Nancy D. Borson

Temporal sequence of transcription of perforin, fas ligand, and tumor necrosis factor-α genes in rejecting skin allografts

BACKGROUND Perforin, Fas ligand (FasL), and tumor necrosis factor-alpha (TNF-alpha) have been implicated in cytolytic T lymphocyte (CTL) effector function. However, the relative roles of these effector molecules in allograft rejection are unclear, and there has been no rigorous quantitation of transcription of the respective genes throughout the period from transplantation to rejection. METHODS We orthotopically transplanted mouse tail skin allografts and estimated the numbers of transcripts of these genes expressed by graft-infiltrating T cells with rigorous quantitative, competitive reverse transcribed PCR (QC-RT-PCR) that enabled the comparison of transcription of different genes. RESULTS Perforin and FasL mRNA levels correlated closely with the rejection of allografts by normal hosts over the 4 days preceding rejection. Antibody-mediated depletion of host CD4+ T cells retarded perforin transcription and significantly suppressed FasL transcription, suggesting FasL was not required for allograft rejection. TNF-alpha transcription was the highest of these genes in this time period, but these levels were dwarfed by TNF-alpha transcription at 24 hr posttransplant when transcription in both autografts and allografts was 30-fold higher than in allografts on the day before rejection. Elimination of the function of these single or paired genes through genetic mutation or antibody treatment had no significant effect on the speed of rejection. CONCLUSIONS The levels of perforin and FasL transcription appeared to be related to the process of allograft rejection in normal hosts. However, TNF-alpha transcription was highest during the posttransplant period suggesting that the principal role of TNF-alpha is in wound-healing rather than the effector phase of rejection.

The application of real-time PCR to the analysis of T cell repertoires

The diversity of T-cell populations is determined by the spectrum of antigen-specific T-cell receptors (TCRs) that are heterodimers of α and β subunits encoded by rearranged combinations of variable (AV and BV), joining (AJ and BJ), and constant region genes (AC and BC). We have developed a novel approach for analysis of β transcript diversity in mice with a real-time PCR-based method that uses a matrix of BV- and BJ-specific primers to amplify 240 distinct BV–BJ combinations. Defined endpoints (Ct values) and dissociation curves are generated for each BV–BJ combination and the Ct values are consolidated in a matrix that characterizes the β transcript diversity of each RNA sample. Relative diversities of BV–BJ combinations in individual RNA samples are further described by estimates of scaled entropy. A skin allograft system was used to demonstrate that dissection of repertoires into 240 BV–BJ combinations increases efficiency of identifying and sequencing β transcripts that are overrepresented at inflammatory sites. These BV–BJ matrices should generate greater investigation in laboratory and clinical settings due to increased throughput, resolution and identification of overrepresented TCR transcripts.

MHC Class II Epitope Nesting Modulates Dendritic Cell Function and Improves Generation of Antigen-Specific CD4 Helper T Cells

CD4 Th cells are critical to the development of coordinated immune responses to infections and tumors. Th cells are activated through interactions of the TCR with MHC class II complexed with peptide. T cell activation is dependent on the density of MHC peptide complexes as well as the duration of interaction of the TCR with APCs. In this study, we sought to determine whether MHC class II peptides could be modified with amino acid sequences that facilitated uptake and presentation with the goal of improving Th cell activation in vitro and in vivo. A model epitope derived from the murine folate receptor α, a self- and tumor Ag, was modified at its carboxyl terminus with the invariant chain-derived Ii-Key peptide and at its N terminus with a peptide that enhances uptake of Ag by APC. Modification of a peptide resulted in enhanced generation of high-avidity murine folate receptor α T cells that persisted in vivo and homed to sites of Ag deposition. The nesting approach was epitope and species independent and specifically excluded expansion of CD4 regulatory T cells. The resulting Th cells were therapeutic, enhanced in vivo helper activity and had an increased ability to resist tolerizing immune microenvironments. In addition to improved immunoadjuvants, this epitope modification strategy may be useful for enhancing ex vivo and in vivo generation of Th cells for preventing and treating diseases.

