Lack of benefit of protease inhibitors on HCV viremia in HIV-infected patients.
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Adult↗
Biomedical subjects
R Bravo
Publications and source records attributed to R Bravo.
Possibilities and limitations in miniaturized sensor design for uric acid.
Uric acid (UA) has been under intensive investigation by electrochemists owing to its important role as a metabolite in biological fluids. One of the major problems in biological determinations of uric acid comes from electrochemical interferences such as ascorbic acid (AA), which has a similar oxidation potential, E1/2 approximately 200 mV versus SCE, at graphite electrodes, and is present at high concentrations in biological systems. UA undergoes a 2 H+, 2 e- oxidation in aqueous buffers. The oxidation product, a diimine, is an unstable intermediate with a half-life of less than 22 ms. A follow-up hydration reaction converts the diimine to an imine alcohol. Results of previous work show that UA weakly adsorbs and undergoes a fast electron transfer reaction, ks = 54 s-1, at carbon fiber electrodes. These characteristics make UA an excellent candidate for fast scan voltammetric (FSV) determinations. This paper presents the results of FSV at bare carbon fiber electrodes. The results show good selectivity and sensitivity in the determination of low concentrations of UA in the presence of high concentrations of AA. By increasing the scan rate above 500 V s-1, voltammograms of UA in the presence of AA can be resolved because of the kinetic differences in the response of the two anions, without the need for a permselective film on the electrode. Results are also presented that demonstrate an effective way to reach a stable background current at bare carbon fiber electrodes, which is required in FSV because the signal from the analyte is smaller than the electrochemical signal from the background current. Signal-to-noise ratios at bare carbon fiber electrodes in FSV are improved, because the high temporal resolution in fast scan methods allows the acquisition of a large number of scans that can be signal averaged in a short period of time. In addition, large signals can be measured because the voltammetric peak current increases with increase in scan rate.
Electrochemistry↗
Expression of constitutively active IkappaB beta in T cells of transgenic mice: persistent NF-kappaB activity is required for T-cell immune responses.
The transcription factor NF-kappaB is normally sequestered in the cytoplasm by members of the IkappaB family, including IkappaB alpha, IkappaB beta, and the recently cloned IkappaB epsilon. Upon cellular activation, these inhibitors are rapidly phosphorylated on two amino-terminal serines, ubiquitinated, and degraded by the 26S proteasome, releasing a functional NF-kappaB. To determine the importance of IkappaB beta in NF-kappaB regulation in T cells, we generated transgenic mice expressing a constitutively active IkappaB beta mutant (mIkappaB beta) under the control of the lck promoter. The transgene contains the two critical N-terminal serine residues mutated to alanines and therefore no longer susceptible to degradation upon cell activation. mIkappaB beta is unable to totally displace IkappaB alpha from RelA-containing complexes, thus allowing a transient activation of NF-kappaB upon T-cell stimulation. However, mIkappaB beta completely blocks NF-kappaB activity after IkappaB alpha degradation. In addition, as a consequence of this inhibition, ikba expression is down regulated, along with that of other NF-kappaB-regulated genes. These transgenic mice have a significant reduction in the peripheral T-cell population, especially CD8+ cells. The remaining T cells have impaired proliferation in response to phorbol 12-myristate 13-acetate plus phytohemagglutinin or calcium ionophore but not to anti-CD3/anti-CD28 costimulation. As a result of these alterations, transgenic animals present defects in immune responses such as delayed-type hypersensitivity and the generation of specific antibodies against T-cell-dependent antigens. These results show that in nonstimulated T cells, IkappaB beta cannot efficiently displace IkappaB alpha bound to RelA-containing complexes and that persistent NF-kappaB activity is required for proper T-cell responses in vivo.
Animals↗
[Electronic forum of Revista de neurología: 'Internet, Medicine and Neuroglia'].
INTRODUCTION: In recent years Internet has drastically transformed the world of telecommunications. Questions about whether Internet and the global communication accessibility may be considered as a fundamental aspect in the medicine of today and tomorrow have been brought up. OBJECTIVE: Discuss current and future applications of Internet to the field of Medicine and Neurology. METHODS: Bibliographical review based on Medline and standard Web search engines. Forum sessions performed through Internet by means of Web-Chat. Forum coordination entirely performed by electronic mail. RESULTS: There are several opinions regarding the influence of Internet on Medicine and Neurology. Currently, the communication and collaboration between teams is improving. It allows scientific publications on-line, improves continuous remote training and facilitates information to patients. New applications like telemedicine need more development. CONCLUSIONS: Currently, Internet represents an efficient tool for collaboration, training and distribution of information in the field of Medicine and Neurology.
