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J R Rodríguez

Publications and source records attributed to J R Rodríguez.

At least 19 recordsLinked to original sources
PubMed2001

Protective immune response against cutaneous leishmaniasis by prime/booster immunization regimens with vaccinia virus recombinants expressing Leishmania infantum p36/LACK and IL-12 in combination with purified p36.

In susceptible mice Leishmania infection triggers a CD4(+) Th2 response that has been correlated with evasion of the host immune system. To develop approaches that might trigger a Th1 response leading to protection against Leishmania we generated vaccinia virus recombinants (VVr) expressing the relevant p36/LACK protein of Leishmania infantum (VVp36) or co-expressing p36/LACK and interleukin-12 (VVp36IL12). Susceptible BALB/c mice were immunized with the VVr in various prime/booster protocols that included purified p36/LACK protein, followed 3 weeks later by a challenge with live L. major promastigotes. The course of the infection was monitored by measuring lesion development, parasite load and immunological parameters (IFN-gamma and IL-10 secretion by in vitro-stimulated lymphocytes, and specific IgG isotypes), before and after challenge. We found protocols of prime/booster immunization (VVp36/VVp36; VVp36IL12/p36; p36/VVp36IL12) that elicited different levels of protection in infected animals. The protocol of priming with purified p36 followed by a booster with VVp36IL12 induced 52% reduction in lesion size and a two-log unit reduction in parasite load. This partial protection correlated with activation of a specific Th1 type of immune response. These protocols could be of interest in the prophylaxis against Leishmania spp. and other parasitic diseases.

Animals↗
PubMed2001

The major core protein P4a (A10L gene) of vaccinia virus is essential for correct assembly of viral DNA into the nucleoprotein complex to form immature viral particles.

The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.

Animals↗
PubMed2000

Chimeras between the human immunodeficiency virus (HIV-1) Env and vaccinia virus immunogenic proteins p14 and p39 generate in mice broadly reactive antibodies and specific activation of CD8+ T cell responses to Env.

A vaccine based on the envelope protein (Env) of the human immunodeficiency virus type 1 (HIV-1) that triggers widely reactive antibodies might be a desirable approach to control virus infection. To expose epitopes which could induce broadly reactive antibodies against HIV-1 Env, we have generated vaccinia virus (VV) recombinants that express Env fused at its N- or C-terminus with two major antigenic proteins of VV, p14 (A27L gene) and p39 (A4L gene). Biochemical analysis of the chimeric proteins in cell cultures revealed that, in all cases, recombinant viruses expressed the correct fusion proteins. When p14 or p39 are fused at the N-terminus of Env the chimeric proteins are poorly glycosylated but when p14 or p39 are fused at the C-terminus of Env, the chimeric proteins are fully glycosylated. In Balb/c mice, immunisation with the referred VV recombinants induced similar levels of CD8+ T cell specific responses to Env as immunisation with the entire Env protein. The humoral immune response triggered by the fusion proteins was broader than in animals immunised with VV expressing the entire Env (VVEnv1), and was directed to epitopes outside of the V3 loop (V1/V2, C1, C2, C4). One of the chimeric constructs induced a better neutralising antibody response than VVEnv1. We conclude that fusing VV proteins p14 or p39 to Env provides an effective means to induce broadly reactive antibodies and CD8+ T cell responses to Env. This approach might have utility against HIV and other pathogens.

AIDS Vaccines↗
PubMed2000

Long-term evolution of local, proximal and remote astrocyte responses after diverse nucleus basalis lesioning (an experimental Alzheimer model): GFAP immunocytochemical study.

