Miyagi, Tohko1; Hikino, Hajime1; Hua, Yan1; Hirohisa, Saito1; Gold, Daniel P.1; Li, Xiao-K.1; Fujino, Masayuki1; Taga, Tetsuo2; Amemiya, Hiroshi1; Suzuki, Seiichi; Kokubo, Toshio3; Kimura, Hiromitsu; Robb, Lorraine4 Author Information

Enhanced green fluorescence protein (EGFP) has been widely applied to gene transduction in cellular and molecular biology as a reporter element. When applied to cell transplantation, it raises fundamental issues concerning cell-associated antigens, in particular, a model of minor histocompatibility antigen(s). Although it is well known that immunological behavior of minor histocompatibility antigens mimic tumor associated antigens (TAA), identified genes coding minor histocompatibility antigens are few and far between. Inasmuch as immunity and tolerance to TAA are provided by immunological behavior of minor histocompatibility antigen such as histocompatibility antigen of the Y chromosome, H–Y, it occurs to us that transgenic as well as transduced EGFP provides a useful model system to be applied to tumor immunology. In this respect, genetic modification of specialized antigen-presenting cells (APC), i.e., dendritic cells (DC), such as gene transduction of EGFP into DC, would provide one of the most important strategies in transplantation as well as tumor immunology inasmuch as DC play a key role in initiating primary immune responses, As far as gene transduction into DC is concerned, others have reported that successful gene transduction occurs in DC by adenoviral vector systems. However, our previous studies concerning EGFP transduction into DC suggested that this view should be carefully examined and interpreted.Employing adenoviral and lentiviral vector systems as well as specialized APC of rat DC and peritoneal exudate macrophages (PEM), EGFP-transduced APC were examined to determine whether and to what extent the EGFP-transduced APC were able to sensitize non-transgenic littermates against transgenic EGFP as antigen(s). Thus EGFP-transgenic cardiac isografts were transplanted to non-transgenic littermates and examined to determine if sensitization of non-transgenic littermate recipients with the EGFP-transduced APC was able to reject the test grafts in an accelerated manner.In this study, we examined this and provide further evidence that widely used viral vector systems are unable to transfer the reporter gene EGFP into mature rat DC generated from bone marrow cells (BMC), driven by Flt3/Flk2 ligand and IL-6. Nevertheless, successful gene transduction was obtained by either applying a lentiviral vector system to the developing DC progenitor cells during a long-term culture of rat BMC or by applying an adenoviral vector system to PEM. Thus, successful gene transduction into specialized APC was verified by in vivo priming of non-transgenic littermates with the EGFP-transduced APC, followed by accelerated rejection of EGFP-transgenic cardiac isografts.

The diversity of Ag-specific receptors on T cells homing to an inflammatory infiltrate in the central nervous system has been analyzed. Experimental autoimmune encephalomyelitis, a T cell-mediated inflammatory disease of the central nervous system, was induced in Lewis rats with a CD4+, CD8- T cell line specific for peptide 68-86 of myelin basic protein. Within the line a wide array of TCR Vbeta genes was transcribed including the Vbeta8, Vbeta10, Vbeta15, Vbeta16, and Vbeta19 families. Accumulation of T cells at the site of inflammation was determined by using RNA-polymerase chain reaction amplification of rearranged TCR Vbeta transcripts derived from brain. By 8 to 1 0 h after i.p. infusion of the pathogenic T cell line, TCR Vbeta transcripts, including mainly Vbeta families that were predominantly rearranged by the line, could be identified in brains. Restricted TCR V gene transcripts with predominance of the Vbeta8 family were identified in brain 48 h after injection, before onsetof disease. Paralysis was apparent by 4 to 5 days after injection. At this time diverse Vbeta gene transcripts were detected in brain, reaching a maximum by day 9, when paralyzed rats have recovered. By day 14 a second stage of limited heterogeneity in the T cell infiltrate could be identified with predominant expression of Vbeta8, Vbeta9, Vbeta10, and Vbeta19. Interestingly, three out of these four Vbeta families were predominantly expressed within the encephalitogenic line. Thus, T cell migration to brain in experimental autoimmune encephalomyelitis is characterized by a rapid penetration of T cells followed by a selective trapping of T cells before the clinical manifestations of disease. When clinical disease was present the T cell infiltrate was diverse, whereas in the post-acute phase of disease the T cells in the central nervous system had limited heterogeneity with selective accumulation of T cells transcribing the same V regions that were detected in the line that incited disease.

