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Biomedical subjects

A M Prince

Publications and source records attributed to A M Prince.

At least 91 records · Page 5Linked to original sources
PubMed1991

Genomic structure of the human prototype strain H of hepatitis C virus: comparison with American and Japanese isolates.

Genomic RNA from the human prototype strain H of the hepatitis C virus (HCV-H) has been molecularly cloned and sequenced. The HCV-H sequence reported consists of 9416 nucleotides including the 5' and 3' untranslated regions. HCV-H shows 96% amino acid identity with the American isolate HCV-1 but only 84.9% with the Japanese isolates HCV-J and HCV-BK. In addition to the hypervariable region (region V) previously identified in the putative E2 domain, three other variable domains were identified: region V1 (putative E1), region V2 (putative E2), and region V3 (putative NS5). These regions appear rather conserved (86-100%) among the American isolates (HCV-1 and HC-J1) or among various Japanese isolates (HCV-J, HCV-BK, HCV-JH, and HC-J4) but show striking heterogeneity when the two subgroups are compared (42-87.5% amino acid difference). A structural similarity between the 5'-terminal hairpin structure of HCV and of poliovirus was observed. This study further suggests the existence of at least two genomic subtypes of HCV and confirms a distant relationship between HCV and pestiviruses.

Amino Acid Sequence↗
PubMed1991

Prevention of HIV-1 IIIB infection in chimpanzees by CD4 immunoadhesin.

The first step in infection by the human immunodeficiency virus (HIV) is the specific binding of gp120, the envelope glycoprotein of HIV, to its cellular receptor, CD4. To inhibit this interaction, soluble CD4 analogues that compete for gp120 binding and block HIV infection in vitro have been developed. To determine whether these analogues can protect an uninfected individual from challenge with HIV, we used the chimpanzee model system of cell-free HIV infection. Chimpanzees are readily infected with the IIIB strain of HIV-1, becoming viraemic within about 4-6 weeks of challenge, although they do not develop the profound CD4+ T-cell depletion and immunodeficiency characteristic of HIV infection in humans. CD4 immunoadhesin (CD4-IgG), a chimaeric molecule consisting of the N-terminal two immunoglobulin-like regions of CD4 joined to the Fc region of human IgG1, was selected as the CD4 analogue for testing because it has a longer half-life than CD4, contributed by the IgG Fc portion of the molecule. In humans, this difference results in a 25-fold increased concentration of CD4-IgG in the blood compared with recombinant CD4. Here we report that pretreatment with CD4-IgG can prevent the infection of chimpanzees with HIV-1. The need for a preventative agent is particularly acute in perinatal HIV transmission. As recombinant CD4-IgG, like the parent IgG molecule, efficiently crosses the primate placenta, it may be possible to set up an immune state in a fetus before HIV transfer occurs, thus preventing infection.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Identification of an immunodominant epitope within the capsid protein of hepatitis C virus.

We have isolated cDNA clones from the 5' end of the Hutchinson strain of hepatitis C virus. Sequences encoding various segments of the HCV structural region were fused to the gene for glutathione S-transferase and analyzed for the expression of hepatitis C virus-capsid fusion proteins. With a set of these fusion proteins, both human and chimpanzee immune responses to capsid were studied. An immunodominant epitope was located within the amino-terminal portion of capsid that is preferentially recognized by antibodies in both human and chimpanzee hepatitis C virus-positive sera. In addition, analyses of sequential serum samples taken from humans and chimpanzees with either chronic or apparently self-limited infections revealed that a strong anti-capsid response develops rapidly after onset of infection.

Amino Acid Sequence↗
PubMed1991

Characterization of an Onchocerca volvulus cDNA clone encoding a genus specific antigen present in infective larvae and adult worms.