Origins of an Anasazi scarlet macaw feather artifact

An artifact ascribed to the Anasazi culture (dated here to 920 ± 35 B.P.) is unique in its integrity, construction technique, style, and materials, including multiple yucca ropes with attached adult scarlet macaw feathers joined to a Sciurus aberti (tas-sel-eared squirrel) pelt and hide straps. We applied methods from anthropology and molecular biology to ascertain the origins of materials and manufacturing technique. The cytochrome b gene from the ancient DNA of the pelt was sequenced in its entirety. This gene was unique as defined by new nucleotide substitutions that distinguished it from the other S. aberti alleles. Phylogenetic trees constructed by both neighbor-joining and maximum parsimony methods are consistent with this unique allele being most closely related to genes from two extant American Southwest S. aberti subspecies and more distantly related to Mexican S. aberti genes. Our observations support the conclusion that the entire artifact was constructed in the American Southwest using native materials, including the squirrel pelt and scarlet macaw feathers. This contradicts a prior hypothesis that the feather rope component was assembled before being traded north from Mexico.

Cysteine-Tailed Class I-Binding Peptides Bind to CpG Adjuvant and Enhance Primary CTL Responses

Immunostimulatory CpG motifs in synthetic oligonucleotides can be effective adjuvants for the priming of CTLs. We first observed that a single male-specific peptide (KCSRNRQYL) (HY2) was more efficient than another male-specific peptide (WMHHNMDLI) (HY1) at priming IFN-γ-secreting CTLs in vivo when combined with lipid A and CpG and that it also visibly precipitated CpG. The addition of the six N-terminal residues (KCSRNR) from HY2 to HY1 yielded a peptide, KCSRNR-HY1, that both precipitated CpG and primed increased numbers of HY1-specific CTLs. We refer to this type of peptide as a primotope that includes a class I binding peptide tailed with amino acids that increase priming. Ala residues were substituted for the Arg/Lys residues (ACSANA-HY1), and these substitutions did not reduce in vivo priming potential. However, the substitution of Ala for Cys (KASRNR-HY1) resulted in the complete loss of priming, demonstrating the importance of Cys for in vivo priming when mixed with CpG. This result suggested that increased priming was based in disulfide bonding between Cys residues and internal phosphorothioate groups of synthetic CpG. The addition of Cys-bearing primotopes to radiolabeled CpG with a single thioate group resulted in the appearance of a new band that was inhibited by 1) Cys > Ala substitution and 2) reduction and alkylation of CpG. These results reveal a novel mechanism for complexing class I binding peptides and CpG adjuvant for development of new peptide-adjuvant combinations for vaccines for cancer and infectious diseases.

Expression of mRNA for a newly identified Pax5 exon is reduced in multiple myeloma

Pax5 is a transcription factor that is critical in the bone marrow for differentiation and proliferation of B cells until the plasma cell stage. In Pax5−/− mice, B-cell development stalls at the pro-B-cell stage. Messenger RNA profiles of alternatively spliced isoforms of Pax5 in bone marrow frequently differ between multiple myeloma (MM) patients and healthy donors. We sought to determine if Pax5 mRNA profiles also differed in blood and unexpectedly detected the presence of a previously unreported exon that alters the amino acid code for the transactivating domain of Pax5 in CD138− B cells. This unique exon escapes detection by conventional analyses of RT-PCR products and may serve as a prototype for other exons, in other genes, that escape RT-PCR detection. Eight percent of tested human subjects were heterozygous for an allele with a nonsynonymous nucleotide substitution in the new exon, and one MM patient was homozygotic for this base difference. Subsequent analysis of plasma and B-cell populations from bone marrow revealed a markedly reduced mRNA expression of the new isoform in cells from MM patients when compared to cells from normal subjects.

Development and immunophenotyping of squamous cell carcinoma xenografts: tools for translational immunology.

Objectives/Hypothesis: The objectives of this study were to delineate methods for the development of primary squamous cell carcinoma (SCCHN) xenografts and to define human leukocyte antigen (HLA), melanoma‐associated antigen (MAGE)‐A3, and human papilloma virus (HPV) 16 antigenic expression in resultant cellular products.

