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J C Howard

Publications and source records attributed to J C Howard.

At least 37 records · Page 2Linked to original sources
PubMed1996

Rat MHC-linked peptide transporter alleles strongly influence peptide binding by HLA-B27 but not B27-associated inflammatory disease.

Rats transgenic for the human MHC molecule HLA-B27 were used to study the effect of two alleles, cima and cimb, which are associated with peptide transport by the MHC-encoded Tap2 transporter, on the function of HLA-B27 as a restriction element for CTL recognition of the male H-Y minor H Ag and on the multisystem inflammatory disease characteristic of B27 transgenic rats. Anti-H-Y CTL generated in cima B27 transgenic rats lysed male B27 cimb/b targets significantly less well than cima/a or cima/b targets. Addition of exogenous H-Y peptides to male B27 cimb/b targets increased susceptibility to lysis to the level of cima/a targets. Male B27 cimb/b cells were less efficient than cima/a cells in competitively inhibiting CTL lysis of female B27 cima/a targets sensitized with exogenous H-Y peptides. 3H-Labeled peptides eluted from B27 molecules of lymphoblasts from rats of two cimb and three cima RT1 haplotypes showed that the cimb peptide pool favors comparatively longer and/or more hydrophobic peptides. These results indicate that RT1-linked Tap2 polymorphism in the rat strongly influences peptide loading of HLA-B27. Nonetheless, the prevalence and severity of multisystem inflammatory lesions were comparable in backcross rats bearing either cima/b or cimb/b. It thus appears either that binding of specific peptides to B27 is unimportant in the pathogenesis of B27-associated disease or that the critical peptides, unlike H-Y and many others, are not influenced by Tap transporter polymorphism.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

The rat cim effect: TAP allele-dependent changes in a class I MHC anchor motif and evidence against C-terminal trimming of peptides in the ER.

Functional polymorphism in the rat peptide transporter associated with antigen processing (TAP) changes the peptide pool available for binding and presentation by a class I MHC allele, RT1.Aa. The peptide binding motif for RT1.Aa, determined by stabilization with synthetic peptides, included a strong preference for arginine at the peptide C terminus. Analysis of natural peptides bound to RT1.Aa by both pool sequencing and anhydrotrypsin chromatography revealed that TAP polymorphism determined the presence or absence of arginine as the peptide C-terminal residue. This result highlights the in vivo impact of TAP-peptide selectivity, and provides evidence against a high rate of generation of new C termini by protease activity in the endoplasmic reticulum.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Supply and transport of peptides presented by class I MHC molecules.

Three observations suggest that the proteasome, transporter associated with antigen processing (TAP) and class I MHC molecules are co-adapted for the generation, transport and loading of specific peptides. Firstly, TAP preferentially transports peptides close in length to the optimum for class I loading; secondly, genetic variation in TAP specificity focusing in the carboxy-terminal of the peptide correlates with preferences among class I molecules for different peptide carboxy-termini; thirdly, TAP associates directly with empty class I molecules and is released by successful peptide loading. This conclusion puts in question the significance for class I loading of proteolytic processing and peptide generation in the endoplasmic reticulum.

Animals↗
PubMed1994

Selectivity of MHC-encoded peptide transporters from human, mouse and rat.

Major histocompatibility complex (MHC) class I molecules present peptides from degraded intracellular antigens to CD8+ T cells. These peptides are translocated in an ATP-dependent fashion into the lumen of the endoplasmic reticulum (ER) for binding to class I molecules by means of the MHC-encoded transporters associated with antigen processing, TAP1 and TAP2. These are members of a family of proteins containing an ATP-binding cassette and form heterodimers in the ER membrane. Defects in the genes encoding TAP1 or TAP2 account for impaired class I assembly and antigen presentation in several human and rodent cell lines. Whereas MHC class I molecules select peptides according to binding motifs, it is not clear to what extent the TAP1-TAP2 transporters have peptide sequence and length specificity. Previous studies of the rat MHC class I molecule RT1Aa, suggested a specific conveyance of peptides by rat TAP1-TAP2. Here we substitute the amino- and carboxy-terminal and the penultimate amino-acid residues of model peptides to show that these residues influence the efficiency of transport. Human TAP and rat TAPa translocated peptides with hydrophobic and basic C termini, whereas mouse TAP and rat TAPu preferred peptides with hydrophobic C termini. This pattern correlates with the predominant peptide binding profiles of mouse and human class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

The distribution of Tap2 alleles among laboratory rat RT1 haplotypes.

