Biomedical subjects
M E Miranda
Publications and source records attributed to M E Miranda.
Immunoglobulin G enzyme-linked immunosorbent assay using truncated nucleoproteins of Reston Ebola virus.
We developed an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), using partial recombinant nucleoproteins (rNP) of Reston Ebola virus (EBO-R) and Zaire Ebola virus (EBO-Z). We examined the reaction of 10 sera from cynomolgus macaques naturally infected with EBO-R to each of the partial rNP in the IgG ELISA. All the sera reacted to the C-terminal halves of the rNP of both EBO-R and EBO-Z. Most of the sera reacted to the RdeltaC (amino acid (aa) 360-739), and Rdelta6 (aa 451-551) and/or Rdelta8 (aa 631-739) at a higher dilution than to the corresponding truncated rNPs of EBO-Z. The results indicate that this IgG ELISA is useful for detecting EBO-R specific antibody, and may have a potential to discriminate EBO-R infection from other subtypes.
Genome structure of Ebola virus subtype Reston: differences among Ebola subtypes. Brief report.
We determined the complete genome sequence of Ebola virus subtype Reston (EBO-R) in the Philippines in 1996. The deduced transcriptional signals were highly conserved among Ebola viruses except for the stop signal of L genes. The intergenic regions were composed of 4 to 7 nucleotides, and of 2 characteristic overlaps and a long intergenic region. The glycoprotein (GP) had several amino acid differences from EBO-R isolated in 1989 and 1992. The variety of GP sequences strongly suggests the independent introduction of EBO-R from unknown natural reservoirs in 1996.
Detection of Ebola viral antigen by enzyme-linked immunosorbent assay using a novel monoclonal antibody to nucleoprotein.
With the increase in international traffic, the risk of introducing rare but severe infectious diseases like Ebola hemorrhagic fever is increasing all over the world. However, the system for the diagnosis of Ebola virus infection is available in a limited number of countries. In the present study, we developed an Ebola virus antigen-detection enzyme-linked immunosorbent assay (ELISA) system using a novel monoclonal antibody (MAb) to the nucleoprotein (NP). This antibody recognized an epitope defined by a 26-amino-acid stretch near the C terminus of NP. In a sandwich ELISA system with the MAb, as little as 30 ng of purified recombinant NP (rNP) was detected. Although this MAb was prepared by immunization with rNP of subtype Zaire, it also reacted to the corresponding region of NP derived from the Reston and Sudan subtypes. These results suggest that our ELISA system should work with three of four Ebola subtypes. Furthermore, our ELISA system detected the NP in subtype Reston-infected monkey specimens, while the background level in noninfected specimens was very low, suggesting the usefulness of the ELISA for laboratory diagnosis with clinical specimens.
Multiplex analysis of cytokines in the blood of cynomolgus macaques naturally infected with Ebola virus (Reston serotype).
Ebola virus (EBO) causes the most severe form of viral hemorrhagic fever in humans and nonhuman primates with up to 90% of infections culminating in death. The requirement of maximum containment laboratories for Ebola virus research has limited opportunities to study the pathogenesis of EBO infections. While tissue damage does occur, often it would appear not to be sufficient to explain death, indicating that soluble mediators play an important role in disease progression. In previous studies, fatal human infections with the Zaire subtype of Ebola (EBO-Z) were associated with an increase in the levels of inflammatory cytokines. In this investigation, a new multiplex assay was developed and used to measure circulating levels of cytokines and chemokines in cynomolgus macaques infected with the Reston subtype of EBO (EBO-R). Increased levels of IL-6, TNF-alpha, IFN-gamma, IL-2, IL-4, IL-8, IL-10, and GM-CSF were detected in infected animals, and the increase in circulating cytokines correlated with an increase in circulating viral antigen. Blood samples from animals showing high levels of cytokines were also tested for the chemokines: MCP-1, IL-1beta, MIP-1alpha, MIP-1beta, IP-10, and RANTES. High levels of MCP-1 and MIP-1beta, and RANTES were found in infected primates and, while levels were more variable, IL-1beta was detected only in infected animals.
Biliary ascariasis after Roux-en-Y hepaticojejunostomy.
The authors report on a 6-year-old girl with biliary ascariasis after surgical treatment of a choledochal cyst and biliary-digestive tract reconstruction by Roux-en-Y hepaticojejunostomy. A precise diagnosis can be obtained by ultrasonography. Surgical treatment is required when clinical and endoscopic treatments fail. In countries in which this disease is endemic, biliary ascariasis should be considered in the differential diagnosis of cholangitis after surgery for hepaticojejunostomy.
Use of a plastic hemoderivative bag in the treatment of gastroschisis.