A study on long-term astrocytic responses (from 1 day to 20 months after lesioning in 4-month-old rats, and from 1 day to 6 months in 20-month-old rats) to diverse unilateral damage of the nucleus basalis (nbM) by injection of 40 nmol of ibotenic acid, or 50 or 100 nmols of quisqualic acid was performed using a histochemical method (immunoreactivity against the glial fibrillary acidic protein GFAP). Glial reactivity (i.e., isolated or clustered hypertrophic and/or hyper-reactive astrocytes) was evaluated in several ipsilateral and contralateral brain regions: the 'local response' within the damaged nbM region; the 'proximal response' (a new concept proposed by us) in the non-damaged structures neighbouring the nbM; and the 'remote response' in the ipsilateral brain cortex and in the contralateral cortex and nbM. In 4-month-old animals, the remote cortical glial responses, independent of the involution of cortical cholinergic activity and randomly located in layers I-V of motor and somatosensory cortical regions, were similar in appearance over a long period (13-20 months), with the highest reactivity 45 days after lesioning. The proximal response lasted from 1 day to 13 months and afterwards tended to disappear. Contralateral reactivity and ipsilateral cortical scars were observed. The local (nbM) glial response was maintained throughout the period studied. Subsets of astrocytes of different reactivities were observed, most of their elements being highly intermeshed. In 20-month-old animals, nbM lesions produced less positive, but similar, glial reactive patterns. This glial reactivity was superposed onto the glial reactivity of old age. All these results are discussed. The maintenance of reactive astrocytes many months after lesioning suggests the existence of cellular factors other than those produced by damaged nbM neurons. Taking into account the role of glial cells under pathological conditions, it is possible that these reactive astrocytes in humans could promote neurodegenerative processes, such as amyloid plaque formation and neurodegeneration (Alzheimer's disease). Along this line, nbM cholinergic involution could then originate cortical involution through induced reactive astrocytosis.

Age Factors↗
PubMed2000

Differences between blood flow as indicated by the hemodialysis blood roller pump and blood flow measured by an ultrasonic sensor.

BACKGROUND/AIM: The ultrasonic transit time is currently the best method for measuring the blood flow rate in the extracorporeal hemodialysis circuit. The purpose of this study was to analyze the differences between blood flow as indicated by the hemodialysis blood roller pump (prescribed blood flow) and by an ultrasonic flowmeter (delivered blood flow). METHODS: The ultrasonic blood flow was measured in 20 patients on chronic hemodialysis who were dialyzed through an arteriovenous fistula. During each dialysis session the ultrasonic blood flow was measured at three different blood roller pump flow rates (300, 350, and 400 ml/min). In order to analyze the influence of inflow and outflow pressures on blood flow, this study was conducted during nine consecutive dialysis sessions during which needles of different sizes were used. RESULTS: The ultrasonic flow was always lower than indicated by the blood roller pump: 265+/-12, 304+/-15, and 341+/- 19 ml/min for blood roller pump flow rates of 300, 350, and 400 ml/min, respectively (variability: -11.6, -13.1, and -14.8%, respectively). An univariate regression analysis showed that the reduction in flow recorded ultrasonically correlated with both venous blood line pressure (r = -0.2679, p<0.001) and negative arterial blood line pressure (r = 0. 6773, p<0.001). By multivariate analysis, only the arterial blood line pressure has a predictive value. When all measurements were grouped by arterial blood line pressure ranges, the variability between ultrasonic blood flow and blood roller pump flow was found to be similar in those groups with the same arterial blood line pressure, regardless of the blood roller pump flow rate. CONCLUSIONS: The blood flow indicated by the dialysis blood roller pump is always greater than the delivered blood flow, and this difference is in turn conditioned by the negative pressure induced by the blood roller pump in the arterial blood line.

Blood Flow Velocity↗
PubMed2000

Integrative medicine: a paradigm shift in medical education and practice.

The use of alternative/complementary medicine has been increasing considerably. Conventional medicine must begin to address issues related to the use, safety, regulation, research and education of alternative/complementary medicine. Integrative medicine combines conventional medicine and alternative complementary practices. Integrative medicine is an innovative approach to medicine and medical education. It involves the understanding of the interaction of the mind, body and spirit and how to interpret this relationship in the dynamics of health and disease. Integrative medicine shifts the orientation of the medical practice from disease based approach to a healing based approach. It does not reject conventional medicine nor uncritically accepts unconventional practices. Integrative medicine is an effective, more fulfilling human approach to medicine based on the benefit of the patient by following good medicine practices in a scientific manner.

