Biomedical subjects
Publications and source records attributed to J F Rodriguez.
An 81-year-old man, with a history of pulmonary tuberculosis presented with coma after coughing associated to haemoptysis. The CT-scan revealed several scattered gas bubbles, while the thoracic scan disclosed an old tubercular parenchymatous cave. In spite of several sessions of hyperbaric oxygen treatment, the patient died with multivisceral organ failure. This case report reviews the pathophysiology of gas embolism due to coughing.
Infectious bursal disease virus (IBDV) is the agent of an immune-depressive disease affecting the poultry industry worldwide. Infection of IBDV leads to expression of five mature virus-encoded proteins. Proteolytic processing of the virus-encoded polyprotein generates VP3 which coats the inner surface of the IBDV capsid. In this report, we describe the characterization of the RNA-binding activity of VP3. For these studies, the VP3 coding region was fused to a histidine tag and expressed in insect cells using a recombinant baculovirus. The histidine-tagged VP3 was affinity-purified and used to study its ability to bind RNA molecules using three complementary methods: (i) Northwestern blotting; (ii) binding of VP3 protein-RNA complexes to nitrocellulose membranes; and (iii) electrophoretic mobility shift assays. The results demonstrated that VP3 efficiently bound ssRNA and dsRNA. Under the experimental conditions used in this study, the formation of VP3-RNA complexes did not depend upon the presence of specific RNA sequences. A series of histidine-tagged VP3 deletion mutants spanning the whole VP3 coding region were generated. The use of these mutants revealed that the VP3 RNA-binding domain layed in a highly conserved 69 aa stretch close to the N-terminus of the protein.
The present paper reports of the development and validation of a control strategy for avian influenza infections in poultry. The "DIVA" (Differentiating Infected from Vaccinated Animals) strategy is based on the use of an inactivated oil emulsion vaccine containing the same haemagglutinin (H) subtype as the challenge virus, but a different neuraminidase (N). The possibility of using the heterologous N subtype, to differentiate between vaccinated and naturally infected birds, was investigated through the development of an "ad hoc" serological test based on the detection of specific anti-N1 antibodies. This was achieved using a baculovirus expressing a recombinant N1 protein. The A/ck/Pakistan/H7N3 virus was used as a vaccine and birds were challenged with the HPAI A/ty/Italy/4580/V99/H7N1 strain. The homologous H group ensured a clinical protection of 93% regardless of the vaccination scheme used, and was able to prevent viraemia and muscle colonization in the clinically healthy challenged birds. However, it was not able to prevent viral shedding. The "ad hoc" serological assay was developed as an indirect immunofluorescence test, and was validated using 608 field sera, and showed an "almost perfect agreement" (Kappa value) with the HI test, with relative sensitivity and specificity values of 98.1 and 95.7, respectively. The results of the present investigation suggest that the "DIVA" control strategy may represent a tool for the control of avian influenza infections in poultry.
The development of a discriminatory test, based on the differentiation between N1 and N3 antibodies, to be used in the framework of a vaccination program, based on vaccination with a heterologous H7N3 inactivated vaccine against the Italian H7N1 field virus, is reported. The indirect immunofluorescence antibody (iIFA) assay was based on the expression of the N1 protein in a baculovirus system. HighFive insect cells were transfected with the recombinant virus and used as an antigen in the iIFA test. Preliminary validation on 608 turkey sera yielded relative sensitivity and specificity of 98.1% and 95.7%, respectively, when compared to the HI test with an almost perfect agreement between the two methods (Kappa value = 0.93). It is concluded that the iIFA test is a valid tool for monitoring avian influenza infection in a vaccinated population.
Infectious bursal disease virus (IBDV) capsid is formed by the processing of a large polyprotein and subsequent assembly of VPX/VP2 and VP3. To learn more about the processing of the polyprotein and factors affecting the correct assembly of the viral capsid in vitro, different constructs were made using two baculovirus transfer vectors, pFastBac and pAcYM1. Surprisingly, the expression of the capsid proteins gave rise to different types of particles in each system, as observed by electron microscopy and immunofluorescence. FastBac expression led to the production of only rigid tubular structures, similar to those described as type I in viral infection. Western blot analysis revealed that these rigid tubules are formed exclusively by VPX. These tubules revealed a hexagonal arrangement of units that are trimer clustered, similar to those observed in IBDV virions. In contrast, pAcYM1 expression led to the assembly of virus-like particles (VLPs), flexible tubules, and intermediate assembly products formed by icosahedral caps elongated in tubes, suggesting an aberrant morphogenesis. Processing of VPX to VP2 seems to be a crucial requirement for the proper morphogenesis and assembly of IBDV particles. After immunoelectron microscopy, VPX/VP2 was detected on the surface of tubules and VLPs. We also demonstrated that VP3 is found only on the inner surfaces of VLPs and caps of the tubular structures. In summary, assembly of VLPs requires the internal scaffolding of VP3, which seems to induce the closing of the tubular architecture into VLPs and, thereafter, the subsequent processing of VPX to VP2.
Infectious bursal disease virus (IBDV) encodes a 17-kDa nonstructural polypeptide known as VP5. This polypeptide is not essential for virus replication in vitro but it plays an important role in in vivo dissemination and pathogenesis. We have characterized the expression of VP5 in three eukaryotic systems: (i) IBDV-infected chicken embryo fibroblasts; (ii) BSC-1 cells infected with a recombinant vaccinia virus vector; and (iii) Cos-1 cells transiently transfected with a plasmid vector. Immunofluorescence analyses showed that upon expression VP5 accumulates within the plasma membrane. This finding was consistent with sequence-based topology predictions, indicating that VP5 is a class II membrane protein with a cytoplasmic N-terminus and an extracellular C-terminal domain. Brefeldin A treatment of VP5-expressing cells prevented the accumulation of this polypeptide in the plasma membrane, thus showing the requirement of an active exocytic pathway to reach that compartment. Expression of VP5 was shown to be highly cytotoxic. Induction of VP5 expression resulted in the alteration of cell morphology, the disruption of the plasma membrane, and a drastic reduction of cell viability. VP5-induced cytotoxicity was prevented by blocking its transport to the membrane with Brefeldin A. Our findings suggest that VP5 plays an important role in the release of the IBDV progeny.
The present study was designed to evaluate the relevance of arginine transport in nitric oxide (NO) synthesis in vascular smooth muscle cells. For this purpose, NO synthesis and arginine transport (system B0,+ and y+) were evaluated in cells treated with IL-1beta or angiotensin II (Ang II). In addition, the effects of 5 mM lysine and glutamine, competitive inhibitors of systems y+ and B0,+ respectively, were examined. L-arginine transport was estimated with 3H-labelled arginine and NO was determined with the Griess reagent. These studies were done in control conditions, arginine-starved cells, and in cells incubated in media containing 10 mM arginine. Our data indicate that induction of NO biosynthesis by IL-1beta depends on external arginine when cells are arginine-depleted for 24 hours. The concentration of arginine producing half maximal activation of NO synthesis in arginine-depleted cells ([arginine]i < 10 microM) was 41.1 +/- 18 microM. By contrast, in normal culture conditions, NO synthesis occurred independently of arginine transport. Neither 5 mM lysine or glutamine which abolished arginine transport through systems y+ and B0,+, respectively, reduced nitrite release in cells incubated in normal media. This suggests that the relevance of arginine uptake to NO synthesis depends on the status of intracellular arginine pools. Intracellular arginine concentrations were not affected by the stimulation of NO production using IL-1beta or its inhibition using Ang II, but were markedly reduced by arginine starvation for 48h. Aspartate levels were also reduced by arginine-depletion, but were not affected in cells incubated with 10 mM arginine. By contrast, glutamate levels were reduced in arginine-starved cells and were increased in cells incubated in arginine-supplemented medium. Ornithine levels were markedly increased by arginine supplementation. Altogether, these findings indicate that NO synthesis is normally independent of membrane transport. However in arginine-depleted cells, membrane transport is essential for NO synthesis. It is concluded that arginine transport is required for the long-term maintenance of intracellular arginine pools.