The isolation and characterization of a recombinant cDNA clone (OV7) expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Using chimpanzee antiserum generated against irradiated infective larvae, we isolated a cDNA clone from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA. The open reading frame encodes 131 amino acids corresponding to a 15.2-kDa protein. Affinity purified antibodies which bound specifically to OV7 fusion polypeptide recognized a single antigen with an apparent molecular weight of 17,000 in extracts of L3, L4 and adult worms. Immunoelectron microscopy established that the antigen encoded by this clone is present in the hypodermis and the basal layer of the cuticle of L3 and female adult worm, and in the egg shell around developing microfilariae. Since the OV7 fusion polypeptide is onchocerca-specific and is recognized specifically by sera from onchocerciasis patients, and sera from non-patent but infected chimpanzees, and not by sera from patients with other filarial parasites, it may have potential as an antigenic component in a test for detection of non-patent and patent infections of O. volvulus. The OV7 amino acid sequence contains residues that have a probable homology with the cysteine proteinase inhibitor superfamily.

Amino Acid Sequence↗
PubMed1991

In vitro and in vivo replication capacity of the precore region defective hepatitis B virus variants.

Although the precore region defective hepatitis B virus variants have been implicated in chronic liver disease and fulminant hepatitis, our knowledge on the molecular biology of these variants is still limited. Using an in vitro transfection assay, we confirmed the replication competent but HBeAg-negative nature of the major variants containing a TAG stop codon in the distal precore region associated with one or two point mutations. Transfection of the two-point-mutated variant into a chimpanzee induced serological responses including anti-HBc and anti-HBs. Interestingly, anti-HBe response was found in the absence of HBeAg antigenemia, suggesting that anti-HBe can be stimulated by degraded HBc. Using the rabbit reticulocyte system the possible effect of the different precore region mutations on the expression of HBcAg from precore- and core-mRNAs was also studied.

Animals↗
PubMed1991

Use of conserved sequences from hepatitis C virus for the detection of viral RNA in infected sera by polymerase chain reaction.

Three oligonucleotide primer combinations selected from the 5' noncoding, the nucleocapsid and the putative nonstructural regions of the hepatitis C virus genome were compared in a nested polymerase chain reaction assay with respect to sensitivity and specificity for the detection of viral RNA in chimpanzee-infected and human-infected sera. Sera from both the acute and the chronic phase of the infection were obtained from 13 animals inoculated with five different non-A, non-B hepatitis strains and from seven cardiac surgery patients who had non-A, non-B hepatitis develop after transfusion and who had been tested in parallel for the presence of hepatitis C virus RNA and anti-C 100-3. A total of 90% of the acute-phase and 100% of the chronic-phase sera tested positive for hepatitis C virus RNA when the 5' noncoding-derived or the nucleocapsid-derived combinations were used; only 58% and 56%, respectively, gave positive results with the putative nonstructural primers, whereas 33% and 71%, respectively, scored positive for C100-3. Thus polymerase chain reaction primers selected from either the highly conserved 5' noncoding or nucleocapsid-regions appear to provide the sensitivity and the specificity necessary to detect low levels of hepatitis C virus RNA in both chimpanzee-infected and human-infected sera.

Animals↗
PubMed1991

Inactivation of viruses in blood with aluminum phthalocyanine derivatives.

The inactivation of viruses added to whole blood and a red cell concentrate with aluminum phthalocyanine and its sulfonated derivatives was studied. A cell-free form of vesicular stomatitis virus (VSV), used as a model, was completely inactivated (greater than 10(4) infectious units; TCID50) on treatment of whole blood with 10 microM (10 mumol/L) aluminum phthalocyanine chloride (AIPs) and visible light dosage of 88 to 176 J per cm2. At 44 J per cm2, complete VSV inactivation was achieved on raising the concentration of AIPc to 25 microM (25 mumol/L). Results at least as good were achieved on similar treatment of a red cell concentrate. Also inactivated were a cell-associated form of VSV and both cell-free and cell-associated forms of human immunodeficiency virus; encephalomyocarditis virus, used as a model for non-lipid-enveloped viruses, was not inactivated by this procedure. This inactivation of cell-free VSV suggests that a similar degree of inactivation could be achieved with a lower concentration of the sulfonated forms of aluminum phthalocyanine. Throughout the above studies, red cell integrity was well maintained, as judged by the absence of hemoglobin release (less than or equal to 2%) during the course of treatment or on subsequent storage. Red cell osmotic fragility was decreased on treatment of whole blood with AIPc. This study indicates that AIPc may be a promising method for the inactivation of viruses in cellular blood products.