Complementary Therapies↗
PubMed1999

The vaccinia virus 39-kDa protein forms a stable complex with the p4a/4a major core protein early in morphogenesis.

The vaccinia virus (VV) 39-kDa protein, the product of the A4L gene, is a highly antigenic protein of the viral core. Pulse-chase and immunoprecipitation experiments have shown that the 39-kDa protein interacts with p4a (encoded by the A10L gene), the precursor of the most abundant virion protein. This interaction is maintained with the processed 4a form that arises during virion maturation. The controlled disruption of mature viral particles showed that the 39-kDa and 4a proteins are tightly bound within the virion. Immunoelectron microscopy showed that both proteins first localize within the cytoplasm and later accumulate inside the viral factories, reaching these locations via a mechanism apparently unrelated to cellular membranes. Double labeling experiments showed a colocalization of both proteins in all virus-induced structures.

Animals↗
PubMed1999

IL-12 delivery from recombinant vaccinia virus attenuates the vector and enhances the cellular immune response against HIV-1 Env in a dose-dependent manner.

To develop vaccination strategies against HIV-1 infection aimed to specifically enhance the cell-mediated immunity (CMI), we have engineered vaccinia virus (VV) recombinants expressing HIV-1 Env (rVVenv) and murine IL-12 (rVVlucIL-12) genes or coexpressing both genes (rVVenvIL-12). In mice inoculated with rVVlucIL-12 there is a rapid clearance of the virus, and this correlates with the induction of high levels of IL-12 and IFN-gamma in serum and spleen early after infection. Enzyme-linked immunospot analysis of mice inoculated with rVVlucIL-12, revealed a nearly 2-fold increase in the number of specific anti-VV CD8+ T cells compared with that in mice given control rVV, and the serum Ab response was biased in favor of a Th1 response. An enhancement of about 2-fold in the number of anti-gp160 IFN-gamma-secreting CD8+ T cells was observed in mice inoculated with rVVenvIL-12, when a dose of 1 x 107 PFU/mouse was used, but this enhancement was not observed when mice were given 5 x 107 PFU. This variation with virus dosage was confirmed in mice immunized simultaneously with different multiplicities of rVV expressing singly the env or IL-12 genes. The highest specific CMI was obtained in mice coadministered a low dose (2 x 104 PFU) of rVVlucIL-12 and 1 x 107 PFU of rVVenv. Our findings provide evidence for specific enhancement of the CMI to HIV-1 Env by the differential expression of IL-12 and env genes delivered from VV recombinants. This approach can be of wide vaccination interest as a means to improve immune responses to other Ags.

Adjuvants, Immunologic↗
PubMed1999

Transport of CSF antibodies to Galpha subunits across neural membranes requires binding to the target protein and protein kinase C activity.

In the light of functional studies, it has been suggested that antibodies directed to alpha subunits of G-proteins delivered into cerebrospinal fluid (CSF) reached and blocked the function of neural transducer proteins. Current understanding indicates that IgGs do not move freely across plasma membranes. Therefore, to characterize the uptake of these antibodies by neural cells, anti-Gi2alpha IgGs were labeled with 125I, fluorescein or with gold particles. The expression of Galpha subunits was also reduced by blocking their mRNA with antisense oligodeoxynucleotides (ODN). Following intracerebroventricular (icv) injection of gold-conjugated anti-Gi2alpha IgGs, electrondense particles entered and became distributed in the cytoplasm and plasma membranes of neural cells. Scattered particles were also found in dendrites and nuclei. Unlabeled IgGs diminished cerebral signals of fluorescein-labeled anti-Galpha IgGs, indicating that this uptake can be saturated. Cerebral radiostaining promoted by in vivo anti-Gi2alpha 125I-IgGs was almost absent in Gi2alpha knocked-down mice, but not after decreasing the quantity of Gzalpha subunits. The immunosignals of CSF anti-Galpha 125I-IgGs, as well as the impairment of opioid-evoked antinociception, were increased by agonist-induced activation of G protein-coupled receptors. The impairing effect of the antibodies on opioid-evoked antinociception was prevented by agents blocking the cellular uptake of proteins, i.e., cytochalasin B, BSA, DMSO, H7, and by down regulation of protein kinase Cbeta1 (PKCbeta1). In mice treated with an ODN to PKCbeta1 mRNA, 125I-IgGs to Gi2alpha subunits remained bound to periventricular structures and did not spread to deeper areas of the CNS. These results indicate that IgGs delivered into the CSF show a saturable binding to Galpha subunits that translocate to the external side of the neural membrane before being internalized by a PKCbeta1-dependent mechanism.

