Biomedical subjects

A M Prince

Publications and source records attributed to A M Prince.

At least 19 recordsLinked to original sources

PubMed2005

Schistosoma infection inhibits cellular immune responses to core HCV peptides.

Patients coinfected with hepatitis C virus (HCV) and the trematode, Schistosoma mansoni, have an increased incidence of viral persistence and accelerated fibrosis. To investigate immunological mechanisms responsible for this more aggressive natural history of HCV, the core HCV-specific T-cell responses were analysed in 44 donated blood units rejected because they had antibodies to HCV (anti-HCV). Half also had anti-S. mansoni antibodies, evidence of past or active infection. HCV-specific ELISPOT responses were examined using pools of 180 overlapping 9-mer peptides with offsets of one covering the core of HCV genotype 4a. Comparison of T-cell responses in blood units positive for both anti-HCV and anti-Schistosoma antibodies with blood units positive only for anti-HCV antibodies showed a significant decrease in core-specific T-cell IFN-gamma (505+/- 46 vs. 803 +/- 66 ISC/10(6) cells, P < 0.001), IL-4 (2 +/- 108 vs. 641 +/- 131 ISC/10(6) cells, P < 0.001), and IL-10 (159 +/- 105 vs. 466 +/- 407 ISC/10(6) cells, P < 0.002) responses. In contrast, there was no significant difference in cell-mediated immune response (CMI) to PHA mitogen between these two groups. Therefore, we concluded T cells from persons with anti-Schistosoma have reduced IFN-gamma, IL-4, and IL-10 secreting HCV-specific T-cell responses. This may explain why Schistosoma coinfection increases persistence and severity of HCV infection.

Animals↗

PubMed2004

Hepatitis C virus replication kinetics in chimpanzees with self-limited and chronic infections.

The availability of molecular beacon-based, real time polymerase chain reaction (PCR) and a semi-automated sample extraction procedure have made it possible for us to retrospectively examine HCV replication kinetics in HCV naive chimpanzees infected during the past 20 years. We compared these in 17 animals that developed chronic infection, and in 21 that developed self-limited infection. No differences were found in infecting dose, or replication kinetics in the acute phase between these two types of infection. An unanticipated finding was the fact that 10 of 17 animals developing chronic infection partially controlled virus replication for 48 +/- 48 weeks after typical acute phase viraemia, and prior to development of chronic infection. Twenty-nine out of 30 (29/30) sera, which were negative by quantitative PCR during the downregulated period, were, however, positive by the more sensitive Genprobe isothermal transcription-mediated amplification (TMA) assay. Thus, downregulation was not complete. Ten animals showing self-limited infection showed complete resolution of viraemia by TMA assay. Quasispecies analysis revealed that in all, except one case, the virus reappearing after downregulation was essentially identical to that of the originally infecting virus.

Animals↗

PubMed2001

Antisense suppression of p107 inhibits 3T3-L1 adipocyte differentiation.

The murine 3T3-L1 preadipocyte cell line is well characterized for its capacity to undergo differentiation into adipocytes under appropriate hormonal stimulation. p107, a member of the retinoblastoma tumor suppressor gene family has been shown to be dramatically upregulated during the early requisite clonal expansion phase of 3T3-L1 adipogenesis; however, a functional consequence has yet to be described. A phosphorothioate antisense RNA approach was utilized to determine if inhibition of p107 expression would block or perturb adipocyte differentiation. A series of three phosphorothioate oligonucleotides in antisense orientation was generated, designated AS1, AS2, and AS3 along with a sense control oligonucleotide complementary to AS1 and added to postconfluent cells at a concentration of 20 and 50 microM throughout hormonally stimulated differentiation. Treatment of cells with either concentration of the sense, AS1, AS2, or 20 microM AS3 oligonucleotides had little effect on either Oil Red O lipid accumulation or induction of p107 protein levels. In contrast, treatment with 50 microM AS3 inhibited the increase in p107 protein levels and led to a complete block in differentiation as detected by Oil Red O lipid accumulation and inhibition of adipocyte-specific mRNA expression. In addition, treatment with AS3 led to a significant inhibition of cellular proliferation associated with clonal expansion. Combined, these results provide strong evidence supporting a functional role for p107 in 3T3-L1 adipocyte differentiation.

3T3 Cells↗

PubMed2001

Role of the oncogenic Raf-1 in orchestration of discrete nuclear factor-kappaB-activating pathways.