Antiviral Agents↗
PubMed1991

Prevention of HIV infection by passive immunization with HIV immunoglobulin.

The use of a human immunodeficiency virus (HIV) immune globulin (HIVIG) in prevention of HIV infection in chimpanzees was investigated in the hope of ultimate application to interruption of vertical transmission. In previous experiments, no protection was observed when relatively high challenge doses were used. This study shows that HIVIG protected against a challenge dose (10 CID50) tenfold lower than that used previously. The protected animal remained free of HIV infection as determined by cocultivation and by polymerase chain reaction (PCR), and did not mount a primary immune response detectable by enzyme-linked immunosorbant assay (ELISA) and neutralization assays. These results imply that HIV vaccines should induce neutralizing antibody and may not need to induce cell-mediated immunity in order to be protective against exposure to HIV. They also provide an experimental basis for the conduct of clinical trials to evaluate prevention of maternal-infant transmission by HIVIG.

Animals↗
PubMed1991

Isolation of a simian immunodeficiency virus related to human immunodeficiency virus type 2 from a west African pet sooty mangabey.

Two of 25 healthy pet sooty mangabey (SM) monkeys (Cercocebus atys) living in West Africa were seropositive by immunoblot when surveyed for antibody to simian immunodeficiency virus of macaques (SIVmac). SIVsmLIB1 was isolated from one of the pet sooty mangabeys. Nucleotide sequence data showed that this isolate is a member of the SIVsm/human immunodeficiecy virus type 2 (HIV-2)/SIVmac group of primate lentiviruses. Furthermore, sequence comparisons revealed extensive genetic diversity among SIVsm isolates similar to that observed previously in SIV isolates from naturally infected African green monkeys. These observations provide additional evidence for monkey-human cross-species transmission of SIVsm as the source of HIV-2 infection of human.

Animals↗
PubMed1991

Elimination of pyridoxylated polyhemoglobin after partial exchange transfusion in chimpanzees.

Partial exchange transfusion with 8.5% pyridoxylated polyhemoglobin solution [PolyHb-PPa] was performed in five male chimpanzees weighing 22-30 kg. Serial blood and urine samples were obtained for 3 days. Percutaneous liver biopsies were performed on the 3rd to 4th, and the 9th to 11th days after PolyHb-PPa administration. Mean exchange volume was 42.5 +/- 10.7 ml/kg BW (26.8-54.6 ml/kg), mean Hb dose 3.7 +/- 0.9 g PolyHb-PPa/kg BW (2.4-4.8 g/kg), mean exchange rate 56.7 +/- 7.1% (48.2-67.4%). All animals survived long-term. Analysis of the plasma Hb concentration-time data showed a first order decline at a plasma level of 3.7 +/- 0.9 g PolyHb-PPa/kg BW. Mean intravascular half-life was 14.6 +/- 3.2 h. Total renal elimination of PolyHb-PPa was about 7%. PolyHb-PPa was absorbed and stored by Kupffer cells and transformed into hemosiderin. Siderosis of Kupffer cells and renal tubules had largely subsided 10 days after PolyHb-PPa indicating subsequent in vivo degradation and metabolization of the polymerized Hb fractions.

Animals↗
PubMed1991

Hemodynamic effects after partial exchange transfusion with pyridoxylated polyhemoglobin in chimpanzees.

