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Biomedical subjects

R Bravo

Publications and source records attributed to R Bravo.

At least 73 records · Page 4Linked to original sources
PubMed1996

Expression of FosB during mouse development: normal development of FosB knockout mice.

FosB, one of the members of the Fos family, is rapidly induced in many cell types upon stimulation and has a stimulatory effect on the proliferation of cultured cells. To understand the tissue distribution of FosB, we have studied its expression pattern by immunohistochemistry in newborn and late embryonic stage mice. These results show that FosB is widely expressed with the highest levels of expression observed in both bony and cartilagenous regions of developing bone. FosB is also detected within whisker follicles, liver, and epidermal tissue. To study the role of FosB in mammalian development we generated embryonic stem (ES) cells, mice and mouse embryo fibroblasts (MEFs) that are deficient for FosB. FosB -/- mice are born at a normal frequency, are fertile and present no obvious phenotypic or histologic abnormalities. FosB-deficient ES cells and MEFs proliferate and enter the S phase normally and we do not find upregulation of other fos family genes to compensate for the lack of FosB. However, we do find that the induction of two AP-1 containing genes is reduced after stimulation of FosB-deficient cells, demonstrating that FosB does indeed play a functional role in transcriptional regulation.

Animals↗
PubMed1996

Molecular mechanisms of TNFalpha cytotoxicity: activation of NF-kappaB and nuclear translocation.

The murine fibrosarcoma cell line WEHI 164 is well known for its susceptibility to tumor necrosis factor (TNFalpha). We have studied the activation of the transcription factor NF-kappaB when WEHI 164 cells are challenged with TNFalpha. NF-kappaB is retained in the cytoplasm of unchallenged cells by its inhibitor IkappaB-alpha. Upon cellular stimulation, IkappaB-alpha is functionally inactivated and NF-kappaB translocated to the nucleus. The extent of the cytotoxic effect and that of nuclear translocation of NF-kappaB show the same TNFalpha dependence. TNFalpha induces a rapid and transient activation of NF-kappaB in WEHI 164 cells which is followed by a second, long lasting phase in which the amount of NF-kappaB complex in the nucleus remains at about 50% of maximum. Upon TNFalpha treatment, IkappaB-alpha is rapidly degraded. However, newly synthesized IkappaB-alpha can be demonstrated later in the cell cytosol. A persistent nuclear localization of NF-kappaB is an obligatory step for the cytotoxic effect to take place. Thus, WEHI 164 cells treated with TNFalpha for up to 6 h can be rescued as long as NF-kappa relocalizes to the cytoplasm in its inactive form. On the other hand, TNFalpha treatments as short as 15 min cause the cytotoxic effect provided that NF-kappaB remains in the nucleus. The activation of NF-kappaB is controlled by both phosphorylation and proteolysis. The activation of NF-kappaB can be blocked by the cysteine protease inhibitor calpain inhibitor I and the serine protease inhibitor TPCK. Signal-induced phosphorylation of IkappaB-alpha does not lead to the dissociation of the inhibitor from NF-kappaB. Phosphorylation appears to regulate the inhibitory activity of IkappaB-alpha both positively and negatively. since inhibitors of protein kinases have opposite effects. Thus, treatment of cells with staurosporin induced a partial activation of NF-kappaB and was synergistic with TNFalpha induced activation. Calphostin C, on the other hand, can block the activation of NF-kappaB by TNFalpha, also blocking its proteolytic degradation.

Animals↗
PubMed1996

[The treatment of chronic hepatitis C with interferon in patients infected with the human immunodeficiency virus. The Spanish Group for the Study of Viral Hepatitis in HIV+ Patients].

BACKGROUND: Alfa-interferon (aIFN) is widely recommended for the treatment of chronic hepatitis C (CHC). Hepatitis C virus (HCV) infection is very common in injecting drug users (IDUs), which in Spain represent the large number of HIV-infected persons. Interaction between human immunodeficiency virus (HIV) and HCV in coinfected patients might accelerate the clinical course of HCV-associated liver disease. The efficacy and safety of aIFN therapy in HIV-infected patients with CHC is not well known. PATIENTS AND METHODS: In a multicenter, prospective, open, non randomized and partially controlled study, we compared the efficacy and safety of aIFN therapy in 119 patients with CHC, of whom 90 were HIV-positive and 29 HIV-negative. Interferon was started at 5 mega U tiw for 3 months, followed in responders by 3 megaU tiw for additional 9 months. RESULTS: One hundred seven patients completed the study. A normalization of the aminotransferase values at the end of treatment (complete response, CR) was observed in 26/80 (32.5%) HIV-positive and 10/27 (37.0%) HIV-negative individuals (p = 0.666). Relapses at 12 months of stopping aIFN were seen in 30.8% of HIV-positive subjects and 12.5% of HIV-negatives (p = 0.403). Side effects were uncommon and did not have severity; only one patient required to stop the medication. However, 3 HIV-positive subjects treated with aIFN (3.5% of them) showed an irreversible fall of CD4+ T-cells below half the baseline values. CONCLUSION: HIV-infected patients with CHC seems to respond to aIFN with a similar rate than HIV-negatives. Moreover, the drug is similarly well tolerated in both groups of patients, although a fall of CD4+ T-cells is an unusual side effect of particular relevance observed in HIV-infected patients.