Humans↗
Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor.
Chemokines are a structurally related family of cytokines that are important for leukocyte trafficking. The C-C chemokine monocyte chemoattractant protein-1 (MCP-1) is a potent monocyte activator in vitro and has been associated with monocytic infiltration in several inflammatory diseases. One C-C chemokine receptor, CCR2, has been identified that mediates in vitro responses to MCP-1 and its close structural homologues. CCR2 has also recently been demonstrated to be a fusion cofactor for several HIV isolates. To investigate the normal physiological function of CCR2, we generated mice with a targeted disruption of the ccr2 gene. Mice deficient for CCR2 developed normally and had no hematopoietic abnormalities. However, ccr2(-/-) mice failed to recruit macrophages in an experimental peritoneal inflammation model. In addition, these mice were unable to clear infection by the intracellular bacteria, Listeria monocytogenes. These results suggest that CCR2 has a nonredundant role as a major mediator of macrophage recruitment and host defense against bacterial pathogens and that MCP-1 and other CCR2 ligands are effectors of those functions.
Animals↗
Gastric hyperplasia and increased proliferative responses of lymphocytes in mice lacking the COOH-terminal ankyrin domain of NF-kappaB2.
The nfkb2 gene encodes the p100 precursor which produces the p52 protein after proteolytic cleavage of its COOH-terminal domain. Although the p52 product can act as an alternative subunit of NF-kappaB, the p100 precursor is believed to function as an inhibitor of Rel/NF-kappaB activity by cytoplasmic retention of Rel/NF-kappaB complexes, like other members of the IkappaB family. However, the physiological relevance of the p100 precursor as an IkappaB molecule has not been understood. To assess the role of the precursor in vivo, we generated, by gene targeting, mice lacking p100 but still containing a functional p52 protein. Mice with a homozygous deletion of the COOH-terminal ankyrin repeats of NF-kappaB2 (p100(-/-)) had marked gastric hyperplasia, resulting in early postnatal death. p100(-/-) animals also presented histopathological alterations of hematopoietic tissues, enlarged lymph nodes, increased lymphocyte proliferation in response to several stimuli, and enhanced cytokine production in activated T cells. Dramatic induction of nuclear kappaB-binding activity composed of p52-containing complexes was found in all tissues examined and also in stimulated lymphocytes. Thus, the p100 precursor is essential for the proper regulation of p52-containing Rel/NF-kappaB complexes in various cell types and its absence cannot be efficiently compensated for by other IkappaB proteins.
Animals↗
B cells genetically deficient in the c-Rel transactivation domain have selective defects in germline CH transcription and Ig class switching.
The Ig heavy chain locus contains a number of binding sites for the transcriptional activator, c-Rel. In this study, we evaluated the capacity of B cells from mice made genetically deficient in the C-terminal, transactivation domain of the c-Rel protein (delta c-Rel) to undergo Ig class switching. Flow-cytometric and digestion circularization PCR analyses revealed that delta c-Rel B cells failed to switch to IgG3 in response to LPS alone, or to IgG1 or IgE in response to LPS + IL-4. This failure to switch to IgG3 or IgG1 was associated with a corresponding loss of germline CH gamma 3 or CH gamma 1 RNA. However, the defective switching to IgE in delta c-Rel B cells was associated with normal levels of germline CH epsilon RNA relative to control B cells. The ability of delta c-Rel B cells to switch to IgG1, in response to LPS + IL-4, could be restored through the action(s) of additional stimuli, and this was associated with induction of normal levels of germline CH gamma 1 RNA relative to controls. In contrast, LPS-activated B cells from delta c-Rel mice underwent normal switching to IgA in the presence of TGF-beta, relative to control B cells. This was associated with equivalent steady state levels of germline CH alpha RNA between the two B cell populations. These data are the first to demonstrate a key and selective role for c-Rel in the regulation of Ig class switching. Furthermore, distinct differences are revealed in the Ig isotype induction profiles of B cells lacking c-Rel activity vs those deficient in p50/nuclear factor-kappa B.