Partial exchange transfusion with 8.5% pyridoxylated polyhemoglobin solution [PolyHb-PPa] was performed in five anesthetized spontaneously-breathing male chimpanzees weighing 22-30 kg. Mean exchange volume was 42.5 +/- 10.7 ml/kg BW (26.8-54.6 ml/kg), mean exchange rate 56.7 +/- 7.1% (48.2-67.4%). All animals survived long-term. The chimpanzee's hemodynamics remained stable for the 5 h observation period. Right and left ventricular filling pressures remained constant, mean arterial pressure and mean pulmonary arterial pressure increased by up to 40% after the exchange. Cardiac output remained unaffected by the partial exchange and stroke volume did not change substantially although oxygen capacity and oxygen transport capacity decreased by about a third. The failure of cardiac output to rise after partial exchange transfusion with PolyHb-PPa contrasts with results after isovolemic hemodilution using non-oxygen-carrying blood substitutes and is not adequately explained by the oxygen capacity of 8.5% PolyHb-PPa (9.3 ml O2/dl).

Animals↗
PubMed1991

Onchocerca volvulus: in vitro killing of microfilaria by neutrophils and eosinophils from experimentally infected chimpanzees.

Peripheral blood leukocytes from chimpanzees experimentally infected with third stage larvae, or with nodular microfilaria (mf) of Onchocerca volvulus, were evaluated for their ability to kill nodular mf in vitro. Inoculation of chimpanzees with third stage larvae, and prospective follow up, permitted each animal to be classified as patent, infected but not patent, or post-patent. Neither lymphocytes nor monocytes significantly adhered to, immobilized, or killed mf. However, neutrophils or eosinophils killed mf effectively in the presence of immune serum. Neutrophil and eosinophil enriched cell preparations from control animals were able to kill mf with serum from infected animals, but to a significantly lesser degree than when both cells and serum were taken from infected animals. Chimpanzees inoculated with mf showed a progressive increase in cellular killing of mf after a second inoculation which surpassed that seen with animals inoculated with third stage larvae. Antibody-mediated killing of mf by neutrophils and eosinophils may be important in the termination of patency in this infection.

Animals↗
PubMed1991

Onchocerca volvulus: characterization of monoclonal antibodies reactive with surface components of third-stage larvae.

Five murine monoclonal antibodies (Mab's) have been produced which are reactive with the surface of the third-stage larvae (L3) of Onchocerca volvulus. These were produced from a fusion performed after intrasplenic injection of 10 live O. volvulus L3. Hybridomas were first screened by Elisa using a PBS extract of O. volvulus female adult worms. Elisa negative wells were screened by IFA on whole formalin-fixed L3. Five Mab's were isolated which were reactive with the surface of L3, all were found to be of the IgM isotype. All five Mab's were cross-reactive with the surface of O. lienalis L3, but not with the L3 of Brugia malayi, B. pahangi, Dirofilaria immitis, or Acanthocheilonema viteae. One of the Mab's, OV3-9, reacted with an antigen common to many species of helminths. The other four were genus specific reacting only with O. volvulus and O. lienalis. All 5 Mab's reacted with L2 of O. volvulus, but not with the L4 by IFA on whole fixed larvae. Only Mab's OV3-8 and OV3-9 reacted with cryosections of adult O. volvulus by IFA indicating that the other 3 Mab's are specific for the surface of L2/L3 of the genus Onchocerca. None of the Mab's reacted with detergent dissociated L3 proteins in Western Blot analysis. The ability of L3 to bind the Mab's was abolished by incubating larvae in either proteinase K or trypsin but was unaffected by incubation of larvae in detergents, or by treatment with periodate, indicating the proteinaceous nature of the Mab specific epitopes. Anti-phosphorylcholine Mab (Gib-13) did not react with the surface of L3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Onchocerca volvulus: biochemical and morphological characteristics of the surface of third- and fourth-stage larvae.

The annulated cuticles of third- and fourth-stage larvae of Onchocerca volvulus have the typical structure of other nematodes but the cuticle of fourth-stage larvae was thinner. The surface of the third-stage larva was wrinkled and fuzzy, while that of the fourth-stage was smooth. Intermediate stages in the formation of the new cuticle and epicuticle beneath the old basal layer and of the separation of the cuticles are shown. Monoclonal antibodies specific to the surface of third-stage larvae did not react with the surface of the fourth-stage larvae. Binding of the monoclonal antibodies to the third-stage larvae was abrogated by treatment of the worms with trypsin and proteinase K, but was unaffected by treatment with periodate or the detergents sodium deoxycholate and SDS. The lectins RCA120 and WGA, but not any of the other lectins tested, bound only to the surface of fourth-stage larvae, and not to that of third-stage larvae. The surfaces of third- and fourth-stage larvae were shown to be different and contained stage-specific surface epitopes.