Analgesics↗
PubMed1999

Enhanced CD8+ T cell response to HIV-1 env by combined immunization with influenza and vaccinia virus recombinants.

With the aim to determine if immunization with two different live recombinant viral vectors could lead to an enhancement of the cellular immune response to HIV-1 antigens, we have characterized the CD8+ T cell response elicited against the V3 loop epitope from HIV-1 env protein in Balb/c mice immunized with either: a recombinant influenza virus (Flu-Env) expressing the V3 loop epitope from HIV-1 strain IIIB, a vaccinia virus recombinant (VV-Env) expressing the complete HIV-1-IIIB env protein, or a combination of both. The CD8+ T cell response, measured by the ELISPOT assay, in animals primed with Flu-Env and boosted with VV-Env was 5 to 6 times higher than in animals inoculated with either Flu-Env or VV-Env alone. Similar results were obtained with recombinant viruses expressing the V3 loop epitope or the complete env protein, respectively, from the MN strain of HIV-1. Our results indicate that the use of two different live vectors for priming and boosting has a synergistic effect on the immune response against HIV-1, and could represent a novel vaccination strategy against AIDS.

Amino Acid Sequence↗
PubMed1999

Myr+-Gi2 alpha and Go alpha subunits restore the efficacy of opioids, clonidine and neurotensin giving rise to antinociception in G-protein knock-down mice.

In mice whose Gi/o-protein function had been impaired by antisense 'knock-down' or pertussis toxin treatment, i.c.v. injection of myr+-Gi/o alpha subunits restored the effectiveness of beta-endorphin, morphine, DPDPE, clonidine and neurotensin to produce antinociception. Myr+-G alpha subunits of the class of G-proteins actually impaired were more effective than unlike but related myr+-G alpha subunits. Selectivity was noted in that only exogenous myr+-G alpha subunits affected (enhanced) the activity of agonists in G alpha-deficient signalling systems. This treatment had little effect on agonist potency when the impairment resided at the receptor level. The potential of the opioids, clonidine and R-PIA to increase G alpha-related in vitro hydrolysis of GTP was also re-established after injecting myr+-Gi2 alpha subunits into Gi2-knocked-down mice. Myr+-Gi2 alpha subunits pre-incubated with GTPgammaS or GDPbetaS before i.c.v. injection did not improve the activity of agonists in vivo (antinociception) or in vitro (regulation of low Km GTPase). After impairing the function of PKCbeta1 by antisense treatment or with the inhibitor H7, the effect of myr+-G alpha subunits on agonist potency was prevented. Electron microscope analysis showed the entry of gold-conjugated myr+-G alpha subunits into neural cells. These particles were found in the cytoplasm, associated with the plasma membranes of different neuronal processes and also in synaptic junctions. In cultured neurons and astrocytes myr+-Gi2 alpha-associated fluorescence was internalised in a dose-dependent manner and distributed in the plasma membrane and cytosol, as well as in nuclei of dividing astrocytes. Thus, G alpha subunits in CSF enter into neurons and functionally couple to the receptor-triggered signalling cascade. As G-proteins have been implicated in the pathophysiology of several neural disorders, this finding may be valuable in the therapy of such dysfunctions.