Animals↗
IkappaBalpha overexpression delays tumor formation in v-rel transgenic mice.
We have previously shown that transgenic mice expressing the oncoprotein v-Rel under the control of a T cell-specific promoter develop T cell lymphomas. Tumor formation was correlated with the presence of p50/v-Rel and v-Rel/v-Rel nuclear kappaB-binding activity. Since experimental evidence has led to the suggestion of a potential tumor suppressor activity for IkappaBalpha, we have studied the role of IkappaBalpha in the transforming activity of v-Rel by overexpressing IkappaBalpha in v-rel transgenic mice. Overexpression of IkappaBalpha in v-rel transgenic mice resulted in an extended survival, and the development of cutaneous T cell lymphomas of CD8(+)CD4(-) phenotype. These phenotypic alterations were associated with a dramatic reduction of p50/v-Rel, but not v-Rel/v-Rel nuclear DNA binding activity and an increased expression of the intercellular adhesion molecule 1. Our results indicate that v-Rel homodimers are active in transformation and that the capacity of v-Rel-containing complexes to escape the inhibitory effect of IkappaBalpha may be a key element in its transforming capability.
Animals↗
Absence of a persistently elevated 37 kDa fos-related antigen and AP-1-like DNA-binding activity in the brains of kainic acid-treated fosB null mice.
Chronic stimulation of the nervous system or acute administration of kainic acid results in a persistent increase in AP-1-like DNA-binding activity in the brain. However, the composition and function of these AP-1 complexes remain controversial. By comparing wild-type and fosB-null mice treated with kainic acid, we establish that the complexes comprise JunD in association with an approximately 37 kDa Delta-FosB species. Delta-FosB was expressed persistently in neurons in many areas of the CNS, even though fosB mRNA only increased transiently. This implies that the 37 kDa protein is very stable. fosB-/- mice are predisposed to seizures. Therefore, the chronic expression of Delta-FosB elicited by kainic acid seizures may be indicative of a compensatory/protective role in the pathophysiology of epilepsy.
Animals↗
Multifocal defects in immune responses in RelB-deficient mice.
Mice with a targeted disruption of the Rel/nuclear factor-kappaB family member RelB develop a complex inflammatory phenotype, myeloid hyperplasia, and splenomegaly due to extramedullary hemopoiesis. In this work, we report that RelB-deficient mice, in addition to the pathologic changes, were highly susceptible to infection by the facultative intracellular bacterium Listeria monocytogenes. RelB binds transcriptionally active kappaB motifs in the TNF-alpha promoter in normal cells, and in vitro studies with macrophages isolated from RelB-deficient animals revealed impaired production of TNF-alpha in response to LPS and IFN-gamma. RelB-deficient mice also were unable to mount a protective immune response against lymphocytic choriomeningitis virus. These results indicate a defective T cell-macrophage interaction and cytotoxic T cell response, respectively, in mice lacking RelB. Analysis of resting and specific Ab production demonstrated that while RelB is not required for the secretion of Ig isotypes that result from heavy chain class switching, it is necessary for normal production of Ag-specific IgG in response to T cell-dependent and -independent stimuli. Thus, RelB is not only essential for a normal hemopoietic system in the unchallenged animal, but also involved in various specific and nonspecific immune responses.
Animals↗
Monocyte chemotactic protein-3 (MCP-3)/fibroblast-induced cytokine (FIC) in eosinophilic inflammation of the airways and the inhibitory effects of an anti-MCP-3/FIC antibody.
