide evidence that CYP24-expressing lung tumors eliminate 1,25(OH)(2)D(3) by a CYP24-dependent process in vivo and that CTA091 administration represents a feasible approach to increase tumor exposure to 1,25(OH)(2)D(3).

CYP24 inhibition preserves 1α,25-dihydroxyvitamin D(3) anti-proliferative signaling in lung cancer cells.

Human lung tumors aberrantly express the 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-catabolizing enzyme, CYP24. We hypothesized that CYP24 reduces 1,25(OH)(2)D(3)-mediated transcription and allows lung cancer cells to escape its growth-inhibitory action. To test this, H292 lung cancer cells and the CYP24-selective inhibitor CTA091 were utilized. In H292 cells, CTA091 reduces 1,25(OH)(2)D(3) catabolism, significantly increases 1,25(OH)(2)D(3)-mediated growth inhibition, and increases 1,25(OH)(2)D(3) effects on induced and repressed genes in gene expression profiling studies. Pathway mapping of repressed genes uncovered cell cycle as a predominant 1,25(OH)(2)D(3) target. In H292 cells, 1,25(OH)(2)D(3) significantly decreases cyclin E2 levels and induces G(0)/G(1) arrest. A broader set of cyclins is down-regulated when 1,25(OH)(2)D(3) is combined with CTA091, and cell cycle arrest further increases. Effects of CTA091 on 1,25(OH)(2)D(3) signaling are vitamin D receptor-dependent. These data provide evidence that CYP24 limits 1,25(OH)(2)D(3) anti-proliferative signaling in cancer cells, and suggest that CTA091 may be beneficial in preserving 1,25(OH)(2)D(3) action in lung cancer.

Estrogen receptor beta (ERbeta) subtype-specific ligands increase transcription, p44/p42 mitogen activated protein kinase (MAPK) activation and growth in human non-small cell lung cancer cells.

In non-small cell lung cancer (NSCLC) cells, 17beta-estradiol increases transcription, activates MAPK, and stimulates proliferation. We hypothesize that estrogen receptor beta (ERbeta) mediates these responses because it, but not ERalpha, is detected in our NSCLC cell lines. To test this, we determined the effects of the ERbeta-selective agonists genistein (GEN) and 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) and the ERalpha-selective agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) in 201T cells. The cells were transfected with either an ERalpha or an ERbeta expression vector and an estrogen response element (ERE)-tk-luciferase reporter construct. PPT increased luciferase activity in cells expressing ERalpha but not ERbeta. GEN and DPN selectively increased luciferase activity in ERbeta-transfected cells at concentrations < or =10 nM. Fulvestrant blocked the GEN- and DPN-mediated increases, indicating that transcription was ER-dependent. GEN but not PPT mediated a significant 1.5-fold increase in reporter activity upon transfection with ERE-tk-luciferase alone, demonstrating that endogenous ERbeta activates transcription. PPT and DPN increased MAPK phosphorylation (2.5-fold and 3.7-fold, respectively). However, only DPN stimulated 201T growth in vitro (p=0.008) and in vivo (p=0.05). We conclude that ERbeta mediates genomic and non-genomic responses to estrogen in 201T cells and that activation of both pathways may be necessary for increased proliferation of these cells.

CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer.

1Alpha,25-dihydroxyvitamin D3 (1,25D3) displays potent antiproliferative activity in a variety of tumor model systems and is currently under investigation in clinical trials in cancer. Studies were initiated to explore its potential in nonsmall cell lung cancer (NSCLC), as effective approaches to the treatment of that disease are needed. In evaluating factors that may affect activity in NSCLC, the authors found that CYP24 (25-hydroxyvitamin D3-24-hydroxylase), the enzyme that catabolizes 1,25D3, is frequently expressed in NSCLC cell lines but not in the nontumorigenic bronchial epithelial cell line, Beas2B. CYP24 expression by RT-PCR was also detected in 10/18 primary lung tumors but in only 1/11 normal lung tissue specimens. Tumor-specific CYP24 upregulation was confirmed at the protein level via immunoblot analysis of patient-matched normal lung tissue and lung tumor extracts. Enzymatically active CYP24 is expected to desensitize NSCLC cells to 1,25D3. The authors therefore implemented a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for 1,25D3 and its CYP24-generated metabolites to determine whether NSCLC cells express active enzyme. Analysis of NSCLC cell cultures revealed time-dependent loss of 1,25D3 coincident with the appearance of CYP24-generated metabolites. MK-24(S)-S(O)(NH)-Ph-1, a specific inhibitor of CYP24, slowed the loss of 1,25D3 and increased 1,25D3 half-life. Furthermore, combination of 1,25D3 with MK-24(S)-S(O)(NH)-Ph-1 resulted in a significant decrease in the concentration of 1,25D3 required to achieve maximum growth inhibition in NSCLC cells. These data suggest that increased CYP24 expression in lung tumors restricts 1,25D3 activity and support the preclinical evaluation of CYP24 inhibitors for lung cancer treatment.

Synaptic localization of a functional NADPH oxidase in the mouse hippocampus.

Superoxide has been shown to be critical for hippocampal long-term potentiation (LTP) and hippocampus-dependent memory function. A possible source for the generation of superoxide during these processes is NADPH oxidase. The active oxidase consists of two membrane proteins, gp91phox and p22phox, and four cytosolic proteins, p40phox, p47phox, p67phox, and Rac. Upon stimulation, the cytosolic proteins translocate to the membrane to form a complex with the membrane components, which results in production of superoxide. Here, we determined the presence, localization, and functionality of a NADPH oxidase in mouse hippocampus by examining the NADPH oxidase proteins as well as the production of superoxide. All of the NADPH oxidase proteins were present in hippocampal homogenates and enriched in synaptoneurosome preparations. Immunocytochemical analysis of cultured hippocampal neurons indicated that all NADPH oxidase proteins were localized in neuronal cell bodies as well as dendrites. Furthermore, double labeling analysis using antibodies to p67phox and the presynaptic marker synaptophysin suggest a close association of the NADPH oxidase subunits with synaptic sites. Finally, stimulation of hippocampal slices with phorbol esters triggered translocation of the cytoplasmic NADPH oxidase proteins to the membrane and an increase in superoxide production that was blocked by inhibitors of NADPH oxidase. Taken together, our data suggest that NADPH oxidase is present in mouse hippocampus and might be the source of superoxide production required for LTP and memory function.

