Thymic selection and adaptability of cytotoxic T lymphocyte responses in transgenic mice expressing a viral protein in the thymus.

Upon primary challenge with lymphocytic choriomeningitis virus (LCMV), H-2d (BALB/cByJ) mice mount a cytotoxic T lymphocyte (CTL) response to a single immunodominant domain of the viral nucleoprotein (NP) but no detectable response to the viral glycoprotein (GP). To manipulate this CTL response, the viral NP gene was expressed in the thymus and peripheral T lymphocytes using the murine Thy1.2 promoter. As a result, such Thy1.2-NP (H-2d) transgenic (tg) mice deleted their high-affinity anti-LCMV-NP CTL, but generated equal numbers of lower-affinity NP CTL. Further, they made an alternative anti-LCMV-GP CTL response that is not normally found in non-tg mice indicating a hierarchial control of the CTL response. Unlike the H-2d mice, H-2b (C57Bl/6J) mice normally mount a CTL response to both LCMV-GP and -NP. When the LCMV-NP was expressed using the Thy1.2 promoter in these H-2b mice, the LCMV-NP-specific CTL response was completely aborted and no CTL to new, alternative viral epitopes were generated. Dilutions of H-2b or H-2d NP peptides indicated that 3-4 logs less H-2b NP peptide was required to sensitize syngeneic target cells for CTL-specific lysis, suggesting that the differing affinities of H-2b and H-2d major histocompatibility complex molecules for their peptides likely account for the total removal of NP CTL in the H-2b mice but only partial removal in H-2d mice made to express thymic NP. Thymic grafting experiments done with thymi from newborn Thy1.2-NP tg mice show that selection processes studied in this model are of central (thymic) origin and are not caused by Thy1.2-positive LCMV-NP-expressing T lymphocytes in the periphery.

Neuron-specific expression of a hamster prion protein minigene in transgenic mice induces susceptibility to hamster scrapie agent.

To study the effect of cell type-restricted hamster PrP expression on susceptibility to the hamster scrapie agent, we generated transgenic mice using a 1 kb hamster cDNA clone containing the 0.76 kb HPrP open reading frame under control of the neuron-specific enolase promoter. In these mice, expression of HPrP was detected only in brain tissue, with highest levels found in neurons of the cerebellum, hippocampus, thalamus, and cerebral cortex. These transgenic mice were susceptible to infection by the 263K strain of hamster scrapie with an average incubation period of 93 days, compared to 72 days in normal hamsters. In contrast, nontransgenic mice were not susceptible to this agent. These results indicate that neuron-specific expression of the 1 kb HPrP minigene including the HPrP open-reading frame is sufficient to mediate susceptibility to hamster scrapie, and that HPrP expression in nonneuronal brain cells is not necessary to overcome the TSE species barrier.

Evaluation of a new molecular assay for detection of human immunodeficiency virus type 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA.

BACKGROUND: Rapid, sensitive, specific, and cost-effective screening of donated blood to prevent transmission of infectious agents remains challenging. In recent years, incorporation of nucleic acid testing for HIV-1 and HCV RNA improved blood safety by reducing the window period between infection and serologic detection. For HBV infection, this window period with most serologic assays is 50-60 days. Adding a nucleic acid test (NAT) for HBV DNA with existing NATs for HIV-1 and HCV RNA would further improve blood safety and blood screening efficiency. OBJECTIVE: To evaluate the Procleix Ultrio Assay for simultaneous detection of HIV-1 and HCV RNA and HBV DNA and corresponding discriminatory assays. STUDY DESIGN: The performance of these assays, which utilize the same technology and assay format as the Procleix HIV-1/HCV assay, was determined using relevant clinical specimens and analytical sensitivity and specificity panels. RESULTS: The Procleix Ultrio Assay demonstrated specificity of > or =99.5% in healthy donor blood specimens and in plasma containing potentially interfering substances or other blood-borne pathogens. Assay sensitivity demonstrated >95% detection of 100copies/mL, 30IU/mL, and 15IU/mL for HIV-1 and HCV RNA, and HBV DNA, respectively. The assay detects all known HIV-1 subtypes and HCV and HBV genotypes and is highly reproducible. Statistical analysis using receiver operating characteristic plots demonstrated wide analyte cutoff values for each assay associated with assay specificity and sensitivity of > or =99.5%. CONCLUSIONS: In this investigational study, the Procleix Ultrio Assay sensitivity and specificity were similar to existing NATs used in blood-bank settings to detect HIV-1 and HCV RNA and provided equivalent sensitivity and specificity for detection of HBV DNA. Using this combination assay, blood safety may be improved and the multiplex format enhances blood screening efficiency. The throughput capability of this assay is compatible with large volume processing and the chemistry is adaptable to full automation.