We are reporting the cDNA sequences of Tap2 from two cima and two cimb rat strains. Comparison of the cDNA sequences shows that these alleles fall into two groups, which we refer to as Tap2-A and Tap2-B. We found that alleles from the Tap2-B group are more closely related to the mouse homologue than are Tap2-A alleles, and among the 48 nucleotides which differ between the Tap2-A and Tap2-B cDNAs, three affect restriction sites. We defined pairs of oligonucleotides which allow amplification of the regions bearing these restriction sites from genomic DNA or cDNA, and this technique has been successful for the genotyping of all of the 56 laboratory strains of Rattus norvegicus tested and for five cell lines tested so far. All 14 known RT1 standard haplotypes were tested, and 7 found to belong to the Tap2-B group, and 7 to Tap2-A. We also found that intron sizes among the alleles of the Tap2-B group fall into two subgroups, providing further insight into the phylogeny of these various haplotypes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Complement-dependent synergistic effects of rat monoclonal IgG antibodies in vivo.

We describe studies aimed at maximizing the effector mechanisms responsible for eliminating target erythrocytes from the circulation in a fully homologous opsonization system in vivo. The effects on the subsequent fate of target erythrocytes were examined in both normal and decomplemented rats preinjected with a variety of rat IgG monoclonal antibodies (mAb) directed against different epitopes on the RT1Aa, the classical class I major histocompatibiliy complex antigen of the DA rat. In general, the clearance of both DA and (DA x PVG)F1 erythrocytes in normal rats preinjected with various pairs of noncompetitive mAb was very rapid when compared with the overall clearance patterns seen with individual antibodies. With all mAb combinations containing IgG2b or IgG2a, an intact complement system was an essential requirement for augmenting the initial clearance and promoting hepatic sequestration of these target cells. The removal of (DA x PVG)F1 erythrocytes, expressing half as much antigen, was considerably slower than the DA cells for each antibody pair tested although a notable degree of heterogeneity was observed in the overall behavior of both types of target cells with different mAb combinations. Our results suggest that the limiting effects of low antigen density on the target cells combined with the use of mAb of an isotype like the rat IgG2a can be overcome using pairs of mAb that recognize different epitopes on the same target antigen.

Animals↗
PubMed1993

Differential effect of transporter Tap 2 gene introduction into RMA-S cells on viral antigen processing.

The protein products of the Tap (Transporter associated with antigen processing) 1 and 2 genes are presumed to deliver peptides across the endoplasmic reticulum (ER) for assembly with major histocompatibility complex (MHC) class I molecules. The antigen processing-defective cell line RMA-S (H-2b) has a premature stop in the Tap 2 gene and probably therefore fails to deliver peptides into the ER, which leads to a low level of cell surface MHC class I molecules. Transfection of a Tap 2 gene restores to RMA-S both MHC class I molecule expression and the ability to present influenza viral antigens. We investigated the ability of RMA-S cells transfected with a Tap 2 gene to process and present alloantigens, Sendai and Rauscher viral antigens to allogeneic and virus-specific cytotoxic T lymphocytes. We found that allogeneic peptides as well as Rauscher and Sendai viral peptides can be processed and presented by RMA-S but at reduced levels. Transfection of a Tap 2 gene of mouse (BALB/c, H-2d) or rat origin into RMA-S increased the presentation of Sendai viral antigens and partially restored the presentation of allogeneic antigens. The already low level of Rauscher viral peptides presented by RMA-S is not elevated by transfection of either Tap 2 gene into RMA-S. This indicates a differential effect of transfection of a Tap 2 gene of rat or allogeneic mouse origin into RMA-S on viral antigen processing.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Speeding up necropsy histology reports in a teaching hospital.

A programme of repeated personal reminders substantially reduced the delay in producing final histological reports for necropsy cases. The proportion delayed for over four weeks was reduced from 69.7% to 46.7%, and the proportion delayed for over 10 weeks was reduced from 38.6% to 11.7%. This improvement may contribute to halting and reversing the now established trend in the decline of necropsy rates.

Autopsy↗
PubMed1993

Chimeric drift in blood cell populations of chimeric rats constructed between congenic strains.