Animals↗
PubMed1990

The use of tri(n-butyl)phosphate detergent mixtures to inactivate hepatitis viruses and human immunodeficiency virus in plasma and plasma's subsequent fractionation.

The treatment of plasma with organic solvent/detergent mixtures at the time of plasma collection or pooling could reduce the exposure of technical staff to infectious viruses and enhance the viral safety of the final product. Treatment of plasma for 4 hours with 2-percent tri(n-butyl)phosphate (TNBP) at 37 degrees C, with 1-percent TNBP and 1-percent polyoxyethylensorbitan monooleate (Tween 80) at 30 degrees C, or with 1-percent TNBP and 1-percent polyoxyethylene ethers, (Triton X-45) at 30 degrees C resulted in the rapid and complete inactivation of greater than or equal to 10(4) tissue culture-infectious doses (TCID50) of vesicular stomatitis and Sindbis viruses, which are used as surrogates. Treatment of plasma with TNBP and TNBP and Tween-80 was shown to inactivate greater than or equal to 10(4) TCID50 of human immunodeficiency virus. TNBP treatment of plasma contaminated with 10(6) chimpanzee-infectious doses (CID50) of hepatitis B virus and 10(5) CID50 of non-A,non-B hepatitis virus prevented the transmission of hepatitis to chimpanzees. Immediately after treatment of plasma with 2-percent TNBP, the recovery of factors VIII, IX, and V and antithrombin III was 80, 90, 40, and 100 percent, respectively. Recovery of all factors was greater than or equal to 90 percent after treatment with TNBP and detergent mixtures. Treated plasma was fractionated by standard techniques into antihemophilic factor and prothrombin complex concentrates, immune globulin, and albumin. Prior treatment with TNBP or TNBP and detergent did not affect the separations of desired proteins. Therefore, it appears possible to inactivate viruses in plasma before the execution of standard fractionation procedures.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Proteins↗
PubMed1990

An economical procedure for screening of hybridoma supernatants for surface reactive antibodies to filarial larvae.

Third- and fourth-stage larvae of Onchocerca volvulus can be attached to the lids of 96-well microtiter plates to facilitate the screening of hybridoma supernatants reactive with filarial larvae. Plates are coated with a solution consisting of 0.1% gelatin, and 0.01% chrome alum. Following coating of plate lids one larva per depression is placed on the lid. An immunofluorescence assay (IFA) was conducted in which there were two 45 minute incubations and eight separate washes by immersion in PBS. At the completion of the assay over 90% of the larvae remain attached. The background in IFA is minimal and the results are consistent with those obtained using a suspension IFA. This technique permits rapid identification of antibody producing hybridomas with small numbers of larvae.

Animals↗
PubMed1989

Absence of perinatal transmission of blood-borne non-A, non-B hepatitis virus by chimpanzees with acute and chronic infection.

Two chimpanzees were born to parents with chronic non-A, non-B hepatitis and remained with their mothers until 12 and 18 months, respectively. The infants were followed from 7 to 8 weeks of age with biweekly or monthly blood samples and with monthly liver biopsies from 4 to 7 months after birth. Another chimpanzee, along with both of its parents, was held throughout the parents' acute infection with non-A, non-B hepatitis; at this time the infant was 14-16 months of age, and it was followed with bi-weekly blood samples and monthly biopsies from the time of potential exposure for 20 months. No abnormalities indicative of non-A, non-B hepatitis were detected in these animals. During the 29 to 35 months of follow-up, alanine aminotransferases and gamma glutamyl-transferases (GGPT) levels remained well within normal range for animals held in the same facility. Histologic and electron microscopic examination of liver tissue revealed no abnormalities.

Acute Disease↗