Detection of HIV-1 in alternative specimen types using the APTIMA HIV-1 RNA Qualitative Assay.

Peripheral blood mononuclear cells (PBMCs), saliva, seminal plasma, and dried blood spots were evaluated as specimen types for the APTIMA HIV-1 RNA Qualitative Assay (APTIMA HIV-1 Assay), which employs a target capture step to recover HIV-1-specific sequences from complex specimen types. Analytical sensitivity studies were carried out using samples that were either diluted or eluted with a buffered detergent and spiked with different concentrations of HIV-1 ranging from 1 to 10,000 copies/mL. PBMC samples spiked with HIV-1 had comparable analytical sensitivity to HIV-1 spiked plasma with a 95% limit of detection of 13.1 and 17.2 copies/mL, respectively. Analytical sensitivity in seminal plasma specimens diluted 1:5 and saliva diluted 1:2 was comparable to HIV-1 spiked dilution buffer alone. Whole blood and dried blood spot specimens spiked with HIV-1 had equivalent reactivity at 250 copies/spot (5000 copies/mL). However, the 95% limit of detection values were significantly different (293.7 copies/mL for whole blood and 2384 copies/mL for dried blood spot specimens). No significant effect on analytical sensitivity was observed when one HIV-1 positive dried blood spot punch was pooled with up to 9 HIV-1 negative dried blood spot punches. Together, these studies demonstrate that the APTIMA HIV-1 RNA Qualitative Assay can be used to process a diverse array of specimen types with minimal impact on analytical sensitivity for most specimen types.

The course of hepatitis C viraemia in transfusion recipients prior to availability of antiviral therapy.

Knowing the likely distribution of intervals from hepatitis C infection to first RNA-negativity is important in deciding about therapeutic intervention. Prospectively collected sera and data from the Transfusion-transmitted Viruses Study (1974-1980) provide specific dates of infection and pattern of alanine aminotransferase (ALT) elevations. We examined frequency, timing and correlates of spontaneous resolution for 94 acutely infected transfusion recipients followed for a median of 9.5 months. Later, follow-up sera (>10 years) were available for 27 of the 94 cases from a Veterans Administration (VA) Study (1989-1990). Twenty-five (27%) of the 94 cases were classified as probably resolved during the episode itself. First RNA negativity occurred at 6-50 weeks (median, 19.5 weeks) after infection, and 5-43 weeks (median, 11 weeks) after ALT elevation. Thirteen of the 25 cases remained RNA-negative subsequently; 12 others had 1-6 RNA-positive sera intercalated between first and last RNA-negative results. RNA negativity, therefore, began variably and was interrupted in 12 cases of 25 (48%) by transient RNA-positive sera. Five of these 25 patients who were RNA-negative in the last study specimen had late, Veterans Administration Study follow-up; none showed viraemia. Of the remaining 69 transfusion transmitted virus study recipients, whose last serum was RNA-positive, two cleared viraemia after the last study serum but before late follow-up. Eleven (16%) had 23 intercalated RNA-negative sera before last positivity. RNA status, therefore, needs monitoring for many months before judging the spontaneous outcome as transient negativity may occur. Resolution was significantly more common in women and symptomatic cases; it was not associated with viral load in the infectious donation, HCV genotype, or the recipient's age.

Mentoring in biomedical science graduate programs: a student's perspective.