Raf-1, a key kinase in the Ras signaling pathway, plays critical roles in cell differentiation, proliferation, and tumorigenesis. However, knowledge of the Raf-1 in inflammation is limited. Using an inducible oncogenic Raf-1, we show that the Raf-1 orchestrates the discrete NF-kappaB activating pathways. While the Raf-1 activation induces a modest IkappaB degradation by enhancing the basal IkappaB kinase activity, it contradictorily suppresses the proinflammatory cytokine inducible IkappaB kinase complex, leading to an inhibition of TNF-alpha- and IL-1beta-induced NF-kappaB activation. Despite considerable degrees of overlap, LPS signaling is not affected by Raf-1. By either conditionally reducing Raf-1 activity or completely disrupting the Raf-1 signaling by PD98059, a specific inhibitor of MEK1, the otherwise inhibited cytokine responses can be restored. Moreover, when the activity of Raf-1 is up-regulated during the cell cycle progression from the G(0) phase to the late G(1) phase, the enhanced Raf-1 activity suffices to shift the TNF-alpha response from the sensitive to the insensitive state. Together, these studies elucidate a mechanism by which signaling outputs are shaped by the intracellular Raf-1, thus explaining the "cellular context"-dependent cytokine response.

Animals↗

PubMed2001

Human milk contains detectable levels of immunoreactive leptin.

Leptin, the recently cloned product of the obese (ob) gene, is a 16 kDa-protein that acts as a circulating satiety factor. It also serves to regulate energy expenditure and may act as a counter regulatory hormone to insulin. Initially thought to be exclusively produced by mature adipocytes, its mRNA has now been identified in significant levels in the placenta as well as the fetus raising speculation regarding its importance as a growth factor. Given studies demonstrating that exclusively breast-fed infants are leaner due to decreased energy intakes than formula-fed infants, we hypothesized that the presence of leptin in human milk could participate in mediating the earlier satiety of those infants fed human milk. We undertook this initial study to qualitatively examine the presence of leptin in human milk utilizing an immunoblot approach. Random milk samples during the first 2 weeks of lactation were available for study from 4 mothers delivering at term. Milk samples were centrifuged, the aqueous layer removed, and the protein content quantitated. One-hundred micrograms of total protein were separated by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and immunoblotted with an antileptin antibody. As controls, recombinant human leptin alone and a sample of milk containing added leptin were similarly electrophoresed and immunoblotted. Labeled proteins were visualized by chemiluminescence. Significant amounts of leptin protein were identified in all milk samples examined. No difference in protein detection was identified in fresh milk vs. frozen milk, and little difference was apparent in foremilk samples vs. hindmilk samples. These preliminary data reveal the presence of leptin in term human milk and suggest that further studies to document bioactivity of milk-derived leptin are warranted.

Female↗

PubMed2001

Immunoprophylaxis of hepatitis C virus infection.

Because hepatitis C virus is etiologically involved in about half the cases of the world's most common cancer, hepatocellular carcinoma, and because this virus is likely to continue to spread in most of the developing world for many years, the authors believe that development of a prophylactic vaccine is imperative. Numerous approaches are available to overcome the many impediments which make the development of an HCV vaccine difficult. Such impediments include the many viral genotypes and quasispecies of HCV and the association of virions with host lipids. It is likely that overcoming these impediments will require a vaccine which induces a strong cell-mediated response. The most promising approach seems to be DNA-based immunization or a prime-boost regimen with DNA priming and boosting with a viral vector. Potentiation of responses with adjuvant strategies will probably be necessary. Hepatitis C virus immunization is in an early stage of development. Given the explosive growth in the understanding of immunology, progress should be rapid.

Animals↗

PubMed2001

Perspectives on prophylactic and therapeutic immunization against hepatitis B and C viruses.

Hepatitis B and C viruses are the etiologic agents of most cases of the world's most common cancer: hepatocellular carcinoma (HCC). The incidence of this cancer is rising globally, due largely to the epidemic spread of HCV infection. It is thus essential that means be found to prevent this lethal disease by prophylactic and therapeutic immunization. DNA-based immunization has the ability to induce both cell-mediated and humoral immunity, and thus lends itself to therapeutic immunization strategies. DNA-based immunization also lends itself to the design of multivalent immunogens targeted at various pathogens. This strategy will facilitate economical immunization in the developing world. DNA-based immunization has protected chimpanzees against HBV challenge and, in combination with recombinant canarypox boosters, has downregulated chronic HBV infection in a chimpanzee. DNA-based immunization is still in its infancy for HCV infections. Substantial immunogenicity has been demonstrated, particularly in mice; however, enhancement of immunogenicity will be required to achieve prophylactic or therapeutic efficacy.