The authors report their experience with the use of a polyvinyl chloride (PVC) bag for blood-derivative transfer as a prosthesis for the creation of a silo for surgical treatment of gastroschisis (GS) in seven newborn infants. The bag is sterile, impermeable to micro-organisms, transparent, flexible, resistant, internally smooth, does not adhere to the bowel loops, readily available, and inexpensive, properties that make it an excellent alternative as a prosthesis for staged surgical treatment of congenital anomalies of the abdominal wall such as GS and omphalocele. The importance of a multidisciplinary team for the care of newborns with GS is also emphasized for a good postoperative outcome.
A monoclonal antibody recognizing an epitope shared by receptors for growth hormone, prolactin, interleukin 2 and interleukin 6.
Monoclonal antibody (MAb) termed R7B4 was generated throughout the idiotypic-anti-idiotypic network from mice immunized with human and bovine growth hormones (GH). The Ab was selected on the basis that it did not recognize human GH (hGH) neither insolubilized nor in solution but inhibited 125I-hGH binding to receptors from rat and rabbit liver and from Nb2-cell membranes. Since it inhibited Nb2-cell mitogenesis stimulated by hGH, prolactins or placental lactogens, MAb R7B4 behaved as an antagonist of lactogenic hormones. Furthermore, the Ab impaired proliferative activity of interleukin 2 (IL-2) on Nb2 cells as well as growth of 7TD1 cells, an interleukin 6 (IL-6) dependent hybridoma not expressing GH receptors. Biotin-labeled MAb R7B4 specifically bound to rat liver microsomes, and the Ab was able to recognize Nb2 and 7TD1-cell membranes as shown by flow cytometry experiments. However, MAb binding was not hampered by hGH, indicating that the Ab did not mimic GH binding site to receptors. Immunoblot assays indicated that rat and rabbit liver as well as Nb2-cells membrane antigens recognized by MAb R7B4 were similar to those revealed by a MAb directed to prolactin receptors. In addition, MAb R7B4 was able to detect two bands probably corresponding to the somatogenic receptor in rabbit liver microsomes as well as three different proteins in 7TD1-cells showing molecular weights similar to those of the IL-6 receptor complex. Results suggest that MAb R7B4 is directed to an epitope shared by receptors for lactogenic and somatogenic hormones, IL-2 and IL-6. To our knowledge, these data are the first experimental evidence of the existence of structural similarity between some of the receptors grouped in the cytokine receptor superfamily.
Ovine placental lactogen and human growth hormone bind to different regions of the same receptors.
Anti-human growth hormone (hGH) polyclonal and monoclonal antibodies (MAb) failed to recognize ovine placental lactogen (oPL), indicating that the antigenic topographies of both hormones are different. Binding assays showed that oPL completely inhibited hGH binding to lactogenic receptors from Nb2-cells and to somatogenic receptors from rabbit or sheep liver; in contrast, oPL only bound to a subpopulation of rat liver receptors. Zinc ion increased hGH and oPL binding to Nb2-cell receptors and slightly inhibited both hormones' recognition by somatogenic receptors. However, ZnCl(2) increased hGH binding to rat liver microsomes but prevented that of oPL. Furthermore, MAb R7B4, recognizing lactogenic as well as somatogenic receptors, entirely blocked hGH binding to the various receptor systems but not affected oPL binding. Therefore, results presented in this paper suggest that oPL and hGH bind to different regions of the same receptors.
Detection and molecular characterization of Ebola viruses causing disease in human and nonhuman primates.
Ebola (EBO) viruses were detected in specimens obtained during the hemorrhagic fever outbreak among humans in Kikwit, Democratic Republic of the Congo (DRC), in 1995 (subtype Zaire) and during an outbreak of disease in cynomolgus macaques in Alice, Texas, and the Philippines in 1996 (subtype Reston). Reverse transcriptase-polymerase chain reaction assays were developed and proven effective for detecting viral RNA in body fluids and tissues of infected individuals. Little change was seen in the nucleotide or deduced amino acid sequences of the glycoprotein (GP) of these EBO virus subtypes compared with those of their original representatives (i.e., the 1976 Yambuku, DRC, EBO isolate [subtype Zaire] and the 1989 Philippines and Reston, Virginia, isolates [subtype Reston]). The nonstructural secreted GP (SGP), the primary product of the GP gene, was more highly conserved than the structural GP, indicating different functional roles or evolutionary constraints for these proteins. Significant amounts of SGP were detected in acutely infected humans.
Epidemiology of Ebola (subtype Reston) virus in the Philippines, 1996.