Analgesics↗
PubMed1999

Treatment of secondary hyperparathyroidism in hemodialyzed patients with high-dose calcium carbonate without vitamin D3 supplements.

BACKGROUND: Vitamin D compounds are usually indicated for the treatment of secondary hyperparathyroidism in dialysis patients. The possibility to induce a reversal of hyperparathyroidism with calcium supplementation alone is controversial. The present study was conducted to assess if oral calcium carbonate may constitute a therapeutic option for the control of hyperparathyroidism in patients with high PTH concentrations at the beginning of the treatment with chronic hemodialysis. METHODS: Thirty-one patients with end-stage renal failure with an intact PTH concentration above 250 pg/ml at the beginning of chronic hemodialysis therapy were treated with high doses of calcium carbonate; no patient received either aluminium-containing binders or vitamin D compounds. To minimize hypercalcemia, a calcium dialysate concentration of 2.5 mEq/l was used in all patients. The goal of the study was to reduce the intact PTH concentration to 250 pg/ml with oral calcium carbonate supplements alone. RESULTS: Throughout the first year on hemodialysis treatment, the intact PTH concentration decreased from 538 +/- 256 to 251 +/- 218 pg/ml (p < 0.001). By the end of the study, the therapeutic objective was achieved in 22 patients (71%) ('responder' group). The remaining 9 patients were classified as the 'treatment failure' group. The basal intact PTH concentration was not different between both groups (508 +/- 235 vs. 612 +/- 303 pg/ml, respectively, p = n.s.), but 5 'treatment failure' patients admitted to take a dose of calcium carbonate lower than that prescribed. There were 40 episodes of hyperphosphatemia (11% of all measurements) in 7 of 31 patients, 5 of them belonged to the noncompliance 'treatment failure' patients. Only 15 episodes (4% of all measurements) of transient hypercalcemia (range 11.1 - 11.9 mg/dl) were detected in 8 patients. CONCLUSIONS: Secondary hyperparathyroidism in hemodialysis patients can often be reverted by oral calcium carbonate alone. But a good adherence to treatment is absolutely necessary.

Administration, Oral↗
PubMed1998

Conception after iodine-131 therapy for differentiated thyroid cancer.

We reviewed the medical history of 26 women with differentiated thyroid cancer who became pregnant after receiving therapeutic doses of radioactive iodine (131I) in order to document the possibility of a greater risk for disorders in these pregnancies. There were a total of 39 pregnancies, 6 of which occurred during the first year after therapy. In 3 cases the following anomalies were encountered: a male suffering Trisomy 18 (Edward's syndrome), a female with constitutional aplastic anemia, and a male with a congenital hip dysplasia. Of the 33 pregnancies that occurred after the first year post-therapy, there were 2 spontaneous abortions and a male effected by ureteral stenosis. Based on these data, although it cannot be confirmed that these congenital disorders are due to the 131I therapy, we recommend that pregnancy be avoided for the first year after therapy.

Abortion, Spontaneous↗
PubMed1998

Vaccinia virus 15-kilodalton (A14L) protein is essential for assembly and attachment of viral crescents to virosomes.

Early stages in vaccinia virus (VV) assembly involve the recruitment of cellular membranes from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to virus factories (or virosomes). The key viral factors involved in this process are not yet known. We have previously identified and characterized two viral proteins, of 21 kDa (A17L gene) and 15 kDa (A14L gene), that associate with tubulovesicular elements related to the ERGIC and are localized in viral membranes at all stages of virion assembly. We showed that the 21-kDa protein is not responsible for the recruitment of membranes from the ERGIC to viral factories. However, it appears to be essential for the organization of viral membranes. In this investigation we have generated a VV recombinant, VVindA14L, in which the expression of the A14L gene is inducibly regulated by the Escherichia coli lacI operator-repressor system. Repression of 15-kDa protein synthesis has a dramatic effect on virus yields and severely impairs plaque formation. Compared to wild-type VV, reduced amounts of 15-kDa protein are produced in VVindA14L-infected cells in the presence of IPTG (isopropyl-beta-D-thiogalactoside), and this correlates with a small-plaque phenotype and reduced VVindA14L yields under these conditions. In the absence of the 15-kDa protein, early and late viral protein syntheses proceed normally; however, proteolytic cleavage of the major core precursors is inhibited. Electron microscopic examination of cells infected with VVindA14L under nonpermissive conditions reveals the presence of numerous membranous elements that look like unfinished or disassembled crescents interspersed between electron-dense masses. These abnormal membrane elements are usually well separated from the surfaces of the dense structures. These findings show that the 15-kDa protein is essential for VV morphogenesis and indicate that this polypeptide is necessary both for the correct assembly of viral crescents and for their stable attachment to the surfaces of viral factories.

