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Biomedical subjects

A M Prince

Publications and source records attributed to A M Prince.

At least 73 records · Page 4Linked to original sources
PubMed1993

Patterns and prevalence of hepatitis C virus infection in posttransfusion non-A, non-B hepatitis.

Improved serologic and polymerase chain reaction (PCR)-based tests for hepatitis C virus (HCV) infection provided an opportunity to reexamine a posttransfusion follow-up study done from 1969 to 1972. A total of 213 cardiac surgery patients was prospectively followed after receiving an average of 18 units of blood, 24% of which was from paid donors. Serial sera were tested for antibody to recombinant DNA-derived C100-3 and capsid polypeptides; selected cases were also tested against synthetic peptides derived from different regions of the HCV sequence. PCR and RIBA II immunoblot assays were done on selected sera. Each of 55 probable and 5 of 11 possible hepatitis cases who were seronegative before transfusion seroconverted. Anti-HCV seroconversion also occurred in 6 (4%) of 148 subjects without hepatitis. Among subjects followed > 1 year, PCR positivity persisted in 14 (82%) of 17. If the results of this study can be generalized, all bloodborne non-A, non-B hepatitis may be due to HCV.

Base Sequence↗
PubMed1993

Passive immunotherapy in the treatment of advanced human immunodeficiency virus infection.

To evaluate the safety and efficacy of passive immunotherapy for advanced human immunodeficiency virus (HIV) infection, a randomized, double-blind, controlled trial of human anti-HIV hyperimmune plasma was conducted. Sixty-three subjects with stage IV HIV disease (AIDS) were randomized to received 250 mL of either HIV-immune plasma or HIV antibody-negative plasma every 4 weeks. Although nonsignificant trends toward improved survival and delayed occurrence of a new opportunistic infection were noted, no significant effects on absolute CD4 lymphocyte counts or quantitative HIV viremia were seen. The only notable toxicity was the allergenicity to be expected from infusing plasma products, usually manifesting as urticaria. Thus, results do not rule out the potential usefulness of passive immunization with different preparations, but did fail to demonstrate clinical benefit of the product studied.

Adult↗
PubMed1993

Enhancement in the safety of immune globulins prepared from high-risk plasma.

Hyperimmune gamma-globulins have proven efficacious in the prevention and treatment of viral infections, including those caused by hepatitis A and B viruses, cytomegalovirus, parvovirus. Interest in the prevention and/or treatment of infections caused by human immunodeficiency virus (HIV) has led to clinical trials with anti-HIV immune plasma and purified immune globulin prepared from donors who are actively infected with HIV. The handling and fractionation of this or other infectious plasma requires the construction and operation of virus containment facilities designed to protect fractionation employees and the immediate environment. This requirement would be reduced substantially by applying virucidal procedures prior to or during plasma pooling. We have shown that heating plasma at 56 degrees C for 1 h followed by treatment with 1% tri(n-butyl) phosphate (TNBP) and 1% Triton X-100 for 4 h at 30 degrees C resulted in the inactivation of > or = 10(12.1) tissue culture infectious doses (TCID50) of HIV. With this treatment, the recovery of IgG was 87 +/- 3%. Fractionation of treated plasma by cold ethanol precipitation proceeded normally, and overall recovery, purity, and potency against selected viral markers were unaffected. The additional treatment of plasma with 15 g/l Aerosil for 4 h at 45 degrees C removed 10(4.5) TCID50 of HIV but resulted in substantial IgG losses both prior to and following fractionation. We conclude that potentially infectious plasma can be treated at 56 degrees C for 1 h and by TNBP/Triton X-100 at 30 degrees C for 4 h prior to fractionation. These steps appear sufficient to assure safety and to permit routine fractionation of plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1993

Viral safety of solvent-detergent treated blood products.