The traditional model for the mentoring of graduate students has been for the student to receive all formal mentoring from the thesis advisor, the laboratory principal investigator (PI). While this continues to be a successful model for some students, other students find that they need or desire additional mentors during their graduate career. Graduate programs have a responsibility to provide their students with increased mentoring opportunities. Three means that graduate programs could use to serve the diverse needs of students are discussed as well as the potential benefits to the program and the students.

Sclerotome induction and differentiation.

Inductive events in the development of the sclerotome and their possible underlying mechanisms were reviewed from the primary literature. A brief review of morphological and anatomical aspects of sclerotome development was given. The importance of the notochord and neural tube in sclerotome induction and somite chondrogenesis in vivo and in vitro was established. The functions and patterns of expression of different sclerotome markers were discussed. Shh and Noggin were discussed as two molecules produced by the neural tube and notochord that appear to maintain and initiate the sclerotome, respectively. While the abilities of the axial organs and Shh and Noggin to induce sclerotome marker expression in the somite was not disputed, the exact nature of these inductions was discussed with regard to possible effects on gene expression, effects on cell survival, and physical effects on the cells and it was argued that the fundamental nature of inductive events in the sclerotome is still unknown.

Determination of sclerotome to the cartilage fate.

When the somite first forms the cells appear to be equivalent in potential. In order to understand the lineage diversification of the somite, the determination of sclerotome cells to the cartilage fate was tested using an in vivo challenge assay in which quail sclerotome fragments were grafted into a dorsal position in a chick host. Grafts containing undetermined cells were expected to differentiate into other tissues while grafts containing determined chondrocyte precursors were expected to consistently give rise to cartilage. We found that grafted sclerotome fragments from somite stages V-XX were capable of giving rise to integrated muscle and dermis and that it was not until fragments from stage XII somites were grafted that cartilage was consistently produced in the assay. Sclerotomal tissue from embryonic day 4-6 embryos remained as morphologically unintegrated mesenchyme when grafted into an embryonic day 2 host, but formed only cartilage when placed into an identically aged host. Vertebral body cartilage from embryonic day 7 and embryonic day 8 embryos formed exclusively ectopic cartilage in an embryonic day 2 host. We conclude that cells determined to the cartilage fate do not appear until somite stage XII, but that not all sclerotome cells are determined at this time. The effect of host age on the differentiation and morphogenetic behavior of sclerotome fragment grafts in this assay indicate the existence of developmental eras within the embryo.

Dorsoventral axis determination in the somite: a re-examination.

We have repeated classic dorsoventral somite rotation experiments (Aoyama and Asamoto, 1988, Development 104, 15-28) and included dorsal and ventral gene expression markers for the somitogenic tissue types, myotome and sclerotome, respectively. While the histological results are consistent with those previously published, gene expression analysis indicates that cells previously thought to be 'sclerotome' no longer express Pax1 mRNA, a sclerotome marker. These results, together with recent quail-chick transplantation experiments indicating that even very late sclerotome tissue fragments are multipotential (Dockter and Ordahl, 1998, Development 125, 2113-2124), lead to the conclusion that sclerotome tissue remains phenotypically and morphogenetically plastic during early embryonic somitogenesis. Myotome precursor cells, by contrast, appear to be determined within hours after somite epithelization; a finding consistent with recent reports (Williams and Ordahl, 1997, Development 124, 4983-4997). Therefore, while these findings support a central conclusion of Aoyama and Asamoto, that axis determination begins to occur within hours after somite epithelialization, the identity of the responding tissues, myotome versus sclerotome, differs. A simple model proposed to reconcile these observations supports the general hypothesis that determinative aspects of early paraxial mesoderm growth and morphogenesis occur in early and late phases that are governed principally by the myotome and sclerotome, respectively.

How virus induces a rapid or slow onset insulin-dependent diabetes mellitus in a transgenic model.