Chimeric drift is the shift in the proportion over time of the two cell lineages which comprise a chimera (genetic mosaic). Chimeric drift in blood cell populations is determined by both the probability of proliferation from stem cell pools of one or the other of the cell lineages which constitute the chimera and the effects of life span in circulating blood cells. Previous evidence suggests that while chimeric drift occurs in chimeras between genetically disparate strains, it does not occur when the strains used are closely related. No information is available from chimeras between congenic strains. In the present study, chimeric rats were produced between strains with distinguishable class I major histocompatibility complex haplotypes, PVG-RT1a and PVG (which express the haplotype RT1c). PVG-RT1a-specific monoclonal antibodies were used to establish the mosaic patterns in the cell populations of peripheral blood by fluorescein-activated cell sorting. Mosaic cell lineage of red blood cells, white blood cells, lymphocytes, monocytes and neutrophil populations were analyzed weekly over a period of 6 weeks. The ratio of cells of the PVG-RT1a lineage to cells of the PVG lineage shifted either in favor of PVG-RT1a or PVG in cellular components of peripheral blood. The percentage of PVG-RT1a cells in peripheral blood of chimeras changes by as much as 54, 28, 21, 19 and 23% in red blood cell, white blood cell, lymphocyte, monocyte and neutrophil populations, respectively. The shifts in the percentage of PVG-RT1a cells appears to occur in a cyclic fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Temporal order and functional analysis of mutations within the Fli-1 and p53 genes during the erythroleukemias induced by F-MuLV.

The erythroleukemias induced by Friend murine leukemia virus (F-MuLV) result from the accumulation of a number of genetic changes, including activation of the Fli-1 proto-oncogene and inactivation of the p53 tumor suppressor gene. We have determined the temporal order of mutation of the genes involved in this multistage malignancy, by serial in vivo transplantation of F-MuLV induced primary erythroleukemias into syngenic Balb/c mice. These primary tumors are capable of growing when transplanted into syngenic mice, but die after several days of in vitro culture. From the transplanted tumors grown in syngenic mice, erythropoietin-dependent cell lines were established in culture that are clonally related to cells in the primary tumors. We show that retroviral insertional activation of the Fli-1 ets family member is the first detectable genetic event in F-MuLV induced primary erythroleukemias. Mutations in the p53 gene were observed in the Epo-dependent cell lines but not in the transplanted erythroleukemias used to establish these cell lines in culture. These data suggest that activation of Fli-1 plays an important role in the early stages of F-MuLV-induced leukemia, perhaps by altering the self-renewal probabilities of erythroid progenitor cells and that p53 mutations immortalize these cells, enabling them to grow in vitro in the presence of Epo.

Animals↗
PubMed1992

Proteasome subunits encoded by the major histocompatibility complex are not essential for antigen presentation.

Major histocompatibility complex (MHC) class I molecules bind and deliver peptides derived from endogenously synthesized proteins to the cell surface for survey by cytotoxic T lymphocytes. It is believed that endogenous antigens are generally degraded in the cytosol, the resulting peptides being translocated into the endoplasmic reticulum where they bind to MHC class I molecules. Transporters containing an ATP-binding cassette encoded by the MHC class II region seem to be responsible for this transport. Genes coding for two subunits of the '20S' proteasome (a multicatalytic proteinase) have been found in the vicinity of the two transporter genes in the MHC class II region, indicating that the proteasome could be the unknown proteolytic entity in the cytosol involved in the generation of MHC class I-binding peptides. By introducing rat genes encoding the MHC-linked transporters into a human cell line lacking both transporter and proteasome subunit genes, we show here that the MHC-encoded proteasome subunit are not essential for stable MHC class I surface expression, or for processing and presentation of antigenic peptides from influenza virus and an intracellular protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Cloning and expression of class I major histocompatibility complex genes of the rat.

Little is known about the organization of class I genes in the rat although there is prima facie evidence that it is distinct from that of the mouse. We report the cloning of 61 nonclassical rat class I genes into cosmid clusters with a total mapped length of 1,264 kb. It is certain that the total number of class I genes in the rat must exceed this number. From restriction maps it is possible to identify substantial regions of duplication. By transfection of cosmids into mouse L cells, it has been possible to demonstrate at least seven different nonclassical rat class I genes that are expressible on the cell surface. Crossreaction of a single mouse monoclonal antibody with all of these class I molecules is consistent with sequence homogenization within the rat nonclassical system. Attempts to find rat homologues of the mouse Tla genes by crosshybridization of rat cosmids with a range of different TLa-specific probes were unsuccessful, suggesting that this large group of divergent class I genes is absent or nearly so from the rat. The large number of class I genes in the rat appears to have arisen by expansion of genes more closely related to the classical sequence.

Animals↗