Diabetes-prone BB rats spontaneously develop type 1 diabetes due to a T-cell-dependent destruction of insulin-producing beta-islet cells. A number of T-cell abnormalities including lymphopenia, poor cell-mediated responsiveness to alloantigen, and an absence of an RT6+ T-cell subset are associated with disease susceptibility. Our previous studies have implicated the thymic antigen-presenting cell in influencing disease potential and responsiveness to alloantigen. Since this cell type is also known to influence T-cell receptor expression in developing thymocytes, we examined the thymic and peripheral T-cell receptor beta chain variable region repertoire in diabetes-prone and diabetes-resistant rats. Our findings indicate that animals susceptible to diabetes induction have a characteristic and limited peripheral beta chain variable region repertoire that differs markedly from that expressed in the thymus.

Antigen receptors on the surface of the thymus-derived (T) lymphocytes are associated with small integral membrane proteins called the T3 (CD3) gamma, delta, epsilon, and zeta chains. After interaction of the T-cell receptor with antigen, the T3 proteins are believed to transfer an activation signal to the intracellular compartment. In previous studies, the human gamma, epsilon, and delta chains have been cloned along with the mouse delta chain, but a relationship between these sequences and known molecular families has not been established. We now report the molecular cloning and characterization of the murine T3-epsilon protein and a sequence and structural analysis of the relationships between all the T3 chains and the immunoglobulin superfamily. It is established that the T3 chains are immunoglobulin-related and a particular relationship to the neural cell adhesion molecule (N-CAM) is noted. This sequence relationship adds interest to previous findings that the T3 chains are genetically linked to N-CAM and Thy-1 antigen on band q23 of human chromosome 11.

T cell acute lymphoblastic leukemias (T-ALL) can be regarded as the malignant counterparts of cells in various T cell differentiation stages. To study the expression of the human T cell receptor (TcR)-CD3 complex during the early stages of T cell differentiation, we have analyzed 22 T-ALL at the cell membrane level and the DNA level and 12 of them at the RNA level. According to their immunologic phenotype, the T-ALL could be divided into three main groups: 10 immature T-ALL (CD1-/CD3-), seven common thymocytic T-ALL (CD1+/CD3-or+), and five mature T-ALL (CD1-/CD3+). Among the 10 immature T-ALL three appeared to express the immunologic phenotype of the putative prothymocyte (TdT+/HLA-DR+/CD7+/CD2+/CD5-/CD1-/CD3-), whereas the other seven T-ALL appeared to be immature thymocytic (TdT+/HLA-DR-/CD7+/CD2+/CD5+/CD1-/CD3-). Transcripts of the CD3-delta and CD3-epsilon genes were present in all CD3- and CD3+ T-ALL tested, including prothymocytic T-ALL. However, prothymocytic T-ALL had germline TcR-beta genes and were not rearranged to the characterized TcR-gamma joining regions. The presence of CD3 transcripts and absence of TcR gene rearrangements in prothymocytic T-ALL supports their immature T cell character. Two immature thymocytic T-ALL also had germline TcR-gamma genes and one of them had germline TcR-beta genes. In all other T-ALL the TcR-gamma and TcR-beta genes were rearranged. The presumptive functional 1.3-kilobase TcR-beta transcripts were detected in the majority of T-ALL with rearranged TcR-beta genes. Distinct levels of TcR-gamma transcripts appeared to be present only in some thymocytic T-ALL, i.e., some immature thymocytic T-ALL and common thymocytic T-ALL. TcR-alpha mRNA could only be detected in CD3+ mature T-ALL, but was absent in all CD3+ common thymocytic T-ALL tested. Our data indicate that CD3 gene transcription is one of the earliest events during T cell differentiation and already occurs in prothymocytes. The TcR-gamma and TcR-beta genes rearrange early during thymocytic differentiation and can subsequently be transcribed. High levels of TcR-gamma gene transcription may only occur in a part of the T cells during thymic differentiation, while TcR-beta gene transcription continues during further differentiation. TcR-alpha gene transcription may be the final step in the production of the complete set of TcR and CD3 proteins, resulting in the expression of the TcR alpha beta-CD3 complex at the cell surface of mature T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The T3 complex is composed of three polypeptide chains that are both structurally and functionally associated with the receptor for antigen on the surface of human T lymphocytes. In a series of experiments utilizing both somatic cell hybrids and chromosomal hybridization in situ, the genes encoding two members of the human T3 complex, T3-delta and T3-epsilon, were found to reside on the long arm of chromosome 11 in band q23. The murine T3-epsilon gene was localized to chromosome 9. The location of the T3-delta and T3-epsilon genes with respect to the Hu-ets-1 gene, which is also located in 11q23, is discussed. Recent assignments of several genes, preferentially expressed in human cells of hematopoietic and neuroectodermal origins, to band q23 of human chromosome 11 and the murine equivalents to murine chromosome 9 may define a conserved gene cluster important in cell proliferation and differentiation.

Nature 321, 431–434 (1986). THREE sequencing errors have been found in the human T3-e nucleotide sequence published in Fig. 3b. These errors have been corrected in the sequence shown below at the positions indicated by vertical arrows. The putative transmembrane region is underlined. The predicted amino acid sequence ends after residue 185 instead of the reported residue 211.