Quantitative und objektive topo-metrische analyse von drusen-papillen mit dem Heidelberg-Retina-Tomographen (HRT)

ZusammenfassungHintergrund und Ziele. Drusen des Sehnervenkopfes stellen eine der häufigsten Ursachen für kongenitale Papillenschwellungen dar. Sie führen zu Defekten der Nervenfaserschicht und zu Gesichtsfeldausfällen. Mittels Heidelberg-Retina-Tomographen (HRT) ist eine dreidimensionale topometrische Analyse von Papillenprominenzen und Messung der peripapillären retinalen Nervenfaserschicht (RNFL) möglich. Patienten und Methoden. Mittels HRT wurde bei 18 Augen von 9 Patienten mit sonographisch nachgewiesenen Drusen eine Topometrie der Papille durchgeführt. Als Vergleich diente eine Gruppe von 18 Augen von 9 altersentsprechenden augengesunden Patienten. Die statistische Auswertung der Daten erfolgte mittels univariater Varianzanalyse. Bei den Patienten mit Drusenpapillen wurde außerdem das Gesichtsfeld mittels 30° Computerperimetrie (Octopus 101), mit Bestimmung der mittleren Empfindlichkeit, des mittleren Defektes und der Verlustvarianz durchgeführt. Aufgrund unzuverlässiger Angaben (Zuverlässigkeitsfaktors >10) in der Computerperimetrie bei 4 von 18 Augen wurden nur die Daten von 14 Augen berücksichtigt. Es wurde die Korrelation von Gesichtsfeldbefunden mit HRT-Parametern durch Bestimmung des Pearson-Korrelationskoeffizienten ermittelt. Ergebnisse. Die peripapilläre “mean retinal nerve fiber layer (RNFL) thickness” der Patienten mit Drusenpapillen unterschied sich signifikant (p<,05) von den Werten der Augengesunden. In der Gruppe der Patienten mit Drusenpapille wurde ein negativer Korrelationskoeffizient (r) zwischen der “mean RNFL thickness” und der Verlustvarianz (r=−0,50; p=0,03) sowie zwischen der “RNFL cross section area” und der Verlustvarianz (r=−0,47; p=0,04) festgestellt. Schlussfolgerung. Mittels HRT konnte eine verminderte peripapilläre “mean retinal nerve fiber layer (RNFL) thickness” bei Drusenpapillen nachgewiesen werden. Diese korrelierte mit der Verlustvarianz des Gesichtsfeldes. Um eine quantitative und objektive topometrische Analyse bei Drusenpapillen durchzuführen, sollte der HRT verwendet werden.AbstractBackground and purpose. Optic nerve head drusen (ONHD) are one of the most frequent causes for congenital swelling of the optic nerve head. Visual field and retinal nerve fiber layer defects are reported in cases of ONHD. The Heidelberg Retina Tomograph (HRT) allows a 3-dimensional topometric analysis of the optic nerve disc and measurement of the peripapillary mean retinal nerve fiber layer. Patients and methods. A total of 18 eyes from 9 patients with sonographically confirmed drusen were analyzed with the HRT. Data were compared to a control group of 18 eyes from 9 matched healthy individuals. Statistical analyses were performed by using ANOVA (univariate). All patients with ONHD underwent a computerised visual field test (30°, Octopus 101). Due to a bad reliability factor of over 10 in the visual fields by 4 out of 18 eyes, only measurements from 14 eyes were included in the study. We correlated visual field and HRT parameters and calculated the Pearsons correlation coefficient (r). Results. We found a significant difference in the measured parameter mean retinal nerve fiber layer (RNFL) thickness (p<0.05) between the two groups. In the ONHD group a negative correlation coefficient was found between the peripapillary mean RNFL thickness and the loss variance (r=−0.50, p=0.03) as well as between the peripapillary RNFL cross-sectional area and the loss variance (r=−0.47, p=0.04). Conclusions. The HRT is able to detect a peripapillary RNFL thinning in cases with ONHD. The mean RNFL thickness correlated with the loss variance. The HRT should be used to perform a quantitative and objective topometric analysis in cases with ONHD.