Highly active antiretroviral therapy (HAART) is the standard treatment for infection with human immunodeficiency virus (HIV). The most common HAART regimen consists of the combination of at least one protease inhibitor (PI) with two nucleoside reverse transcriptase inhibitors (NRTIs). Contrary to PIs, NRTIs require intracellular activation from the parent compound of their triphosphate moiety to suppress HIV replication. Simultaneous intracellular determination of two NRTI triphosphates is difficult to accomplish due to their relatively small concentrations in peripheral blood mononuclear cells (PBMCs), requiring large amounts of blood from HIV-positive patients. Recently, we described a method to determine intracellular zidovudine triphosphate (ZDV-TP) concentrations in HIV-infected patients by using solid-phase extraction and tandem mass spectrometry. The limit of quantitation (LOQ) for ZDV-TP was 0.10 pmol, and the method was successfully used for the determination of ZDV-TP in HIV-positive patients. In this study, we enhanced the aforementioned method by the simultaneous quantitation of ZDV-TP and lamivudine triphosphate (3TC-TP) in PBMCs from HIV-infected patients. The LOQ for 3TC-TP was 4.0 pmol, with an interassay coefficient of variation and an accuracy of 7 and 12%, respectively. This method was successfully applied to the simultaneous in vivo determination of the ZDV-TP and 3TC-TP pharmacokinetic profiles from HIV-infected patients receiving HAART.
The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions.
In recent years, several hypotheses have emerged to explain the toxicologic activity of particulate matter. Organic compounds, ultrafine particles, biologic components, and transition metals are some of the constituents that reportedly exert some type of adverse effect on human health. A considerable fraction of the urban particulate matter consists of carbon compounds, which originate mostly from anthropogenic sources. The toxicity of organic fractions from particulate matter have been mainly evaluated by considering their mutagenic activity. This research expands on the toxicologic profile of organic compounds adsorbed to particulate matter, specifically in Puerto Rico, by using the cytotoxic neutral red bioassay (NRB). The NRB uses normal human epidermal keratinocytes or other types of cells to measure the effect on cell viability when exposed to organic compounds associated to the particles in the air. We validated the NRB for particulate matter by using a standard reference material (SRM 1649). We used the NRB to determine toxicologic differences of extracts between an urban industrialized site with anthropogenic activity versus a coastal region with less human activity. The cytotoxicity associated with organic compounds in particulate matter collected at the urban industrialized site was detected in both the particulate matter (3/4) 10 microm in aerodynamic diameter (PM(10)) and particulate matter (3/4) 100 microm in aerodynamic diameter (PM(100)). Greater toxic effects were observed in PM(10) extracts than in PM(100) extracts, but PM(10) toxic effects were not significantly different from those in PM(100). The extracts from the industrialized site were more cytotoxic than the extracts from coastal reference site, although in the summer, extracts from both sites were significantly cytotoxic to normal human epidermal keratinocytes. In addition, the nonpolar extracts of both PM(10) and PM(100) exerted the greatest cytotoxicity, followed by the polar, and, finally, the moderately polar extract. This study demonstrates that extracts from the Guaynabo industrialized site were more toxic than similar extracts obtained from a reference coastal site in Fajardo, Puerto Rico.
The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of an immune depressive disease that affects domesticated and wild avian species. The expression strategy of IBDV includes the synthesis of a 110-kDa polyprotein containing the capsid precursor polypeptides. The polyprotein is autocatalitically processed rendering three polypeptides: NH2-VPX-VP4-VP3-COOH. We have carried out a systematic analysis, using a series of plasmids encoding polyproteins containing either deletions or single amino acid substitutions, to identify the processing sites. The results obtained showed the existence of two sites, 511LAA513 and 754MAA756, that are essential for the processing of the VPX-VP4 and VP4-VP3 precursors, respectively. These sequences are highly conserved among IBDV strains form serotypes 1 and 2. A secondary VPX-VP4 processing site was detected in a 19-amino acid stretch located upstream of the 511LAA513 site. Analyses using versions of the 754MAA756 VP4-VP3 processing site containing conservative and nonconservative amino acid substitutions demonstrated that the specificity of the cleavage is dictated by the conserved AA dipeptide.