Ebola (subtype Reston [EBO-R]) virus infection was detected in macaques imported into the United States from the Philippines in March 1996. Studies were initiated in the Philippines to identify the source of the virus among monkey-breeding and export facilities, to establish surveillance and testing, and to assess the risk and significance of EBO-R infections in humans who work in these facilities. Over a 5-month period, acutely infected animals were found at only one facility, as determined using Ebola antigen detection. Three of 1732 monkeys and 1 of 246 animal handlers tested had detectable antibodies; all were from the same facility, which was the source of infected monkeys imported to the United States. Virus transmission, which was facilitated by poor infection-control practices, continued for several months in one facility and was stopped only when the facility was depopulated. None of the 246 employees of the facilities or 4 contacts of previously antibody-positive individuals reported an Ebola-like illness. This investigation suggests that human EBO-R infection is rare.
Accessibility of SSA/Ro and SSB/La antigens to maternal autoantibodies in apoptotic human fetal cardiac myocytes.
Access of intracellular Ags SSA/Ro and SSB/La to cognate maternal autoantibodies is unexplained despite their strong association with congenital heart block. To investigate the hypothesis that apoptosis facilitates surface accessibility of these Ags, human fetal cardiac myocytes from 16- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusing the aorta with collagenase. Confirmation of cardiac myocytes included positive staining with antisarcomeric alpha-actinin and contractility induced by 1.8 mM calcium. Incubation with 0.5 microM staurosporine or 0.3 mM 2,3-dimethoxy-1,4-naphthoquinone induced the characteristic morphologic and biochemical changes of apoptosis. The cellular topology of Ro and La was evaluated with confocal microscopy and determined in nonapoptotic and apoptotic cardiocytes by indirect immunofluorescence. In permeabilized nonapoptotic cardiocytes, Ro and La were predominantly nuclear, and propidium iodide (PI) stained the nucleus. In early apoptotic cardiocytes, condensation of the PI- and Ro- or La-stained nucleus was observed, accompanied by Ro/La fluorescence around the cell periphery. In later stages of apoptosis, nuclear Ro and La staining became weaker, and PI demonstrated nuclear fragmentation. Ro/La-stained blebs emerged from the cell membrane, a finding observed in nonpermeabilized cells, supporting an Ab-Ag interaction at the cell surface. In summary, induction of apoptosis in cultured cardiocytes results in surface translocation of Ro/La and recognition by Abs. Although apoptotic cells are programmed to die and do not characteristically evoke inflammation, binding of maternal Abs and subsequent influx of leukocytes could damage surrounding healthy fetal cardiocytes.
Density estimates of rural dog populations and an assessment of marking methods during a rabies vaccination campaign in the Philippines.
We estimated the population density of dogs by distance sampling and assessed the potential utility of two marking methods for capture-mark-recapture applications following a mass canine rabies-vaccination campaign in Sorsogon Province, the Republic of the Philippines. Thirty villages selected to assess vaccine coverage and for dog surveys were visited 1 to 11 days after the vaccinating team. Measurements of the distance of dogs or groups of dogs from transect lines were obtained in 1088 instances (N = 1278 dogs; mean group size = 1.2). Various functions modelling the probability of detection were fitted to a truncated distribution of distances of dogs from transect lines. A hazard rate model provided the best fit and an overall estimate of dog-population density of 468/km2 (95% confidence interval, 359 to 611). At vaccination, most dogs were marked with either a paint stick or a black plastic collar. Overall, 34.8% of 2167 and 28.5% of 2115 dogs could be accurately identified as wearing a collar or showing a paint mark; 49.1% of the dogs had either mark. Increasing time interval between vaccination-team visit and dog survey and increasing distance from transect line were inversely associated with the probability of observing a paint mark. Probability of observing a collar was positively associated with increasing estimated density of the dog population in a given village and with animals not associated with a house. The data indicate that distance sampling is a relatively simple and adaptable method for estimating dog-population density and is not prone to problems associated with meeting some model assumptions inherent to mark-recapture estimators.
Haemophilus species bacteremia in adults. The importance of the human immunodeficiency virus epidemic.