Animals↗

PubMed2001

Infectivity of blood from PCR-positive, HBsAg-negative, anti-HBs-positive cases of resolved hepatitis B infection.

BACKGROUND: Numerous reports have noted the existence of sera, particularly from resolving cases of HBV infection, that are positive for HBV DNA by PCR, despite being negative for HBsAg and IgM anti-HBc. If such blood is infective and detectable by HBV NAT screening, it seems desirable to introduce such screening for transfused blood. STUDY DESIGN AND METHODS: Three chimpanzees were inoculated with serum and lymphocytes from three patients who were HBV DNA PCR positive, but HBsAg negative. The animals were tested over a period of 15 months for HBsAg, anti-HBs, anti-HBc, and HBV DNA by PCR. RESULTS: All animals remained uninfected. CONCLUSION: Small amounts of plasma and MNCs from HBV DNA-positive HBsAg-negative blood do not appear to be infectious; however, further studies with larger volumes of inoculum should be conducted.

Animals↗

PubMed2001

Automation of nucleic acid extraction for NAT screening of individual blood units.

BACKGROUND: Automation of NAT for single units of blood is currently hampered by the labor-intensive steps involved in the extraction of nucleic acids from samples before the amplification procedures. A new method has been developed for the automation of these steps using hydrophilic polyvinylidene fluoride (PVDF) filter plates. STUDY DESIGN AND METHODS: Quantitative nucleic acid recoveries from sera containing HCV, HIV, HBV, HAV, and human parvovirus B19 and from 3H-labeled HCV RNA were determined in parallel by the semi-automated PVDF method and a single-column method (Qiagen). Quantitative PCR was performed. RESULTS: Similar recoveries of HCV, HIV, and HBV (with silica beads) were observed with the PVDF method and with the Qiagen single-column method. The sensitivity of the PVDF-based PCR assay for HCV, HIV, and HBV in serially diluted serum samples was always within two serial dilutions of that obtained when the Qiagen single-column method was used in the same assays. With the use of 3H-labeled HCV RNA, recoveries of approximately 70 percent were found by both methods. CONCLUSION: The PVDF method will permit full automation of the simultaneous extraction of nucleic acid from sera containing HCV, HIV, and HBV. This procedure will permit NAT screening of individual units of blood, will replace the current screening of pools, and will achieve improved blood safety with reduced labor and costs.

Blood Transfusion↗

PubMed2001

DNA prime/canarypox boost-based immunotherapy of chronic hepatitis B virus infection in a chimpanzee.

There are about 200 million chronic hepatitis B virus (HBV) carriers at high risk of development of cirrhosis and hepatocellular carcinoma. Termination of the carrier state may avert these risks. We have investigated immunotherapy for chronic HBV infection in a chimpanzee HBV carrier using recombinant DNA-based immunization followed by a recombinant canarypox booster. One week after the booster, HBV DNA declined greater than 400-fold and remained undetectable by the quantitative polymerase chain reaction (PCR) assay for 186 weeks. Plasma levels of hepatitis B surface antigen (HBsAg) declined for only a short time. The decline in HBV DNA correlated with a boost in gamma interferon production without a corresponding boost in cytotoxic T lymphocyte levels, and decline in the transcriptional template or covalently closed circular DNA level. Confirmation of these findings requires further studies in chimpanzees and/or in humans.

Alanine Transaminase↗

PubMed2001

Perspectives on hepatitis B studies with chimpanzees.

Chimpanzees have been shown to be exquisitely susceptible to human hepatitis viruses, without themselves developing clinical illness, thus providing an important model for studies on these agents. Chimpanzees have contributed substantially to human welfare by making possible the development of hepatitis B vaccines, which now prevent development of cirrhosis and hepatocellular carcinoma in millions of people. They have provided a means to evaluate the efficacy of virus inactivation strategies, which have made blood derivatives formerly contaminated with blood-borne viruses (hepatitis B, C, and human immunodeficiency viruses) safe with respect to their transmission. In exchange for these contributions, humans owe chimpanzees lifelong retirement in sanctuaries that offer socialization and environmental enrichment.