HeLa Cells↗
PubMed1997

The apoptosis pathway triggered by the interferon-induced protein kinase PKR requires the third basic domain, initiates upstream of Bcl-2, and involves ICE-like proteases.

The interferon-induced double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase which exerts antiviral and anticellular functions. The antiviral effect of PKR is mediated by the phosphorylation of the alpha subunit of the translational initiation factor elF-2 alpha, while it is not known whether the anticellular effect is due to phosphorylation of elF-2 alpha, l kappa B, or other unknown substrates. We have previously shown that activation of PKR during infection of cells with a vaccinia virus recombinant expressing the wild-type kinase resulted in a complete inhibition of viral and cellular protein synthesis and in the induction of apoptosis. Here, we report that expression of the human proto-oncogene bcl-2 blocks PKR-induced apoptosis but not PKR-induced inhibition of translation. In addition, PKR-induced apoptosis resulted in a cleavage of the death substrate poly(ADP-ribose) polymerase (PARP). Moreover, induction of apoptosis by PKR was not observed with a mutant lacking the third basic region (aa 234-272). Taken together, these results suggest that the third basic region of PKR is required for PKR-induced apoptosis, the process is initiated upstream of bcl-2 and involves activation of a cellular protease, CPP32, or its family members that cleave PARP.

Apoptosis↗
PubMed1997

Characterization of early stages in vaccinia virus membrane biogenesis: implications of the 21-kilodalton protein and a newly identified 15-kilodalton envelope protein.

Vaccinia virus (VV) membrane biogenesis is a poorly understood process. It has been proposed that cellular membranes derived from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) are incorporated in the early stages of virion assembly. We have recently shown that the VV 21-kDa (A17L gene) envelope protein is essential for the formation of viral membranes. In the present work, we identify a 15-kDa VV membrane protein encoded by the A14L gene. This protein is phosphorylated and myristylated during infection and is incorporated into the virion envelope. Both the 21- and 15-kDa proteins are found associated with cellular tubulovesicular elements related to the ERGIC, suggesting that these proteins are transported in these membranes to the nascent viral factories. When synthesis of the 21-kDa protein is repressed, organized membranes are not formed but numerous ERGIC-derived tubulovesicular structures containing the 15-kDa protein accumulate in the boundaries of the precursors of the viral factories. These data suggest that the 21-kDa protein is involved in organizing the recruited viral membranes, while the 15-kDa protein appears to be one of the viral elements participating in the membrane recruitment process from the ERGIC, to initiate virus formation.

Amino Acid Sequence↗
PubMed1997

Folate supplementation and neural tube defects: a review of a public health issue.

Folate deficiency is one of today's most common vitamin deficiencies in women. Women who consume a low level of folate during pregnancy are at risk for poor pregnancy outcomes including neural tube defects (NTD). However, other factors such as heredity, social class, maternal age, birth order, maternal diet, length of time between pregnancies, maternal zinc deficiency, use of anticonvulsant drugs, abnormal homocysteine metabolism and the use of oral contraceptives, have also been implicated as causes of NTD. Animal studies have shown that fetuses are highly dependent on the folate status of the mother. In addition, several retrospective and prospective human studies have provided evidence that folate lowers the incidence of NTD.

Animals↗