BACKGROUND: Until the late 1970s, invasive infections caused by Haemophilus species were thought to occur mainly in children and only infrequently in adults. OBJECTIVE: To report the largest series to date of Haemophilus species bacteremia (HB) from a single center. DESIGN: Retrospective. SETTING: Large, tertiary care, general teaching hospital. METHODS: We reviewed the charts of adult patients with HB detected from January 1, 1986, to December 31, 1994. Haemophilus strains were serotyped, and the antimicrobial resistance pattern was analyzed. RESULTS: One hundred sixteen patients had HB (0.26 cases per 1000 admissions). Thirty-eight children and 16 adults were excluded. Human immunodeficiency virus (HIV) infection was the most common underlying condition (n = 18 [29%]), followed by malignant neoplasms (n = 12 [19%]) and chronic obstructive pulmonary disease (n = 12 [19%]). Prevalence in HIV-positive patients was 5 cases per 1000 admissions vs 0.2 cases per 1000 admissions in HIV-negative patients. Infection was nosocomial in 16 patients (26%). Focal diseases were pneumonia in 41 patients (66%), cholangitis in 5 patients (8%), endocarditis in 3 patients (5%), meningitis and septic arthritis each in 1 patient (2%), and primary bacteremia in 9 patients (14%). The HIV-positive patients were significantly younger and presented more frequently with pneumonia (P < .05). Overall, 14 patients died (22%). Bacteremia was polymicrobial in 11 patients (18%). Haemophilus influenzae was isolated in 53 patients (85%). Rates of antimicrobial resistance were 11% to chloramphenicol sodium succinate, 48% to ampicillin sodium, 78% to erythromycin stearate, 76% to combined sulfamethoxazole and trimethoprim, 15% to rifampin, and 57% to clarithromycin. CONCLUSIONS: Infection with HIV has become the most common underlying disease in adults with HB in our hospital. Therapeutic approaches must take into account the high rate of antimicrobial resistance.
Transplantation and organ donation in Hospital de Especialidades CMN SXXI in Mexico City.
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Evaluation of tolerability, safety, and efficacy of cyclosporine microemulsion in renal transplant recipients.
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Evaluation of a canine rabies vaccination campaign and characterization of owned-dog populations in the Philippines.
A mass canine rabies vaccination campaign in Sorsogon Province, the Republic of the Philippines, was conducted in April and May 1993. From 1 to 14 days following visits by vaccination teams to 30 selected villages (barangays), survey teams revisited the barangays to assess vaccine coverage. Modified cluster survey methods were used to gather information about vaccine coverage in the owned-dog population (210 households) and about characteristics of owned-dogs and factors influencing owner willingness to participate in the campaign. Vaccinated dogs were identified by asking owners about receipt of certificates given by the vaccinating teams and examining each animal for a special collar or paint mark placed on the animal at the time of vaccination. Survey results indicated that 73% (178/243) of eligible dogs were vaccinated and 82% of vaccinated dogs were marked with a collar or paint. Dogs were owned by 69% of households and ranged in age from 3 days to 13 years (median = 1 year), and the ratio of male to females dogs was 1:1. The dog-to-human ratio was 1:3.8, with an average of 1.4 dogs per household or 2.1 dogs per dog-owning household. Most dogs were kept as guards (83%) and most were free-ranging (85%). The most common reasons dogs were not vaccinated included they could not be restrained (11/64), the owner was not home (10/64), and fear of injury resulting from vaccination (10/64). The owners of 20% of vaccinated dogs reported some adverse reaction in their pet. Improved vaccine coverage was significantly associated with restrained dogs kept primarily for guard functions by owners who received information about the vaccination campaign from multiple sources. Vaccine coverage was sufficiently high to potentially control rabies transmission among dogs through herd immunity and indicated a successful vaccine campaign.
[Clinical spectrum of hypoxanthine-guanine phosphoribosyltransferase deficiency: study of 12 cases].
BACKGROUND: The hypoxanthine-guanine phosphoribosyltransferase deficiency (HGPRT) may have two clinical forms: that of the Lesch-Nyhan syndrome (complete HGPRT deficiency) and that of the Kelley-Seegmiller syndrome (partial HGPRT deficiency). The clinical and biochemical features of the HGPRT deficiency are not completely known. METHODS: A series of 12 patients, 8 with the Lesch-Nyhan syndrome and 4 with the Kelley-Seegmiller syndrome are described. The plasma and urine concentrations of hypoxanthine, xanthine and uric acid were compared with those obtained in 20 normal subjects and 41 patients with primary gout. The molecular defect which determines the deficient HGPRT activity was studied in one patient with the Kelley-Seegmiller syndrome. RESULTS: The 8 patients with the Lesch-Nyhan syndrome presented choreoathetosis, corticospinal motor system dysfunction, mental retardation and signs of self mutilation. The neurologic manifestations of the patients with the Kelley-Seegmiller syndrome were very heterogeneous: two patients had psychomotor retardation with spastic movement, one was mentally retarded with generalized dystonia and one patient only had gout with no neurologic manifestations. The erythrocytic HGPRT activity ranged between 0.28 and < 0.01 nmol/h and mg of hemoglobin in all the patients. Plasma and urine purine concentrations were very high, being greater than those in normal subjects and patients with gout (p < 0.01). A mutation was identified in exon 3 (substitution of guanine with thymine) conditioning the substitution of the normal glycine amino acid by valine (HGPRT Madrid) on molecular study. CONCLUSIONS: The hypoxanthine-guanine phosphoribosyltransferase deficiency has a heterogeneous clinical expression. The activity of this enzyme in erythrocytes and the results of the metabolism of the purines do not allow prediction of the severity of the clinical manifestations.