Animal Welfare↗

PubMed2001

Pharmacokinetics, safety, and antiviral effects of hypericin, a derivative of St. John's wort plant, in patients with chronic hepatitis C virus infection.

Hypericin is a natural derivative of the common St. Johns wort plant, Hypericum perforatum. It has in vitro activity against several viruses, including bovine diarrhea virus, a pestivirus with structural similarities to hepatitis C virus (HCV). We conducted a phase I dose escalation study to determine the safety and antiviral activity of hypericin in patients with chronic HCV infection. The first 12 patients received an 8-week course of 0.05 mg of hypericin per kg of body weight orally once a day; 7 patients received an 8-week course of 0.10 mg/kg orally once a day. At the end of the 8-week period of treatment, no subject had a change of plasma HCV RNA level of more than 1.0 log(10). Five of 12 subjects receiving the 0.05-mg/kg/day dosing schedule and 6 of 7 subjects receiving the 0.10-mg/kg/day dosing schedule developed phototoxic reactions. No other serious adverse events associated with hypericin use occurred. The pharmacokinetic data revealed a long elimination half-life (mean values of 36.1 and 33.8 h, respectively, for the doses of 0.05 and 0.1 mg/kg) and mean area under the curve determinations of 1.5 and 3.1 microg/ml x hr, respectively. In sum, hypericin given orally in doses of 0.05 and 0.10 mg/kg/d caused considerable phototoxicity and had no detectable anti-HCV activity in patients with chronic HCV infection.

Adolescent↗

PubMed2001

Novel hepatitis B virus strain from a chimpanzee of Central Africa (Pan troglodytes troglodytes) with an unusual antigenicity of the core protein.

We and others have previously reported a hepatitis B virus (HBV)-like hepadnavirus strain which seemed to be indigenous to West African chimpanzees (Pan troglodytes verus). After that, we obtained an HBsAg-positive serum sample from a chimpanzee from Central Africa, named Bassi, belonging to another subspecies (P. troglodytes troglodytes). The full-genome nucleotide sequence of the hepadnavirus from Bassi showed a significant difference (9-26%) from those so far reported from primates including humans, chimpanzees and gorillas, suggesting a novel strain. More interestingly, however, the core antigen (HBcAg) deduced from Bassi's sequence showed only 78-82% similarity to known primate strains at the amino acid level, whereas the other strains shared more than 90% similarity. HBcAg expressed from Bassi HBV failed to react with monoclonal antibodies that were directed at an epitope borne by codons 135-145 of HBcAg of conventional hepadnaviruses. This could explain why Bassi was negative for anti-HBc in a routine test. Here we report the novel HBV strain presumably indigenous to P. troglodytes troglodytes in Central Africa.

Africa, Central↗

PubMed2001

Prevention of respiratory syncytial virus infection in high risk infants.

The incidence of serious RSV illness in premature infants and in infants with CLD can be reduced. Prophylaxis in infants with CHD and cystic fibrosis seem prudent as well, and clinical trials are currently under way to evaluate the use of Synagis in these high-risk groups. Synagis is preferred for most high-risk children because of its ease of administration, safety and effectiveness. The dose for Synagis is 15 mg/kg i.m. monthly during RSV season.

Antibodies, Monoclonal↗

PubMed2000

DNA prime-canarypox boost with polycistronic hepatitis C virus (HCV) genes generates potent immune responses to HCV structural and nonstructural proteins.

DNA vaccination was employed to study immune responses to hepatitis C virus (HCV) proteins. As an immunizing strategy, we studied immune responses of BALB/c (H-2d) and C57BL/6 mice (H-2b) to HCV genes delivered intramuscularly as a polycistronic construct capsid/E1/E2/NS2/NS3 (pRC/C-NS3) encoding 5 structural and nonstructural proteins. We also evaluated canarypox virus containing the same HCV genes as a means for potentiating immune responses to naked DNA. Our results indicate that mice that received a polycistronic pRC/C-NS3 with canarypox booster had enhanced antibody and cellular responses to HCV proteins. Immunodominant CD8(+) T cell responses to several HCV structural and nonstructural proteins, characterized by cytotoxicity and interferon (IFN)-gamma production or IFN-gamma production without significant cytotoxicity, were observed in both strains of mice. The combination of naked DNA with a nonreplicating canarypox booster encoding HCV polycistronic pRC/C-NS3 genes appears to diversify and enhance T cell responses to HCV proteins.