Monocyte chemotactic protein-3 (MCP-3)/fibroblast-induced cytokine (FIC), a CC chemokine, is chemotactic for cells that typically infiltrate the late-phase allergic reaction. We developed a mouse model of airway inflammation to study the role of MCP-3/FIC. The immunization of mice with OVA resulted in Ag-specific IgE Ab production and the expression of mRNA for IL-4 in the lung tissue. Two weeks after immunization mice were challenged with the allergen by inhalation. Lungs were lavaged, and the tissue was examined at 2 or 24 h. Allergen challenge resulted in the increased recovery of leukocytes in the lavage fluid, but saline challenge did not. There was a significant increase in eosinophils (29 +/- 8% vs 1.2 +/- 0.2%) and lymphocytes (25 +/- 4% vs 5 +/- 2%) in the bronchoaveolar lavage fluid. Histologic examination of the lung demonstrated intense airway inflammation following OVA challenge. The expression of MCP-3/FIC and other CC chemokines (MCP-1, macrophage inflammatory protein-1alpha, and RANTES) was investigated by reverse transcription-PCR followed by densitometric analyses. The allergen challenge up-regulated the expression of mRNA for MCP-1, MCP-3/FIC, and macrophage inflammatory protein-1alpha at 2 and/or 24 h. Immunocytochemical staining for MCP-3/FIC showed that the allergen challenge induced the expression of MCP-3/FIC predominantly in the airway epithelium. Pretreatment of mice with an anti-MCP-3/FIC Ab significantly inhibited the OVA-induced airway inflammation and the bronchoalveolar lavage eosinophilia (8 +/- 2% vs 46 +/- 11% after control Ab, p < 0.03). We conclude that MCP-3/FIC plays a significant role in the allergen-induced eosinophilic inflammation of the airways.
Animals↗
p50-NF-kappaB complexes partially compensate for the absence of RelB: severely increased pathology in p50(-/-)relB(-/-) double-knockout mice.
RelB-deficient mice (relB(-/-)) have a complex phenotype including multiorgan inflammation and hematopoietic abnormalities. To examine whether other NF-kappaB/Rel family members are required for the development of this phenotype or have a compensatory role, we have initiated a program to generate double-mutant mice that are deficient in more than one family member. Here we report the phenotypic changes in relB(-/-) mice that also lack the p50 subunit of NF-kappaB (p50(-/-)). The inflammatory phenotype of p50(-/-)relB(-/-) double-mutant mice was markedly increased in both severity and extent of organ involvement, leading to premature death within three to four weeks after birth. Double-knockout mice also had strongly increased myeloid hyperplasia and thymic atrophy. Moreover, B cell development was impaired and, in contrast to relB(-/-) single knockouts, B cells were absent from inflammatory infiltrates. Both p50(-/-) and heterozygous relB(-/+) animals are disease-free. In the absence of the p50, however, relB(-/+) mice (p50(-/-)relB(-/+)) had a mild inflammatory phenotype and moderate myeloid hyperplasia. Neither elevated mRNA levels of other family members, nor increased kappaB-binding activities of NF-kappaB/Rel complexes could be detected in single- or double-mutant mice compared to control animals. These results indicate that the lack of RelB is, in part, compensated by other p50-containing complexes and that the "classical" p50-RelA-NF-kappaB activity is not required for the development of the inflammatory phenotype.
Abnormalities, Multiple↗
Activation of the nuclear factor-kappaB by Rho, CDC42, and Rac-1 proteins.
The Rho family of small GTPases are critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to the cell nucleus, including the JNK/SAPK signaling pathway, the c-fos serum response factor, and the p70 S6 kinase. Here we report a novel signaling pathway activated by the Rho proteins that may be responsible for their biological activities, including cytoskeleton organization, transformation, apoptosis, and metastasis. The human RhoA, CDC42, and Rac-1 proteins efficiently induce the transcriptional activity of nuclear factor kappaB (NF-kappaB) by a mechanism that involves phosphorylation of Ikappa Balpha and translocation of p50/p50 and p50/p65 dimers to the nucleus, but independent of the Ras GTPase and the Raf-1 kinase. We also show that activation of NF-kappaB by TNFalpha depends on CDC42 and RhoA, but not Rac-1 proteins, because this activity is drastically inhibited by their respective dominant-negative mutants. In contrast, activation of NF-kappaB by UV light was not affected by Rho, CDC42, or Rac-1 dominant-negative mutants. Thus, members of the Rho family of GTPases are involved specifically in the regulation of NF-kappaB-dependent transcription.
3T3 Cells↗
[Serotypes of the human immunodeficiency virus type 1 in Madrid].