Using the spheroplast fusion technique, we have introduced the cloned E beta b gene into two d haplotype cell lines, the B lymphoma line A20-2J and the macrophage tumor line P388D1. Analysis with a monoclonal antibody indicates that the product of the transfected E beta b gene associates with the endogenous E alpha chain to form an E alpha dE beta b complex. While expression of E alpha dE beta b is constitutive in A20-2J cells transfected with the E beta b gene, surface expression of E alpha dE beta b is detected in transfected macrophage cells only after treatment of cells with culture supernatants from concanavalin A (Con A)-stimulated T cells. Transfected B lymphoma cells and transfected Con A supernatant-treated macrophage cells have acquired the ability to present antigen to E alpha dE beta b-restricted T-cell hybridomas. The observed inducible expression of the transfected gene in the macrophage host indicates that sequences responsible for regulated expression of the E beta b gene may be associated with the transfected gene. In combination with directed mutagenesis, the system described here provides a means to study (i) E beta b sequences that are important in determining the restriction specificity of the E molecule and (ii) sequences associated with the E beta gene that may be important in the regulation of E beta chain expression.

That some value of the f (sub S 2 ) /f (sub O 2 ) ratio must be exceeded to stabilize sulfide immiscibility in gabbroic magmas is the working hypothesis for the formation of platinum-enriched horizons in the Bushveld and Stillwater Complexes. Intrinsic f (sub O 2 ) measurements from 900 degrees to 1,160 degrees C are given for eight eastern and three western Bushveld samples representative of the chromitites within 970 m of the stratigraphy containing the reef. Similarly, f (sub O 2 ) -T data for five samples of anorthosites and one sample of chromitite are given for 370 m of the stratigraphy enclosing the Stillwater J-M reef platiniferous horizon.In the eastern Bushveld, the f (sub O 2 ) decreases (more reduced) with elevation; in the Merensky Reef footwall about a three order of magnitude drop from f (sub O 2 ) = 10 (super -11) to f (sub O 2 ) = 10 (super -13.7) was measured at 1,150 degrees C for the respective horizons of -869.8 m and -687 m (base of the Merensky Reef as zero datum elevation). From the -687-m horizon to the Merensky Reef, the f (sub O 2 ) values at 1,150 degrees C range between 10 (super -13) to 10 (super -14) with one western Bushveld Merensky Reef sample at 10 (super -15.6) .In the Stillwater Complex, original igneous f (sub O 2 ) values and later serpentinization produce f (sub O 2 ) values at 1,150 degrees C that vary from 10 (super -12.0) (deep footwall) to 10 (super -9.8) (zone of interest). The serpentinization has erased the original igneous redox equilibria for several meters on each side of the zone of interest.Six lines of evidence are reviewed that suggest the Bushveld Merensky event was the result of a system open to volatile composition changes and that the Bushveld chamber may have hovered close to the sulfide immiscibility during 677.3 m of accumulation. In our model, at the Merensky formation time a final perturbation in the f (sub S 2 ) /f (sub O 2 ) ratio triggered the sulfide immiscibility. Spatial constraints and mass balance calculations are presented to test this model.The Merensky Reef and the Stillwater zone of interest both display visual graphite, potholes, and enigmatic platinum-group element concentrations, as well as other discussed similarities that suggest the presented model for the Bushveld may have been originally operative for the Stillwater as well.

Data on cleft lip with or without cleft palate are utilized to evaluate a new method for estimating recurrence risk under the polygenic threshold model. The influence of critical factors on the accuracy of the estimated risks are evaluated. The model is relatively sensitive to the estimate of heritability employed and relatively robust to the population prevalence estimate and to family size. The calculated risks for cleft lip with or without cleft palate are contrasted with the empiric figures from studies on Caucasians and Japanese populations.

THE number density of fission tracks in solids is reduced if the sample is heated before chemical etching, and the effect of annealing must be accounted for before an age date can be assigned to the sample1–3. The extent of the annealing can be observed by a measurement of the reduction in the track parameters (diameter and/or length) and subsequent comparison to unannealed tracks. Correct ages can be obtained by careful calibration studies of track density reduction against track diameter or length reduction at different annealing temperatures and times4,5. For crystalline minerals, however, these correction techniques are not generally valid6. In this study, glass samples were partially annealed and then immersed in liquid nitrogen for various periods and we show that the properties of the glass and/or the track parameters can be altered so as to observe tracks that would normally be erased by annealing.

Previous articleNext article No AccessReviewsThe Mineral Resources of Africa. Nicolas de Kun D. P. GoldD. P. Gold Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Journal of Geology Volume 74, Number 6Nov., 1966 Article DOIhttps://doi.org/10.1086/627223 Views: 1Total views on this site Copyright 1966 The University of ChicagoPDF download Crossref reports no articles citing this article.