Animals↗

PubMed2000

Full-genome sequence analyses of hepatitis B virus (HBV) strains recovered from chimpanzees infected in the wild: implications for an origin of HBV.

Hepatitis B virus (HBV) belongs to the genus Orthohepadnavirus of the family Hepadnaviridae. Having been found in various animals (duck, heron, woodchuck, ground squirrel, and primates), hepadnaviruses must have undergone a long history of evolution and may comprise more members than currently recognized. Chimpanzees may also have their own hepadnavirus, even if it might be very close to HBV. We analyzed HBV-like sequences from three chimpanzees (Pan troglodytes) that were most likely infected during their life in Africa in the wild. Two chimpanzees (Ch256 and Ch258) possessed a viral genome of 3182 nt in length with a 33-nt deletion in the preS1 region, which could not be classified into any of the six genotypes (A-F) of human HBV but was very homologous to a previously reported isolate from a London Zoo chimpanzee. Phylogenetically distinct from the HBV-like sequences from gibbons, orangutans, and a gorilla so far reported, the Ch256 and Ch258 isolates would represent an indigenous chimpanzee HBV (tentatively ChHBV). A third chimpanzee (Ch195) had a 3212-nt genome, classifiable into the genotype E of HBV. Because HBV-E has been found mostly in Africans, Ch195 may have been infected from a human source in Africa. However, an inverse scenario is also possible: a spread of HBV-E might have occurred from chimpanzees to humans a long time ago in Africa. Analysis of the arginine-rich C-terminal region of the core protein, which is well conserved among mammalian hepadnaviruses, indicated that HBV-E/F and nonhuman primate hepadnaviruses are much closer than HBV-A/B/C/D to the hepadnaviruses of woodchuck and ground squirrel. Our results support an "ex-nonhuman primate" hypothesis for the origin of HBV.

Africa↗

PubMed2000

Immune responses to hepatitis C virus structural and nonstructural proteins induced by plasmid DNA immunizations.

DNA-based immunizations have been used to elicit cellular immunity to hepatitis C virus (HCV) proteins in mice. Mice were immunized by intramuscular or intradermal injections of plasmid DNA derived from a near-full-length HCV genotype 1b genomic clone (pRC/B2) or individual genomic clones. These immunizations induced cytotoxic T lymphocytes (CTLs), as revealed in standard chromium-release assays that used syngeneic peptide-pulsed or transfected target cells. These assays identified four CTL epitopes within the capsid, E1, and E2 regions of the polyprotein sequence of HCV genotype 1a that were cross-reactive with HCV genotype 1b. Additionally, CTLs derived from mice immunized with either NS3 or NS5 specifically lysed target cells sensitized to either the genotype 1a or 1b gene products. Nucleic acid immunizations also generated humoral immunity to HCV proteins, as detected by anti-HCV reactivity to NS3 and capsid in ELISAs and immunoblot assays.

Animals↗

PubMed2000

Identification of a human epitope in hepatitis C virus (HCV) core protein using a molecularly cloned antibody repertoire from a non-symptomatic, anti-HCV-positive patient.

Healthy carriers of hepatitis C virus (HCV) infection exhibit a specific antibody response against all HCV antigens, which could play a role in disease control. Generation of panels of human antibodies may permit a thorough characterization of this response and further identify particular antibodies with potential clinical value. To this effect, we have established a human phage-display antibody library from a patient exhibiting a high antibody response against HCV antigens and no clinical symptoms of disease. This library was screened against a recombinant core antigen [amino acids (aa) 1-119] produced in E. coli. Two recombinant Fab-carrying phages (rFabCs) were isolated and characterized. Both rFabC3 and rFabC14 recognize aa 1-48 on core antigen, but rFabC14 is competed out by a synthetic peptide, C(2-20) (aa 1-20), at much lower concentrations than rFabC3. In order to identify more precisely the recognition sites of these antibodies, we produced soluble forms of the rFabs (sFabs), and used them to pan a random phage-display peptide library. A single peptide sequence, QLITKPL, was identified with sFabC3, while two equally represented sequences, HAFPHLH and SAPSSKN, were isolated using sFabC14. The QLITKPL sequence was partially localized between aa 8 and 14 of core protein, but no clear homology was found for the two sFabC14 peptides. However, we confirmed the specificity of these peptides by competition experiments with sFabC14.

Adult↗