BACKGROUND: HIV-1 shows high genetic variability, mainly in the genomic region codifying the envelope proteins, which are the most immunogenic. This fact explains the high heterogeneity of antibodies against HIV-1 epitopes. Both genetic and serologic diversity has allowed to classify HIV-1 variants in several subtypes (genotypes and serotypes, respectively). The clinical and epidemiological significance of infection caused by each subtype remains to be clarified. PATIENTS AND METHODS: Serum samples from 154 HIV-seropositive individuals living in Madrid were studied. Serotyping was performed using 4 peptides belonging to the V3 env region. Epidemiological and clinical variables examined in these patients were the route of infection, the year in which HIV infection occurred, the country of birth, and the rate of disease progression (rapid versus slow). RESULTS: 148 (96.2%) samples could be serotyped, and the B class was recognized in 131 (88.5%) of them. Serotype A/C was found in 9 (6.1%). Two samples (1.3%) reacted to peptide E; however, both were also reactive against the B peptide, suggesting co-infection with B and E subtypes. Six samples were EIA-reactive for HIV-1/2 but were typed as HIV-2 alone. Infection with serotypes A/C was more frequent amongst immigrants, mainly in Africans. There was not association between any subtype and the route of infection neither a different rate of disease progression. CONCLUSION: HIV-1 serotype B is the most frequently found in HIV-seropositive individuals living in Madrid, without association with the route of infection or the clinical course of the disease. Serotypes A/C and E were found sporadically, mainly among immigrants.
Africa↗
Genetic approaches to study Rel/NF-kappa B/I kappa B function in mice.
The generation of animal models in which individual members of a gene family are genetically altered is a particularly attractive way to elucidate their function-Members of the Rel/NF-kappa B/I kappa B family constitute an important network of transcription factors and regulatory proteins that control the expression of numerous cellular and viral genes crucial for a variety of processes. A few examples are developmental pattern formation and immune response in Drosophila, viral replication, and immune, inflammatory, acute phase and stress responses in vertebrates. The findings from knockout and transgenic mice developed to study Rel/NF-kappa B/I kappa B function in vivo are reviewed here. In general, these studies point to the essential role of these factors in the development and function of the vertebrate immune system.
Animals↗
Significance of indeterminate reactivity to human T-cell lymphotropic virus in western blot analysis of individuals at risk.
Current tests to confirm human T-cell lymphotropic virus (HTLV) infection in individuals at risk of retroviral infection commonly yield indeterminate results. To assess the significance of HTLV-seroindeterminate reactivities in a high-risk population, 16 at-risk individuals who had this serologic pattern by Western blot were studied using a polymerase chain reaction (PCR) assay. Human T-cell lymphotropic virus type II infection was confirmed by the presence of virus-specific nucleic acid in four patients. However, PCR analysis was negative in the remaining 12 individuals. These results indicate strongly that all specimens from at-risk individuals with nondiagnostic HTLV reactivity by current Western blot assay should continue to be considered inconclusive, requiring further testing by more sensitive tests.
Antibodies, Viral↗
Co-infection with several HCV genotypes enhances liver damage in patients with chronic hepatitis C. The Hepatitis/HIV Spanish Study Group.
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Genotype↗
A pilot study on the efficacy of escalating dosage of alpha-interferon for chronic hepatitis C in HIV-infected patients. The Hepatitis/HIV Spanish Study Group.
Chronic liver disease caused by hepatitis C virus (HCV) seems to present a more accelerated course in HIV-infected patients, leading to cirrhosis and liver failure in a shorter period of time than in HIV-negative individuals. As efforts to increase life expectancy in HIV-infected people progress, substantial morbidity and mortality from HCV infection is likely to arise amongst subjects infected parenterally, such as injecting drug users, haemophiliacs and transfusion recipients. Preliminary results have suggested that alpha-interferon (IFN) treatment allows higher rates of response in HIV-infected patients with higher CD4+ lymphocyte counts, suggesting a primary dependence of IFN on a preserved immune system in order to act appropriately. In an open, multicentre, prospective trial we analysed whether the use of larger doses of IFN, through an escalating schedule, might overcome the limits imposed by immune dysfunction. An interim analysis performed in 29 patients concluded that escalating the dosage did not improve the rate of response to IFN. In fact, only one (8.3%) out of 12 patients without response after 3 months of being on IFN therapy achieved response after the dosage was increased from 5 MU to 8 MU s.c. three times a week. Moreover, he relapsed 3 months after completion of treatment for 1 year.
Adult↗