Biomarker discovery by plasma proteomics in familial Brugada Syndrome.

Brugada Syndrome (BS) is a polygenic inherited cardiac disease characterized by life-threatening arrhythmias and high incidence of sudden death. In this study, two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (LC-MS/MS) was used to investigate specific changes in the plasma proteome of BS patients and family members sharing the same gene mutation (SCN5AQ1118X), with the aim to identify novel disease biomarkers. Our data demonstrate that the levels of several proteins were significantly altered in BS patients compared with controls. In particular, apolipoprotein E, prothrombin, vitronectin, complement-factor H, vitamin-D-binding protein, voltage-dependent anion-selective channel protein 3 and clusterin were considerably increased in plasma sample of BS patients, whereas alpha-1-antitrypsin, fibrinogen and angiotensinogen were considerably decreased; moreover, post-translational modifications of antithrombin-III were detected in all affected individuals. On the light of these results, we hypothesize that these proteins might be considered as potential markers for the identification of disease status in BS.

Dynamic simulations of pathways downstream of ERBB-family, including mutations and treatments: concordance with experimental results.

The pathways downstream of ErbB-family proteins are very important in BC, especially when considering treatment with onco-protein inhibitors. We studied and implemented dynamic simulations of four downstream pathways and described the fragment of the signaling network we evaluated as a Molecular Interaction Map. Our simulations, enacted using Ordinary Differential Equations, involved 242 modified species and complexes, 279 reversible reactions and 111 catalytic reactions. Mutations within a single pathway tended to be mutually exclusive; only inhibitors acting at, or downstream (not upstream), of a given mutation were active. A double alteration along two distinct pathways required the inhibition of both pathways. We started an analysis of sensitivity/robustness of our network, and we systematically introduced several individual fluctuations of total concentrations of independent molecular species. Only very few cases showed significant sensitivity. We transduced the ErbB2 over-expressing BC line, BT474, with the HRAS (V12) mutant, then treated it with ErbB-family and phosphorylated MEK (MEKPP) inhibitors, Lapatinib and U0126, respectively. Experimental and simulation results were highly concordant, showing statistical significance for both pathways and for two respective endpoints, i.e. phosphorylated active forms of ERK and Akt, p one tailed = .0072 and = .0022, respectively. Working with a complex 39 basic species signaling network region, this technology facilitates both comprehension and effective, efficient and accurate modeling and data interpretation. Dynamic network simulations we performed proved to be both practical and valuable for a posteriori comprehension of biological networks and signaling, thereby greatly facilitating handling, and thus complete exploitation, of biological data.

Prevalence of HTLV-I-associated T-cell lymphoma.

In order to assess the prevalence rate of HTLV-1-associated T-cell lymphomas and human retrovirus infection in general, approximately 21,000 individuals representing various patient populations, retroviral risk groups, and blood donors were examined for HTLV-I, HTLV-II, HIV-1, or HIV-2 infection using serologic and PCR assays. The prevalence rates among volunteer blood donors were 0.02% and 0% for HTLV and HIV, respectively. Significantly increased HTLV prevalence rates were observed among paid blood donors, African American health care clinic patients, Amerindians, recipients of HTLV-positive cellular blood products, intravenous drug users, sexual contacts and family members of HTLV-positive people, and patients with primary thrombocytosis and other-than-low-grade non-Hodgkin's lymphoma (NHL). Among some of these groups there were significant differences in the prevalence of HTLV-I versus HTLV-II. The eight HTLV-positive NHL patients all had mature, high-grade, CD4+ T-cell lymphomas with clonally integrated HTLV-I, for a prevalence of 4% among other-than-low-grade NHL patients. Seven of the eight died from their disease within 2 years despite treatment. Interestingly, two groups at risk for HTLV infection, namely needle stick victims and recipients of HTLV-infected and/or pooled plasma products, showed no evidence for infection. Significantly increased HIV-1 prevalence was observed among paid blood donors, African Americans, homosexuals, female prostitutes, hemophiliacs, and other-than-low-grade NHL patients. Only one patient was infected with HIV-2. Of the nine HIV-positive, other-than-low-grade NHL patients, seven HIV-1 positives had B-cell lymphomas, one HIV-1 positive had an HTLV-I-positive CD4+ T-cell lymphoma, and one infected with HIV-2 had a CD4+ T-cell lymphoma that was HTLV negative. The data indicate that HTLV-I lymphoma, while uncommon, is not necessarily rare among other-than-low-grade NHL cases in the United States and, given its poor prognosis, should probably be studied separately in clinical trials.

Detection of viable oocysts of Cryptosporidium parvum following nucleic acid sequence based amplification.