Inhibition of estrogen receptor alpha-mediated transcription by antiestrogenic 1,1-dichloro-2,2,3-triarylcyclopropanes.

A novel class of pure antiestrogens, 1,1-dichloro-2,2,3-triarylcyclopropanes (DTACs), lack estrogenic activity in a mouse uterotrophic assay and inhibit the growth of estrogen-sensitive MCF-7 breast cancer cells (Day et al., 1991). Here, reporter assays were used to evaluate the effects of the DTACs on estrogen receptor alpha (ERalpha)-mediated transcription from either classic estrogen-response elements (EREs) or nonclassic AP-1 elements. Among the DTACs tested, only the compounds with smaller aromatic substituents, BDRM72 and BDRM81, displayed weak agonist activity on EREs. Their activity was less than that observed for the ER partial agonist, 4-hydroxytamoxifen (ZOHT). In competition experiments, the DTACs blocked estradiol-stimulated transcription from an ERE in a dose-dependent manner and were more effective inhibitors than ZOHT. Each of the DTACs was significantly less active than ZOHT or the pure antiestrogen ICI 182,780 (faslodex) in stimulating transcription from nonclassic AP-1 elements in the presence of ERalpha. DTACs did not modulate either basal or TPA (12-O-tetradecanoylphorbol-13-acetate)-stimulated transcription from an AP-1 element in the absence of ERalpha, indicating that they are not nonspecific inhibitors of transcription and that ERalpha is the drug target. Glutathione S-transferase pull-down assays were used to examine whether DTACs alter the interaction between ERalpha and the p160 coactivator, GRIP1. BDRM35, which has the same dimethylaminomethoxy and phenolic moieties as ZOHT, reduced binding by more than 50%. Thus, disruption of p160 coactivator recruitment by ERalpha may represent one mechanism by which DTACs function as antiestrogens. BDRM35 also suppresses estradiol induction of endogenous target genes c-myc and cyclin D1 in MCF-7 breast cancer cells.

Investigación de péptidos plasmáticos en la insuficiencia renal crónica / Investigation of plasma peptides in chronic renal failure

Se describe una metodología para el fraccionamiento de péptidos y se aplica, en el presente trabajo, al análisis de compuestos péptidos presentes en el plasma de pacientes con insuficiencia renal crónica (IRC). Las muestras de plasma de sujetos controles (C;n=5) y de pacientes con IRC (n-10) fueron ultrafiltradas a través de membranas Diaflo PM 30 e YM 02, obteniéndose 2 fracciones, fracción A con sustancias de masa molecular relativa (M) menor de 1.000 y fracción B con M, aproximada entre 1.000 y 30.000, aunque los ensayos de estandarización mostraron que esta última fracción incluía proteínas de M, semejante a la albúmina. Las fracciones A, estudiadas mediante cromatografía en capa fina, mostraron la presencia de una banda fluorescamina positiva en las muestras de IRC, ausente en las C. Las fracciones B fueron analizadas por electroforesis en gel de poliacrilamida con SDS (SDS-PAGE) y tranferencia con detección inmunológica. Para desarrollar esta última técnica, se obtuvo un antisuero en conejo contra la fracción plasmática B, obtenida a partir de un pool de plasmas de pacientes con IRC. Después de la separación en SDS-PAGE, los compuestos fueron transferidos a papel de nitrocelulosa y detectados mediante una técnica de doble antisuero, empleando como primer antisuero, el anti-fracción IRC obtenido en conejo y luego, un antisuero inti-IgG de conejo, marcado con peroxidasa. Para el revelado final se utilizó una reacción colorimétrica, mediada por peroxidasa. El análisis de SDS-PAGE mostró que las fracciones plasmáticas B de pacientes con IRC contenían mayor número de bandas en el rango de M, 15.000-70.000 que las C, aumentando las diferencias cuali y/o cuantitativas entre los dos grupos, al emplear la técnica de separación electroforética, tranferencia de detección inmunológica. El procedimiento descrito constituye una metodología apropiada para el estudio de péptidos en líquidos biológicos

Diferenciación de proteinurias mediante electroforesis en gel de poliacrilamida / Differentation of proteinurias interceding polyacrylamide gel electrophoresis

El análisis de la distribución de las proteínas urinarias de acuerdo con sus masas moleculares relativas, constituye una información valiosa para el diagnóstico de enfermedades renales y extrarrenales. Se detalla el fraccionamiento de las proteínas presentes en muestras de orina de 24 h, sin concentrar. Se utiliza la técnica de electroforesis vertical en geles de policrilamida 10% con dodecil-sulfato de sodio (SDS-PAGE). El desarrollo electroforético se realiza entre 25 y 50 mA (V max = 200 V), durante 4-5 h,revelando con azul brillante de Coomassie R-250. Los perfiles proteicos obtenidos permiten diferenciar proteinurias tubulares, glomerulares y mixtas con distintos grados de selectividad y proteinurias extrarrenales. La buena resolución y sensibilidad de la técnica permite analizar muestras sin concentrar (concepción proteica > 0,3 g/l) y resulta adecuada para una correcta caracterización de proteinurias.