Systemic and intracellular pharmacokinetics of zidovudine were determined for 28 human immunodeficiency virus type 1-infected pregnant women and their newborn infants. Plasma zidovudine and intracellular zidovudine monophosphate and triphosphate concentrations were determined in serial maternal samples and cord blood at delivery. Higher levels of cord blood zidovudine were associated with lower maternal zidovudine clearance and longer infusion times. Median levels of zidovudine monophosphate and triphosphate in maternal (1556 and 67 fmol/106 cells) and cord (1464 and 70 fmol/106 cells) blood were similar but highly variable. Intersubject pharmacokinetic variability for zidovudine is substantial, but intravenous therapy provides plasma concentrations and intracellular zidovudine triphosphate levels consistent with high antiviral activity. The substantial amount of intracellular zidovudine triphosphate in cord blood provides an explanation for the clinical success of zidovudine in reducing vertical transmission. Studies of simpler oral regimens of zidovudine can now be evaluated regarding the ability to achieve these pharmacologic end points associated with highly effective parenteral therapy.
Nucleoside reverse transcriptase inhibitors (NRTIs) used against the human immunodeficiency virus (HIV) need to be activated intracellularly to their triphosphate moiety to inhibit HIV replication. Intracellular concentrations of these NRTI triphosphates, especially zidovudine triphosphate (ZDV-TP), are relatively low (low numbers of femtomoles per 10(6) cells) in HIV-infected patient peripheral blood mononuclear cells. Recently, several methods have used either high-performance liquid chromatography (HPLC) or solid-phase extraction (SPE) coupled with radioimmunoassay to obtain in vivo measurements of ZDV-TP. The limit of detection (LOD) by these methods ranged from 20 to 200 fmol/10(6) cells. In this report, we describe the development of a method to determine intracellular ZDV-TP concentrations in HIV-infected patients using SPE and HPLC with tandem mass spectrometry for analysis. The LOD by this method is 4.0 fmol/10(6) cells with a linear concentration range of at least 4 orders of magnitude from 4. 0 to 10,000 fmol/10(6) cells. In hispanic HIV-infected patients, ZDV-TP was detectable even when the sampling time after drug administration was 15 h. Intracellular ZDV-TP concentrations in these patients ranged from 41 to 193 fmol/10(6) cells. The low LOD obtained with this method will provide the opportunity for further in vivo pharmacokinetic studies of intracellular ZDV-TP in different HIV-infected populations. Furthermore, this methodology could be used to perform simultaneous detection of two or more NRTIs, such as ZDV-TP and lamivudine triphosphate.
A cDNA corresponding to the coding region of VP1, the putative RNA-dependent RNA polymerase, of infectious bursal disease virus (IBDV) was cloned and inserted into the genome of a vaccinia virus inducible expression vector. The molecular mass and antigenic reactivity of VP1 expressed in mammalian cells are identical to those of its counterpart expressed in IBDV-infected cells. The results presented here demonstrate that VP1 is efficiently incorporated into IBDV virus-like particles (VLPs) produced in mammalian cells coexpressing the IBDV polyprotein and VP1. Incorporation of VP1 into VLPs requires neither the presence of IBDV RNAs nor that of the nonstructural polypeptide VP5. Immunofluorescence, confocal laser scanning microscopy, and immunoprecipitation analyses conclusively showed that VP1 forms complexes with the structural polypeptide VP3. Formation of VP1-VP3 complexes is likely to be a key step for the morphogenesis of IBDV particles.