A reliable method using nucleic acid sequence based amplification (NASBA) with subsequent electrochemiluminescent detection for the specific and sensitive detection of viable oocysts of Cryptosporidium parvum in environmental samples was developed. The target molecule was a 121-nt sequence from the C. parvum heat shock protein hsp70 mRNA. Oocysts of C. parvum were isolated from environmental water via vortex flow filtration and immunomagnetic separation. A brief heat shock was applied to the oocysts and the nucleic acid purified using an optimized very simple but efficient nucleic acid extraction method. The nucleic acid was amplified in a water bath for 60-90 min with NASBA, an isothermal technique that specifically amplifies RNA molecules. Amplified RNA was hybridized with specific DNA probes and quantified with an electrochemiluminescence (ECL) detection system. We optimized the nucleic acid extraction and purification, the NASBA reaction, amplification, and detection probes. We were able to amplify and detect as few as 10 mRNA molecules. The NASBA primers as well as the ECL probes were highly specific for C. parvum in buffer and in environmental samples. Our detection limit was approximately 5 viable oocysts/sample for the assay procedure, including nucleic acid extraction, NASBA, and ECL detection. Nonviable oocysts were not detected.

Increasing smoking cessation counseling by advanced practice nurses.

The magnitude of individual and societal problems caused by tobacco use mandates that all primary care providers identify and advise smokers to quit. However, this topic has received little attention in the nurse practitioner literature. The purpose of this project is to identify effective methods by which advanced practice nurses can increase the identification and counseling of smokers by reviewing research on this topic. The articles for review were obtained through a computerized literature search and a review of related reference lists. The articles were analyzed and categorized into three groups: office-wide interventions to increase provider identification and counseling of smokers, smoking cessation training programs for providers, and studies using the stages of change theory. Provider smoking cessation programs and office-wide reminders increased the identification and counseling of patients who smoke. The stages of change theory helped explain the steps smokers must progress through to cease smoking. Interventions appropriate for various stages in the cessation process are suggested.

[Sudden death due to recuperated ventricular fibrillation: Brugada syndrome?]. / Muerte súbita por fibrilación ventricular recuperada: síndrome de Brugada?

We report a 47 years old male who was recuperated from a sudden death, and in whom the cardiological assessment showed a right bundle branch block and a fluctuating ST segment elevation V1 to V3. During the electrophysiological study, a polymorphic tachycardia and a ventricular fibrillation were induced. Procainamide administration enhanced ST segment alterations in right precordial leads, and isoproterenol normalized the EKG. All these disturbances are similar to the condition described by Brugada brothers. The patient was treated with an internal implantable defibrillator, without the use of antiarrhythmic drugs and is well after four months of follow up.

[Anticoagulation in atrial fibrillation: not yet settled]. / Tratamiento anticoagulante en fibrilación auricular: aún no está todo resuelto.

A great deal of interest has received atrial fibrillation, the most common arrhythmia in adults, due to its complications and difficult treatment its most dreaded complication is atrial thrombi formation with the subsequent risk of embolization. There are several reports defining risk factors for embolic complications and the usefulness of anticoagulants for their prevention. We review the state of the art of anticoagulation in atrial fibrillation not associated to rheumatic valvulopathy. We also give tools to assess embolic risk and to determine the anticoagulant choice for the different presentation forms of atrial fibrillation.

Characteristics of DNA-tagged liposomes allowing their use in capillary-migration, sandwich-hybridization assays.

Liposomes that have been labeled externally with a DNA oligomer are used in a capillary-migration, sandwich-hybridization assay for specific DNA target sequences. The liposomes are used in a DNA detection scheme that produces visually observable results in 10 min. The preparation and covalent attachment of a thiol-activated 22-base oligomer to the external surface of dye-containing liposomes is described, and the specificity of the assay toward perfectly complementary target DNA is demonstrated. Several characteristics of DNA-tagged liposomes that allow the use of increased stringency during hybridization are evaluated. These include the effect of temperature, formamide, and salt concentration on both the sandwich-hybridization assay and the liposomes themselves. The effects of several components of a common hybridization solution are determined with regard to both assay performance and liposome stability. Using a solution of 0.02% sodium dodecyl sulfate in 3X standard saline citrate, a visual detection limit of 200 amol of target DNA was obtained.

Rapid method for visual identification of specific DNA sequences based on DNA-tagged liposomes.

We describe a rapid method for visually determining specific DNA sequences at femtomole concentrations. Liposomes, encapsulating a red dye and labeled with oligonucleotide, were used in a capillary migration-sandwich hybridization assay. Capture probe was immobilized on nitrocellulose strips, and liposomes, migrating along each strip, formed a visually discernible band in the presence of target DNA. One femtomole of synthetic target sequence could be detected in < 10 min. Sufficiently stringent hybridization conditions can be used to allow the discrimination of a 10% mismatch sequence from perfectly complementary DNA. A 366-base PCR product was detected at 200 fmol.