AIDS-Related Opportunistic Infections↗
PubMed1996

Overexpression of RelB in transgenic mice does not affect I kappa B alpha levels: differential regulation of RelA and RelB by the inhibitor protein.

In mouse lymphoid tissues, RelB heterodimers represent the constitutive kappa B-binding activity, whereas RelA and c-Rel complexes most likely are involved in inducible kappa B-binding and gene activation. Our laboratory has previously shown that the potential excess of NF-kappa B activity in transgenic mice overexpressing RelA is counteracted by a dramatic increase in I kappa B alpha, mainly due to its increased stability through association with RelA. As an attempt to elucidate the in vivo mechanisms that lead to the constitutive DNA-binding activity of RelB heterodimers, we have generated mouse lines overexpressing a relB transgene in a position-independent and copy number-dependent manner. Expression of RelB in these transgenic animals is very high in immature thymocytes and restricted to T cell areas in secondary lymphoid tissues. In contrast to the results obtained with RelA-transgenic thymocytes, we demonstrate here that overexpression of RelB results in a dramatic increase in overall kappa B-binding activity. Interestingly, I kappa B alpha protein levels are not altered in the RelB-transgenic animals, indicating that within the same cell type RelA and RelB complexes are differentially regulated by I kappa B alpha.

Animals↗
PubMed1996

Development of a multiplex PCR assay for the simultaneous detection and discrimination of HIV-1, HIV-2, HTLV-I and HTLV-II.

BACKGROUND: Multiplex polymerase chain reaction (PCR) has been established as a general technique for the simultaneous amplification of different target sequences. Uses of multiplex include pathogens identification, linkage analysis and genetic disease diagnosis. The high sensitivity of PCR may produce false-positive results due to contamination with previously amplified material. OBJECTIVES: To develop a multiplex PCR technique that can simultaneously detect and discriminate human immunodeficiency virus types 1 and 2 (HIV-1/2) and human T-lymphotropic virus types 1 and 2 (HTLV-I/II) proviral sequences. Such a method should incorporate a system that prevents the occurrence of false-positive results. STUDY DESIGN: Combinations of four primer pairs, one for each retrovirus, were assayed in order to determine the combination of oligonucleotides as well as the PCR conditions that yield the most specific and sensitive coamplification of proviral sequences. To prevent contamination with DNA from previous PCR amplifications, the uracil N-glycosylase (UNG) system was incorporated into the coamplification format. RESULTS: A combination of primer pairs from the gag region of HIV-1, env of HIV-2, pol of HTLV-I and tax of HTLV-II yielded specific and sensitive coamplification of proviral sequences. The UNG system was incorporated and shown to be efficient in the degradation of contaminating DNA. In the evaluation of a serologically well established panel of singly and dually infected individuals, the assay detected 20/22 HIV-1, 8/10 HIV-2, 8/8 HTLV-I and 8/8 HTLV-II infections.

Cell Line↗
PubMed1996

Interferon alpha for the treatment of chronic hepatitis C in patients infected with human immunodeficiency virus. Hepatitis-HIV Spanish Study Group.

Liver disease secondary to hepatitis C virus (HCV) infection is a rising cause of morbidity and mortality among individuals who have been infected parenterally with human immunodeficiency virus (HIV) such as injection drug users, hemophiliacs, and transfused patients. We analyzed both the efficacy of interferon (IFN) alpha therapy in these patients and the predictors of response to this agent. A total of 119 patients with chronic hepatitis C (90 of whom were infected with HIV and 29 of whom were not) were included in a multicenter, prospective, open, nonrandomized observational study. IFN-alpha was given subcutaneously in a dosage of 5 million units three times a week during a 3-month period; those patients who responded received a dose of 3 million units given subcutaneously three times a week for an additional 9 months. One hundred seven patients completed the study; the level of aminotransferases returned to normal and sera became negative (complete response) for HCV RNA in 26 (32.5%) of 80 HIV-infected patients and 10 (37.0%) of 27 non-HIV-infected patients (P = .666) after completion of the treatment. Two variables were independently associated with a response in HIV-infected patients: a CD4+ T lymphocyte count of > 500 x 10(6)/L and a baseline HCV viremia level of < 10(7) copies/mL. In the 12 months following treatment, relapses occurred in 30.8% of the HIV-infected patients and 12.5% of non-HIV-infected patients (P = .403).

Adult↗
PubMed1996

Constitutive expression of Bc1-3 in thymocytes increases the DNA binding of NF-kappaB1 (p50) homodimers in vivo.

Previous studies have indicated that Bcl-3 interacts through its ankyrin repeats with the transcriptional factors NF-kappaB1 (p50) and NF-kappaB2 (p52), affecting their biological activities. To further investigate the role of Bcl-3 in vivo and its association with the NF-kappaB proteins, we have generated transgenic mice constitutively expressing Bcl-3 in thymocytes. The results indicate that Bcl-3 is associated with endogenous p50 and p52 in nuclear extracts from transgenic animals. Remarkably, constitutive expression of Bcl-3 in these cells augments the DNA binding activity of p52 homodimers. This effect could be reproduced in vitro and is blocked by anti-Bcl-3 antibodies. We have also shown that Bcl-3 is phosphorylated in thymocytes and that its dephosphorylation greatly decreases the effect on p50 homodimers.

Animals↗