BACKGROUND: Glutathione (GSH) is the principal intracellular defense against oxidants, and HIV-infected individuals tend to have subnormal concentrations in plasma. This GSH deficiency may contribute to the pathogenesis of disease progression. In the pediatric population correlations between GSH concentrations with clinical, immunologic and virologic disease profiles are scarce. OBJECTIVES: The main objectives of this study were (1) to compare plasma GSH concentrations of HIV-infected children and healthy controls and (2) to correlate GSH values with clinical, immunologic and virologic disease indices. METHODS: Twenty-four HIV-infected and 24 healthy control children entered the study. Plasma concentrations of total glutathione and related thiols were determined. RESULTS: The difference in mean plasma GSH concentrations between HIV-infected (2.96 +/- 0.31 microM) and control (6.62 +/- 0.58 microM) groups was highly significant (P < 0.0001). Linear regression analyses in HIV-infected patients revealed significant correlations between GSH and both absolute CD4+ cell counts (r = 0.56, P = 0.004) and viral load measured as log HIV-RNA PCR (r = -0.49, P = 0.018). GSH concentrations did not significantly correlate with CDC clinical stage but were lower in HIV-infected patients with growth failure (1.60 +/- 0.54 microM) vs. non-growth failure (3.23 +/- 0.33 microM); P = 0.05. CONCLUSIONS: This study confirmed that HIV-infected children are deficient in plasma GSH concentrations compared with healthy controls. We documented that low GSH concentrations in HIV-infected children are directly correlated with CD4+ cell counts and inversely correlated with viral loads. These findings support a possible role of GSH in the pathogenesis of HIV disease progression.
Protein p54, one of the most antigenic structural African swine fever virus (ASFV) proteins, has been localized by immuno-electron microscopy in the replication factories of infected cells, mainly associated with membranes and immature virus particles. Attempts to inactivate the p54 gene from ASFV by targeted insertion of beta-galactosidase selection marker was uniformly unsuccessful, suggesting that this gene is essential for virus viability. To demonstrate that, we inserted in the TK (thymidine kinase) locus of the virus a construction containing a second copy of the p54 gene and beta-glucuronidase selection marker under the control of p54 and p73 promoters, respectively. Virus mutant clones expressing a second copy of p54 and beta-glucuronidase were used to achieve deletion mutants of the original copy of the gene. Virus mutants expressing only the second inserted copy of p54 and the two selection markers mentioned above were successfully obtained. Therefore, we have demonstrated that the p54 gene product plays an essential role in virus growth, characterizing for the first time in ASFV an essential virus gene.
Nonhuman primates housed in outside cages in rabies enzootic areas should be vaccinated against rabies because of the risk of exposure to rabid wildlife. This article reports that vaccination with an inactivated rabies vaccine for domestic animals induced levels of neutralizing antibodies against rabies in pigtail macaques (Macaca nemestrina) without causing adverse reactions.
We present an analysis of the complete genome of African swine fever virus (ASFV) strain BA71V, including 80 kbp of novel sequence and 90 kbp previously reported by several authors. The viral DNA is 170,101 nucleotides long and contains 151 open reading frames. Structural and/or functional information is available on 113 viral proteins. ASFV encodes five multigene families, putative membrane and secreted proteins, and enzymes involved in nucleotide and nucleic acid metabolism (including DNA repair) and protein modification. Database comparisons have provided clues about genes that may modulate the virus-host interaction, thus, possibly controlling ASFV virulence and persistence. The virus possesses genes similar to CD2, IkappaB, C-type lectins, MyD116/gadd34/gamma, 34.5, bcl-2/bax, iap, NifS, and ERV1, which may allow a viral regulation of cell adhesion, apoptosis, and redox metabolism, as well as of the host immune response against ASFV infection. The proteins encoded by different ASFV isolates are highly similar, the most variable ones being those belonging to multigene families, some membrane proteins, and those containing tandem repeats. DNA sequence data confirm the intermediate characteristics of ASFV between poxviruses and iridoviruses, supporting the notion that ASFV belongs to an independent virus family.