Prototypical HTLV-I/II infection is rare in LGL leukemia.

The etiology of LGL leukemia is not known; however, we recently detected HTLV-II in a patient with LGL leukemia. In this study, we found that sera from 6 of 28 patients with LGL leukemia were positive for HTLV-I/II using a whole virus ELISA; moreover, the ELISA-negative sera were near the positive cut-off value. Therefore, we performed additional studies on these sera using commercially available assays which can confirm and distinguish HTLV-I from HTLV-II infection. Serum from only one patient was confirmed positive using conventional criteria (HTLV-II+). Sera from 25 patients (89%) had indeterminate reactivity on Western blot assays. Of these, sera from 21 (84%) reacted to gag protein p24; 12 (48%) reacted with recombinant env protein p21e, and 10 (40%) reacted with both. We could not detect HTLV-I/II pol or pX gene sequences in these patients using polymerase chain reaction analyses, with the exception of the HTLV-II-infected patient described previously. These data show that most patients with LGL leukemia are not infected with prototypical HTLV-I or HTLV-II. The frequent reactivity of patient sera to HTLV-I/II gag protein p24 and to env protein p21e, however, suggests that a deleted or variant form of HTLV-I/II may be associated with LGL leukemia.

[Prevalence of dental fluorosis in Chile: a pilot study]. / Fluorosis dental endemica en Chile: estudio piloto.

The prevalence of enamel fluorosis and its severity was studied in 118 young men of 2 socio-economic levels. The subjects were born and resided for at least 6 years in Chilean communities with different natural levels of fluoride in drinking water. There was a high prevalence of enamel defect overall (54%) most of it of mild degree (36%). This was not related to the level of fluoride in drinking water, however further studies are needed since Chilean children are receiving fluoride from other sources. A national program to supplement drinking water with fluoride should take this information into account.

Evaluation of a solid-phase immunoassay for the simultaneous detection of antibodies to human immunodeficiency virus type 1 and human T-cell lymphotropic virus type I.

We describe the evaluation of a solid-phase immunoassay developed for the simultaneous detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus types I (HTLV-I) and II (HTLV-II) in human serum. The immunoassay employs a mixture of HIV-1 and HTLV-I whole viral lysates immobilized in the wells of microtiter plates. Evaluation of genetically well-pedigreed specimens along with normal blood donor samples indicated that the performance characteristics of the test were equivalent to the sensitivity and specificity of individual tests licensed by the Food and Drug Administration for antibodies to HIV-1 and HTLV-I. Furthermore, the test was also able to detect the presence of cross-reacting antibodies in HTLV-II-infected individuals. The use of such a test would greatly reduce the continually mounting costs associated with screening transfusable products for infectious agents.

Immunodetection of human T-cell lymphotropic virus type I core protein in biological samples by using a monoclonal antibody immunoassay.

A monoclonal antibody-based enzyme immunoassay (EIA) has been developed for detection of human T-cell lymphotropic virus type I (HTLV-I) core protein. The monoclonal antibody (clone 6.11) specifically recognizes the p19 gag gene-encoded protein of the virus. The EIA was over 100 times more sensitive than reverse transcriptase measurement and was capable of responding to less than 500 pg of whole-virus lysate. The assay exhibited type specificity in that HTLV-II antigens failed to produce a positive signal. In addition, a panel of other viruses demonstrated no antigenic cross-reactivity. These included herpesviruses, measles virus, human immunodeficiency viruses, and others. Viral p19 was followed during the course of density gradient ultracentrifugation in the presence of detergent, where it was noted to associate with viral membrane proteins. In comparison, reverse transcriptase activity localized in fractions of higher density containing envelope-free cores. Of clinical interest, the EIA was used to detect HTLV-I antigen in the viral cultures of patients with HTLV-I-associated myelopathies and from symptom-free individuals with proviral integration.

Monoclonal antibody identifies a highly conserved and immunodominant epitope of the human immunodeficiency virus transmembrane protein.

A murine monoclonal antibody (MAb), 10E9, has been generated which identifies a conserved and immunodominant epitope of the human immunodeficiency virus (HIV) transmembrane protein, gp41. The MAb reacts with the protein backbone of the mature env gene product and also with polyprotein precursor, gp160. Human sera were tested for their ability to competitively inhibit the immunoreactivity of MAb 10E9. Of 100 serum samples obtained from patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC), all showed strong inhibition to the reaction. In contrast, sera obtained from normal donors or those with other viral infections failed to perturb the binding activity of MAb 10E9. The geographic diversity of the AIDS/ARC patients studied provides evidence that the 10E9 epitope of gp41 is highly conserved.