We developed two distinct transgenic mouse models in which virus induced insulin-dependent (type 1) diabetes mellitus (IDDM). In one of these lines, the unique viral transgene was expressed in the islets of Langerhans and also in the thymus, but in the other line, expression was only in the islets. Insertion and expression of the viral (self) gene, per se, did not lead to IDDM, (incidence < 5%). By contrast, induction of an anti-self (anti-viral) CD8+ CTL response to the same virus later in life caused IDDM (incidence < 90%) in both transgenic lines, although the kinetics and requirements for CD4 help, the affinity and avidity of CD8+ CTL differed in each line. Mice not expressing the viral (self) gene in the thymus developed IDDM 10-14 days after infection. CD4+ T cells played no detectable role, since their depletion failed to alter either the kinetics or incidence of IDDM. By contrast, mice that expressed the viral gene in the thymus required significantly more time to develop IDDM. Their anti-self (viral) CD8+ CTL were of lower affinity and avidity than CD8+ CTL generated by nontransgenic controls. Disease was dependent on T cell help, since deletion of CD4+ cells completely circumvented the IDDM.

Competitive selection in vivo by a cell for one variant over another: implications for RNA virus quasispecies in vivo.

Infidelity of genome applications of RNA viruses leads to the generation of viral quasispecies both in vitro and in vivo. However, the biological significance of such generated variants in vivo is largely unknown and controversial. To study this issue, we continued our evaluation of the tropism of a lymphocytic choriomeningitis virus (LCMV) variant termed clone 13 with its parental virus clonal pool ARM 53b (wild-type parent) for neuronal cells in vivo. Earlier in vivo and in vitro studies noted that the wild-type virus contained a Phe at glycoprotein (GP) residue 260 which correlated with neuron tropism compared with LCMV variants containing a Leu at residue 260 which showed selected tropism for cells of the immune system (C.F. Evans, P. Borrow, J. C. de la Torre, and M. B. A. Oldstone J. Virol. 68:7367-7373, 1994; L. Villarete, T. Somasundaram, and R. Ahmed, J. Virol 68:7490-7496, 1994). Here we (i) evaluated the ability of the viral variants with either a Phe or Leu at GP residue 260 to replicate in vivo in the spleen, liver, or brain, (ii) analyzed the ability of these viruses to compete against each other for cell (neuron)-specific selection following a single viral inoculation of different ratios of both viruses, and (iii) utilized genetic reassortants of both viruses to test their ability to replicate in neurons in vivo. We found that viral variants containing either a Phe or Leu at GP residue 260 were equally capable of replicating in neurons, but when inoculated together, neurons selected for the viral population containing Phe at GP residue 260 over viruses containing a Leu at this position. This was in contrast to selection in the liver and spleen that favored viruses with Leu and not Phe at GP residue 260. Analysis of inoculations with viral reassortants indicated that genes encoded on the short RNA (the GP and nucleoprotein, not the L [polymerase] and Z proteins that are encoded by the large RNA) were associated with neurotropism. Since the nucleoprotein sequences of wild-type Armstrong and clone 13 are identical, it is likely that specific cytoplasmic factors of the neurons play a fundamental role in the selection of virus with Phe at GP residue 260.

Islet inflammation and hyperplasia induced by the pancreatic islet-specific overexpression of interleukin-6 in transgenic mice.

Interleukin-6 (IL-6) is thought to be involved in the pathogenesis of autoimmune insulin-dependent diabetes mellitus. To examine this possibility, we developed two lines of transgenic mice (termed RIP-IL6) which overexpressed IL-6 in the pancreatic islet beta cells. RIP-IL6 mice, while showing a modest reduction in body weight, remained normoglycemic throughout their lives. Furthermore, insulin gene expression and glucose tolerance were similar to non-transgenic littermates. Histopathological examination revealed significant changes in the pancreas but not other organs of RIP-IL6 animals, with marked alterations in the architecture of the islets, in the islet cells, and in surrounding tissues. In younger animals these changes included islet hyperplasia with increased mitotic figures, neo-ductular formation, fibrosis, and a scant mononuclear cell infiltration (insulitis). In addition, immunostaining for islet hormones revealed changes in both the topography and density of beta and alpha cells. In older RIP-IL6 mice, a more florid insulitis was observed which was composed predominantly of B220+ B lymphocytes and, to a lesser extent, Mac-1+ macrophages and CD4+ and CD8+ T lymphocytes. Immunostaining for mouse IgG revealed significant numbers of plasma cells in the peri-islet infiltrates, which suggested that IL-6 induced differentiation of the recruited B lymphocytes. Therefore, islet overexpression of IL-6 produces a complex, localized host response implicating this cytokine in not only inflammatory processes that occur in autoimmune diabetes but also cellular neogenesis, which may indicate a role in tissue repair.