Laboratory research commencing in 1982 led to licensing in the United States in 1985 of a solvent/detergent (SD)-treated anti-haemophilic factor (AHF) concentrate. Licensing was based on (a) studies demonstrating the inactivation of several marker viruses [vesicular stomatitis virus (VSV), Sindbis virus, Sendai virus], human immunodeficiency virus (HIV), hepatitis B virus (HBV), and non-A, non-B hepatitis virus [NANBHV; now known to be principally hepatitis C virus (HCV)] added to AHF just before treatment, (b) the realization that the principal viruses of concern in a transfusion setting (e.g. HIV, HBV, NANBHV) were all lipid-enveloped, and (c) laboratory, preclinical and clinical evidence indicating that AHF and other proteins present in the preparation were unaffected. The applicability of the SD method to a wide range of products and preparations, high process recoveries, and a growing body of viral safety information linked with the failure of several other virus inactivation methods to eliminate hepatitis transmission fostered the adoption of SD technology by more than 50 organizations world-wide. SD mixtures are now used in the preparation of products as diverse as intermediate purity and monoclonal antibody purified AHF and other coagulation factor concentrates, fibrin glue, normal and hyperimmune IgG and IgM preparations including those derived from tissue culture, plasma for transfusion, and various diagnostic controls. Over four million doses of SD-treated products have been administered, and numerous laboratory and clinical studies designed to assess virus safety have been conducted. SD treatment has been shown to inactivate > or = 10(9.2) tissue culture infectious doses (TCID50) of VSV, > or = 10(8.8) TCID50 of Sindbis virus, > or = 10(6.0) TCID50 of Sendai virus, > or = 10(7.3) duck infectious doses of duck HBV, > or = 10(11.0) degrees TCID50 of HIV-1, > or = 10(6.0) TCID50 of HIV-2, > or = 10(6.0) chimpanzee infectious doses (CID50) of HBV, > or = 10(5.0) CID50 of HCV, > or = 10(6.0) TCID50 of cytomegalovirus, > or = 10(5.8) TCID50 of herpes simplex virus type 1, > or = 10(4.0) TCID50 of PI-1, > or = 10(6.0) TCID50 of murine leukemia virus (Mov-3), > or = 10(4.0) TCID50 of murine xenotropic virus, and > or = 10(2.0) TCID50 of Rauscher murine leukemia ecotropic virus. Moreover, in ten prospective clinical studies, 0/53, 0/427, and 0/455 patients susceptible to HBV, NANBHV (HCV), and HIV became infected on follow-up.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Products↗
PubMed1992

Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus.

A cDNA clone designated OV7 encodes a polypeptide that corresponds to a highly antigenic Onchocerca volvulus protein. OV7 has significant amino acid sequence homology to the cystatin superfamily of cysteine proteinase inhibitors. In this report we establish that the OV7 recombinant protein is active as a cysteine proteinase inhibitor, and we have named it onchocystatin. It contains a cystatin-like domain that inhibits the activity of cysteine proteinases at physiological concentrations. Recombinant glutathione S-transferase-OV7 (GST-OV7, 1 microM) and maltose-binding protein-OV7 (MBP-OV7, 4 microM) fusion polypeptides inhibit 50% of the enzymatic activity of the bovine cysteine proteinase cathepsin B. Neither fusion polypeptide inhibits serine or metalloproteinases activity. The Ki for GST-OV7 fusion polypeptide is 170 nM for cathepsin B and 70 pM or 25 nM for cysteine proteinases purified from a protozoan parasite Entamoeba histolytica or the free living nematode Caenorhabditis elegans, respectively. The 5' end of the OV7 clone was isolated by polymerase chain reaction and sequenced, thus extending the previous cDNA clone to 736 base pairs. This represents the complete coding sequence of the mature onchocystatin (130 amino acids). A hydrophobic leader sequence of 32 amino acids was found, indicating a possible extracellular function of the onchocerca cysteine proteinase inhibitor.

Amino Acid Sequence↗
PubMed1992

Genomic typing of hepatitis C viruses present in China.

Hepatitis C virus (HCV) genomic clones were obtained from the serum of Chinese HCV carriers using a polymerase chain reaction-based approach. Consensus sequences were derived from (1) the structural region (nt 1-1543) for one carrier, (2) the hypervariable region V (nt 1156-1233) from four carriers and (3) region V3 from four carriers. Region V3, located in the nonstructural domain NS5 (nt 7066-7137), has been previously shown to be a particularly good marker for the genomic typing of HCV isolates [Inchauspe et al., Proc. Natl. Acad. Sci. USA 88 (1991) 10292-10296]. Comparison of these sequences with sequences from geographically distinct HCV isolates indicates that Chinese HCV strains are closely related to, though distinguishable from, Japanese prototype strains. One amino acid motif, GGAA, located in region V, was found to be conserved only among Chinese isolates. This may define a new subgroup among HCV isolates.

Alanine Transaminase↗
PubMed1992

Sensitivity of serological assays to identify blood donors with hepatitis C viraemia.

Blood donors at high risk of hepatitis C virus (HCV) infection were tested for viraemia by the polymerase chain reaction (PCR). PCR results were accepted as positive only if reactive in 3 of 4 tests and if confirmed in an independent laboratory. The sera were also tested by 6 different assays to determine the ability of current serological assays to detect viraemic blood donors. Of 19 PCR-positive sera, only 13 (68%) were detected by the most sensitive of the serological assays. If these results are confirmed, automated PCR assays may be required for blood-donor screening to prevent transmission of HCV.

Blood Donors↗
PubMed1992

Solvent/detergent-treated plasma: a virus-inactivated substitute for fresh frozen plasma.

Fresh frozen plasma (FFP) is prepared in blood banks world-wide as a by-product of red blood cell concentrate preparation. Appropriate clinical use is for coagulation factor disorders where appropriate concentrates are unavailable and when multiple coagulation factor deficits occur such as in surgery. Viral safety depends on donor selection and screening; thus, there continues to be a small but defined risk of viral transmission comparable with that exhibited by whole blood. We have prepared a virus sterilized FFP (S/D-FFP) by treatment of FFP with 1% tri(n-butyl)phosphate (TNBP) and 1% Triton X-100 at 30 degrees C for 4 hours. Added reagents are removed by extraction with soybean oil and chromatography on insolubilized C18 resin. Treatment results in the rapid and complete inactivation of greater than or equal to 10(7.5) infectious doses (ID50) of vesicular stomatitis virus (VSV) and greater than or equal to 10(6.9) ID50 of sindbis virus (used as marker viruses), greater than or equal to 10(6.2) ID50 of human immunodeficiency virus (HIV), greater than or equal to 10(6) chimp infectious doses (CID50) of hepatitis B virus (HBV), and greater than or equal to 10(5) CID50 of hepatitis C virus (HCV). Immunization of rabbits with S/D-FFP and subsequent adsorption of elicited antibodies with untreated FFP confirmed the absence of neoimmungen formation. Coagulation factor content was comparable with that found in FFP. Based on these laboratory and animal studies, together with the extensive history of the successful use of S/D-treated coagulation factor concentrates, we conclude that replacement of FFP with S/D-FFP, prepared in a manufacturing facility, will result in improved virus safety and product uniformity with no loss of efficacy.

Animals↗
PubMed1992

Three different patterns of hepatitis C virus infection in chimpanzees.

The relationship between hepatitis C virus RNA and hepatitis C virus-associated antibodies (antibody against the putative capsid protein and C-100 antibody) was determined by nested polymerase chain reaction and enzyme-linked immunosorbent assay in serial serum samples obtained from eight chimpanzees experimentally infected with hepatitis C virus. Three different patterns emerged from the polymerase chain reaction data: the first (group 1) was acute resolving hepatitis with transient appearance of HCV RNA (two cases). The second (group 2) had chronic hepatitis with persistent hepatitis C virus RNA positivity (four cases) and the third (group 3) had chronic hepatitis with intermittent appearance of hepatitis C virus RNA (two cases). In four of eight animals, hepatitis C virus RNA was first detectable in serum 1 wk after inoculation. Although serum HCV RNA was detected in all infected chimpanzees, two were positive only for antibody against the putative capsid protein, whereas two were positive only for antibody to C-100 antigen. In four of eight cases, antibody against the putative capsid protein appeared earlier than did antibody to C-100 antigen, was detected just before or coincident with rising glutamate pyruvate transaminase values and remained positive for a long time even after recovery. Six of eight animals (75%) were still hepatitis C virus RNA positive 1 yr after inoculation, suggesting that the risk of development of the chronic carrier state is high in hepatitis C virus infection. Furthermore, there did not appear to be a good correlation between antibody titer in serum and hepatitis C virus infectivity titer.

Alanine Transaminase↗
PubMed1992

Onchocerca volvulus: immunization of chimpanzees with X-irradiated third-stage (L3) larvae.

To provide a theoretical basis for the potential development of vaccines against Onchocerca volvulus (Ov) a trial has been conducted to assess the protective efficacy of immunization of chimpanzees with X-irradiated L3 larvae. Approximately 1000 larvae were injected at 0, 1, and 7 months. The immunized animals, and unimmunized controls, were then challenged with 250 live L3. In order to provide possibly protective exposure to the immunologically distinct L4 epicuticle, a radiation dose (45 krad) was chosen which preserved about 50% of the molting ability of unirradiated larvae. Despite the presence of a strong immune response to crude adult worm extracts, and to cloned Ov antigens, at the time of challenge little or no significant protection against patent infection was observed: three of four immunized animals developed patent infection as compared to four of four controls. One immunized animal failed to become patent or to manifest the late antibody response to adult worm antigens seen in both subpatent and patent infections in this model, and may have been protected from infection. The implications of these studies for future attempts to immunize against O. volvulus are discussed.

Animals↗
PubMed1992

Identification and characterization of an Onchocerca volvulus cDNA clone encoding a microfilarial surface-associated antigen.

The identification and characterization of a recombinant cDNA clone (OV103) expressing a microfilarial surface-associated antigen of Onchocerca volvulus is described. OV103 was identified and isolated from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA using a chimpanzee antiserum, taken 2 years after infection with third-stage larvae of O. volvulus. The cDNA clone encodes a 12.5-kDa protein that corresponds to a 15-kDa parasite protein present in microfilariae and adult female worms. The antigen encoded by this clone is located in the basal layer of the cuticle and the hypodermis of the female adult worm, and on the surface of microfilariae. OV103 fusion polypeptide is recognized only by some sera from onchocerciasis infected subjects (57%), but more significantly (89%) by sera from individuals that have low levels of patent infection. In addition, the antibody response to this protein developed before appearance of microfilariae in the skin of chimpanzees that had developed non-patent or low level patent infections, while the antibody response in chimpanzees with high levels of microfilariae appeared later at the time of appearance of microfilariae. Preliminary experiments indicated that affinity purified antibodies directed against OV103 fusion polypeptide mediated killing of nodular microfilariae in vitro in the presence of normal peripheral blood granulocytes.

Amino Acid Sequence↗
PubMed1992

Inactivation of the human immunodeficiency virus by hypericin: evidence for photochemical alterations of p24 and a block in uncoating.

Following attachment and entry of human immunodeficiency virus (HIV) into a host cell, the HIV genomic RNA is reverse transcribed to cDNA. This step may be inhibited by hypericin, a compound that induces alterations of the retroviral capsid. Incubation of HIV with hypericin rendered the virus noninfectious. The replication of HIV was blocked early; HIV cDNA could not be detected in cells challenged with hypericin-treated HIV. Hypericin did not inhibit the binding of recombinant gp120 to CD4+ cells, nor did hypericin inhibit syncytium formation. However, reverse transcriptase activity could not be released from hypericin-treated virions. Western blot analysis revealed altered mobility of the HIV major capsid protein (p24) following hypericin treatment. Hypericin-treated recombinant HIV p24 exhibited similar altered mobility. The inactivation of HIV infectivity and the alterations in p24 mobility required hypericin incubations in the presence of visible light. Collectively, these data suggest that photochemical alterations of the HIV capsid may contribute to the hypericin-mediated inactivation of HIV. Such alterations may inhibit the release of RT activity from treated HIV, and prevent uncoating and subsequent reverse transcription of the HIV genome within a target cell.

Anthracenes↗
PubMed1992

Immunity in hepatitis C infection.

Polymerase chain reaction (PCR) and newer serologic assays for hepatitis C virus (HCV) were used to investigate 19 HCV cross-challenge episodes in chimpanzees. In these cross-challenges, 59% showed seroconversion after challenge, 33% showed reappearance of HCV-associated hepatocellular ultrastructural changes, 5 animals not PCR-positive at the time of challenge showed return of PCR positivity, and 26% developed hepatitis after rechallenge. A total of 74% showed at least one of these signs of reinfection. The frequency of development of serologic and ultrastructural responses was, however, reduced in secondary compared with primary infections (P less than .01). In 10 animals, the cross-challenge was done with heterologous strains, and in 9 with the originally infecting virus. There was no significant difference in the responses to homologous and heterologous challenges. The data suggest relatively weak immunity in HCV infections.

Alanine Transaminase↗
PubMed1992

High-temperature short-time heat inactivation of HIV and other viruses in human blood plasma.

An ultra-short-time heating system was used to process blood plasma spiked with various viruses (HIV, vesicular stomatitis virus, encephalomyocarditis virus). Virus reduction and recovery of plasma proteins were measured at various temperatures from 65 to 85 degrees C. Processing at 77 degrees C and 0.006 s resulted in a high level of virus kill, including greater than or equal to 4.4 log10 HIV, while maintaining protein structure and activity essentially intact.

Blood↗
PubMed1992

PCR: how to kill unwanted DNA.

Avoidance of contamination in the PCR laboratory requires the use of strict precautions. Among these, chemical decontamination of surfaces and equipment is desirable to prevent inadvertent contamination of samples by the gloved hand and by pipettors. We have investigated the use of sodium hypochloride (Clorox), in comparison to concentrated HCl, for PCR sterilization. Ten percent Clorox was found to eliminate all ethidium bromide-stainable DNA and to prevent PCR amplification of a 600-bp DNA segment within one minute of template treatment. RNA was similarly destroyed. By contrast, even 2.0 N HCl did not destroy DNA detectable by PCR within five minutes. Because of its high efficacy, low cost and relatively low corrosiveness, we recommend the use of ten percent Clorox as a decontaminant for elimination of DNA templates in the PCR laboratory.

Animals↗
PubMed1992

Inactivation of viruses in red cell and platelet concentrates with aluminum phthalocyanine (AIPc) sulfonates.

Aluminum phthalocyanine tetrasulfonates (AIPcS) are photoactive compounds with absorption maxima at 665-675 nm. The inactivation of viruses (vesicular stomatitis virus, VSV; human immunodeficiency virus, HIV) added to either whole blood or red blood cell concentrates (RBCC) and platelet concentrates (PC) on treatment with tetrasulfonated AIPc (AIPcS4) was evaluated. Treatment of RBCC with 10 microM AIPcS4 and 44 J/cm2 visible light resulted in the inactivation of greater than or equal to 10(5.5) infectious doses (TCID50) of cell-free VSV, greater than or equal to 10(5.6) TCID50 of cell-associated VSV, and greater than or equal to 10(4.7) TCID50 of cell-free sindbis virus. Both greater than or equal to 10(4.2) TCID50 of cell-free and greater than or equal to 10(3.6) TCID50 of cell-associated forms of HIV were also shown to be inactivated. Encephalomyocarditis virus, used as a model for nonenveloped viruses, was not inactivated. Equivalent virus kill with Photofrin II required a substantially higher concentration of dye and longer exposure to visible light. Following AIPcS4 treatment, red cell integrity was well maintained as judged by the low level (less than 2%) of hemoglobin release immediately following treatment and on subsequent storage, by measurements of erythrocyte osmotic fragility, and by the normal recovery and circulatory survival on infusion of treated, autologous red blood cells in baboons. Treatment of PC with 10 microM AIPcS4 and 44 J/cm2 visible light also resulted in effective virus kill (greater than or equal to 10(5.5) TCID50) of VSV; however, both the rate and extent of platelet aggregation in response to collagen addition declined by at least 50%. Based on these results, further characterization of AIPcS4-treated RBCC is justified.

Animals↗