Separation of human plasma factor IX from HTLV-I or HIV by immunoaffinity chromatography using conformation-specific antibodies.

Immunoaffinity chromatography using conformation-specific antibodies yields pure factor IX from human plasma in a single rapid, facile purification step. We evaluated this technique to determine whether factor IX can be separated from human T cell leukemia virus-I (HTLV-I) and human immunodeficiency virus (HIV) in plasma supplemented with these viruses. Viral content was determined with an enzyme-linked immunosorbent (ELISA) assay sensitive to 50 ng viral protein. Both HTLV-I and HIV coeluted with unbound protein. Neither HTLV-I nor HIV was detected in purified factor IX. We conclude that, to the limits of detection, factor IX purified by this method is free of viral contamination.

Complete amino-acid sequence of bovine seminal ribonuclease, a dimeric protein from seminal plasma.

The complete amino-acid sequence of BS-RNAse, a dimeric ribonuclease isolated from bovine seminal plasma, was determined. The reduced and S-carboxymethylated subunit chain of the enzyme was cleaved by trypsin and chymotrypsin. The resulting peptides, purified by cation-exchange chromatography were sequenced by dansyl-Edman, subtractive Edman degradation and carboxypeptidase A and B digestion. Chymotryptic peptides were used for the alignment. Automated Edman degradation of the native protein, through the N-terminal 41 amino-acid residues, completed the sequence information. The subunit chain of BS-RNAse, composed of 124 amino-acid residues, with a molecular mass of 13,610 Da, is highly homologous (81%) to pancreatic ribonuclease A. A good degree of homology (31%) was also found with human angiogenin. No N-linked carbohydrate-attachment sites, such as Asn-X-Ser/Thr, were found in the protein.

Herpes zoster: a possible early clinical sign for development of acquired immunodeficiency syndrome in high-risk individuals.

Zoster is uncommon before the age of 50 years in immunologically normal individuals, but it occurs with increased frequency in people who are immunosuppressed. A retrospective review of 300 patients with acquired immunodeficiency syndrome associated with Kaposi's sarcoma, revealed that 8% had prior zoster, a rate that is sevenfold greater than historic controls of the same age. We prospectively examined forty-eight patients, with no known immunodeficiency or signs of AIDS or AIDS related complex (ARC), who presented with zoster localized to the thoracic region. Forty-one patients had known risk factors for AIDS and thirty-five had antibody to the AIDS-associated virus (AAV) at the time of presentation. One seropositive subject had no known risk factors. Absolute lymphocyte counts, lymphocyte OKT4/OKT8 ratios, and lymphocyte mitogen responses were all depressed in subjects with antibody to AAV when compared with seronegative individuals. Seven of thirty-three AAV antibody-positive subjects, who could be followed longitudinally, developed AIDS from 1 to 28 months (mean = 13) after zoster. One antibody-negative subject seroconverted to become AAV seropositive 16 months after zoster and developed Kaposi's sarcoma 1 month later. These eight subjects had persistently low lymphocyte OKT4/OKT8 ratios and elevated beta-2 microglobulin. In patients at risk for AIDS, the occurrence of zoster may be one sign that heralds the marked depression of cellular immunity associated with AIDS or ARC.

Quantitative estimation by a standardized enzyme-linked immunosorbent assay of human T-cell lymphotropic virus type I antibodies in adult T-cell leukemia and acquired immune deficiency syndrome.

Sera from patients with adult T-cell leukemia and asymptomatic carriers of human T-cell lymphotropic virus type I (HTLV-I) from widely separated areas of the world reacted strongly in a standardized quantitative enzyme-linked immunosorbent assay procedure with HTLV-I viral antigen prepared from a strain isolated in the United States. There was a sharp differentiation of the values seen in the patients as compared with a normal population. Of the 35 acquired immune deficiency syndrome patients with Kaposi's sarcoma, only 2 were positive for HTLV-I antibodies in this test, and the distribution of the negative assay values in the other acquired immune deficiency syndrome patient sera was similar to that seen in the normal sera. Sera which contained extremely high levels of antibodies to other unrelated viruses (rubella virus, cytomegalovirus, and herpes simplex virus) all showed negative anti-HTLV-I results, in a pattern similar to the normal sera. Sera from patients with several autoimmune disease (systemic lupus erythematosus, rheumatoid arthritis, thyroiditis) as well as those with infectious mononucleosis or myeloma all also showed the normal distribution of negative results, in spite of the presence of very high levels of the autoantibodies, etc., associated with their illnesses.