CD4-deficient mice have reduced levels of memory cytotoxic T lymphocytes after immunization and show diminished resistance to subsequent virus challenge.

Although primary antiviral CD8+ cytotoxic T lymphocytes (CTL) can be induced in mice depleted of CD4+ T cells, the role of CD4+ T lymphocytes in the generation and maintenance of antiviral memory CTL is uncertain. This question, and the consequences upon vaccine-mediated protection, were investigated in transgenic CD4 knockout (CD4ko) mice, which lack CD4+ T lymphocytes. Infection of immunocompetent C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV), or with recombinant vaccinia viruses bearing appropriate LCMV sequences, induces long-lasting protective immunity, mediated mainly by antiviral CD8+ CTL. Here we report two important findings. First, LCMV-specific CD8+ memory CTL are maintained at considerably lower levels in CD4ko mice than in normal C57BL/6J mice; we demonstrate a reduction in precursor CTL evident as soon as 30 days postimmunization and declining, by day 120, to levels 1 to 2 log units below those in normal mice. Thus, CD4+ T cells appear to be important to the generation and maintenance of their CD8+ counterparts. Second, this reduction has an important biological consequence; compared with immunocompetent mice, CD4ko mice immunized with vaccinia virus recombinants expressing nucleoprotein or glycoprotein of LCMV are less effectively protected from subsequent LCMV challenge. Thus, this study underscores the potential importance of CD4+ T lymphocytes in generation of appropriate levels of CD(8+)-cell-mediated immunoprotective memory and has implications for vaccine efficacy in individuals with immune defects in which CD4 levels may be reduced, such as AIDS.

Lookback on donors who are repeatedly reactive on first-generation hepatitis C virus assays:justification and rational implementation.

BACKGROUND: The purpose of this study was to explore strategies to minimize the number of unwarranted consignee notifications resulting from hepatitis C virus (HCV) first-generation (single-antigen) enzyme immunoassay (EIA 1.0) targeted lookback. STUDY DESIGN AND METHOD: The four blood centers participating in this study contributed data on 3753 HCV EIA 1.0-repeatably reactive (RR) donations. The analysis focused on 1) statistical evaluation of HCV EIA 1.0 signal-to-cutoff (S/CO) ratios versus HCV second-generation recombinant immunoblot assay (RIBA 2.0) interpretation from all participating blood centers and 2) RNA testing using transcription-mediated amplification on all HCV EIA 1.0 RR/RIBA 2. 0-positive or -indeterminate specimens and a subset of RIBA 2. 0-negative donations for which specimens were available. RESULTS: Analysis of HCV EIA 1.0 S/CO ratios versus RIBA 2.0 indicated that 1180 (89%) of 1326 RIBA 2.0-positive specimens had an S/CO ratio >2. 5, while 146 (11%) had a ratio 2.5, while 1954 (87%) had a ratio 2.5. HCV RNA was detected in only 2 (1.5%) of 137 HCV EIA 1.0-RR/RIBA 2.0-negative specimens: 1 of these 2 specimens had an S/CO >2.5, while the other had an S/CO 2.5 yielded an 89- percent sensitivity for RIBA 2.0-positive specimens, and donations with an S/CO ratio >2.5 had a 75-percent probability of being RIBA 2.0 positive. A policy recommendation to use the S/CO ratio to triage lookback would prevent unwarranted notification of 87 percent of recipients of blood from RIBA 2.0-negative donors and would result in a failure to notify only 5 to 10 percent of recipients potentially exposed to infectious units.

Highly sensitive multiplex assay for detection of human immunodeficiency virus type 1 and hepatitis C virus RNA.

Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was >or=99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 - specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both >or=99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications.