Characterization of gamma-aminobutyric acid receptor GABAB(1e), a GABAB(1) splice variant encoding a truncated receptor.

We have identified a splice variant encoding only the extracellular ligand-binding domain of the gamma-aminobutyric acid B (GABA(B)) receptor subunit GABA(B(1a)). This isoform, which we have named GABA(B(1e)), is detected in both rats and humans. While GABA(B(1e)) is a minor component of the total pool of GABA(B(1)) transcripts detected in the central nervous system, it is the primary isoform found in all peripheral tissues examined. When expressed in a heterologous system, the truncated receptor is both secreted and membrane associated. However, GABA(B(1e)) lacks the ability to bind the radiolabeled antagonist [(3)H]CGP 54626A, activate G-protein coupled inwardly rectifying potassium channels, or inhibit forskolin-induced cAMP production when it is expressed alone or together with GABA(B(2)). Interestingly, when co-expressed with GABA(B(2)), not only does the truncated receptor heterodimerize with GABA(B(2)), the association is of sufficient avidity to disrupt the normal GABA(B(1a))/GABA(B(2)) association. Despite this strong interaction, GABA(B(1e)) fails to disrupt G-protein coupled inwardly rectifying potassium activation by the full-length heterodimer pair of GABA(B(1a))/GABA(B(2)).

Schlafen, a new family of growth regulatory genes that affect thymocyte development.

The Schlafen (Slfn) family of genes are differentially regulated during thymocyte maturation and are preferentially expressed in the lymphoid tissues. Ectopic expression of the prototype member Slfn1 early in the T lineage profoundly alters cell growth and development. In these mice, the DP thymocytes fail to complete maturation, and, depending on the transgene dosage, the number of thymocytes is reduced to 1%-30% of normal. Furthermore, expression of the Schlafen family members in fibroblasts and thymoma cells either retards or ablates cell growth. The conceptual protein sequences deduced for each of the family members have no similarity to characterized proteins and must therefore participate in a heretofore unknown regulatory mechanism guiding both cell growth and T cell development.

An alternative translational reading frame encodes an immunodominant retroviral CTL determinant expressed by an immunodeficiency-causing retrovirus.

Recognition of virus-infected or transformed cells by CD8+ CTL requires a trimolecular complex composed of MHC class I, beta2-microglobulin, and a specific foreign peptide composed of 8 to 10 linear amino acids. The generation of such CTL epitopes has traditionally been thought to be from the primary open reading frame encoding the viral or tumor-associated proteins. In this report it is demonstrated that a viral CTL epitope can also be generated from an alternative reading frame. Using a combination of synthetic peptides and Sindbis or vaccinia expression systems, MHC class I Kd-restricted BALB/cByJ CTL directed against defective gag gene constructs of the LP-BM5 virus complex that causes murine AIDS were shown to have specificity for the antigenic peptide SYNTGRFPPL. This epitope is generated in a novel fashion from the second open reading frame (ORF2) of both the defective and ecotropic helper virus components of LP-BM5. Importantly, lysis of target cells expressing BM5 ecotropic helper, and/or defective viral gag, demonstrated that the SYNTGRFPPL epitope is generated during the course of a normal retroviral infection. Furthermore, MAIDS-resistant BALB/cByJ mice also generated secondary restimulated CTL specific for SYNTGRFPPL following in vivo priming with the LP-BM5 retroviral complex. These data suggest that retroviruses, and potentially other viruses and foreign genes, are capable of expressing T cell epitopes from alternative open reading frames. If one considers the influence of self peptides on T cell development, these "alternative reading frame-derived" peptides could provide an important additional influence on the functional T cell repertoire.

The influence of the MAPK pathway on T cell lineage commitment.

During development, progenitor thymocytes differentiate into either CD4 or CD8 T cells, and this fate decision depends on the specificity of the T cell antigen receptor (TCR) for MHC class II or class I molecules. Based on the mechanisms of fate specification known for simple metazoan organisms, we sought to determine whether the extracellular signal-related kinases (ERKs) play a role in T cell differentiation and lineage commitment. Using a dominant gain-of-function mutant of the erk2 gene, we show that differentiation into the CD4 lineage is favored. We also show that, conversely, the addition of a pharmacological inhibitor of the ERK pathway favors differentiation into the CD8 lineage. We present a quantitative selection model that incorporates these results as well as those of recent reports on the role of Notch in T cell lineage specification.

Altered collagen metabolism and delayed healing in a novel model of ischemic wounds.

Cellular mechanisms occurring in the healing wound have been well described in various animal models. However, the events associated with wound healing seen in ischemic skin have not been as thoroughly defined. In this series of experiments, we created a novel model of excisional skin wounds under gradient ischemia to study the cellular and extracellular events leading to delayed healing. We hypothesized that altered collagen metabolism accounts for delayed wound healing in ischemic skin. Three pairs of 4 mm punch wounds were made 4 days after bipedicle skin flaps were created on the dorsum of rats. Sham-operated control animals had the same punch wounds without flap creation. The kinetics of excisional wound healing were measured by means of computerized planimetry. In addition, wounds were excised with a 6 mm trephine, radiolabelled with ((3)H)-proline and in vitro collagen synthesis determined as collagenase digestible protein along with quantitation of DNA content. Total collagen deposition was determined as 4-hydroxy-L-proline by high-performance liquid chromatography, and wounds were histologically evaluated. Data was analyzed by means of two-way analysis of variance. Although control wounds healed by day 10, flap wounds consistently had greater surface area on days 2, 4, 6, 8, and 12 (p < 0.001). Relative collagen synthesis (% collagen/noncollagen protein), as measured by an in vitro synthesis method, showed no statistically significant differences between flap and controls wounds. However, the total collagen content (deposition) as measured by 4-hydroxy-l-proline was significantly lower in flap wounds compared with controls on days 7 (p < 0.05) and 9 (p < 0.001). In addition, a significant increase occurred in DNA content in the flap wounds on days 7 (p < 0.05) and 9 (p < 0.001) versus control wounds. These data indicate that, in ischemic wounds, significantly less collagen is deposited despite the inherent ability of the tissue to synthesize appropriate levels of collagen. Because the in vitro collagen synthesis technique only assesses the ability of the tissue to synthesize collagen in a well oxygenated environment, one cannot be assured that the tissue expresses this potential in vivo. However, these data are consistent with the hypothesis that the delay in wound closure is due to an alteration in collagen metabolism which results in a net decrease in collagen accumulation. Because of the observed increase in DNA within the ischemic wounds, we suggest that there is prolonged inflammation in these wounds which may enhance collagen degradation through the release of proteases. In addition, there may be an inability of the tissue to maintain appropriate levels of collagen in this inflammatory wound environment.

Segmental colonic gangrene: a previously unreported complication of adult hemolytic uremic syndrome.

Gastrointestinal manifestations, however slight, must be carefully observed in patients with HUS regardless of age. Both clinical and diagnostic evaluations must be weighed expeditiously to rule out any surgically correctable disease. Although most patients with HUS can be treated nonoperatively, a considerable mortality still exists, especially in adults. Once surgical intervention is required, overall mortality is still favorable, especially with early diagnosis and proper treatment.

CTL responses to the gag polyprotein encoded by the murine AIDS defective retrovirus are strain dependent.

Murine AIDS (MAIDS) is induced by a mixture of retroviruses, of which a replication defective virus is the proximal agent of disease. This defective virus harbors a single intact gene that encodes an aberrant gag polyprotein. Certain mouse strains are genetically resistant to MAIDS, with several genes, including H-2Dd, contributing to this resistance. Because MHC class I gene products present intracellular Ags to CTL, recombinant viruses were used to determine whether gag-specific CTLs mediate the genetic linkage between H-2Dd and resistance. Interestingly, while genetically resistant BALB/cByJ and C57BL/KsJ mice (H-2d) generated gag-specific CD8+ CTLs, a similar response was not detected in susceptible BALB.B and C57BL/6J mice (H-2b). However, this CTL response does not appear to be responsible for genetic resistance because: 1) a vigorous CTL response could also be generated by susceptible (C57BL/6 x BALB/cBy) F1 mice and 2) the relevant epitope is H-2Kd restricted.

Cytotoxic T lymphocytes directed against MAIDS-associated tumors and cells from mice infected by the LP-BM5 MAIDS defective retrovirus.

The LP-BM5 retrovirus, a complex containing ecotropic helper, recombinant MCF, and defective retroviruses, causes an immunodeficiency-termed mouse AIDS (MAIDS). Many disease features of MAIDS resemble those of AIDS, including terminal B cell lymphomas. Previously we generated from MAIDS-susceptible C57BL/6 mice cytolytic T lymphocytes (CTL) specific for MAIDS-associated B cell lymphomas. Data of the present study (1) exclude MCF and establish a role for defective virus in generating C57BL/6 CTL to MAIDS-associated tumors by experiments involving in vitro stimulation with cells from LP-BM5, ecotropic, or ecotropic-rescued defective virus-infected mice and (2) confirm that such CTL are specific for tumors of MAIDS origin. Several approaches testing for direct involvement of defective virus or its gag-encoded polyprotein, however, did not provide evidence that MAIDS tumor-specific CTL were directed to structural virion proteins, suggesting the possibility that such CTL are specific for nonvirion antigens whose expression depends on the action of the defective genome in the MAIDS disease process.

Novel regulation of an MHC class I gene response to interferon-gamma.

The ability of IFN-gamma to increase the expression of MHC class I gene products is likely to enhance cytolytic T lymphocyte recognition of viral pathogens and tumor cells. The murine lymphoma AKR SL3-cl.F AZR (SL3-cl.F) responds aberrantly to treatment with interferon-gamma such that H-2Dk surface expression is augmented, but H-2Kk expression remains at constitutive levels. Somatic cell fusions have been used to demonstrate that the lesion responsible for this phenotype is cis-dominant, implicating a primary lesion within the SL3-cl.F H-2Kk gene. In this communication, we have used PCR to analyze the nucleotide sequence in regions of the SL3-cl.F H-2Kk promoter known to contain interferon-responsive enhancer elements. Comparison of the SL3-cl.F H-2Kk sequences to known consensus elements revealed complete identity. In order to identify the lesion responsible for the SL3-cl.F phenotype, two H-2Kk genomic clones were independently isolated from SL3-cl.F. Each clone exists as a 10.5-kbp EcoRI fragment containing the entire structural gene. The site of transcription initiation is at the center of this fragment; therefore, all regulatory elements within 5 kbp of the transcript start site which could alter steady-state message levels are included. Interestingly, IFN-gamma-augmented expression of the H-2Kk gene was restored following DNA-mediated transfection of either of these clones into fibroblast cell lines and the parental cell line SL3-cl.F. Because isolation of these clones required passage of the DNA through a prokaryotic host, which alters the pattern of DNA methylation, there was the possibility that demethylation was responsible for the newly acquired IFN-gamma-responsive phenotype. Treatment of SL3-cl.F with 5-azacytidine, which inhibits de novo methylation, did not restore IFN-gamma-augmented expression, however, thus excluding H-2Kk specific methylation as a potential mechanism. Collectively, these data demonstrate that the alteration responsible for the phenotype observed in SL3-cl.F does not involve known transcriptional regulatory elements. Potential mechanisms which might account for the mutant phenotype are discussed.

HLA and T cell receptor polymorphisms in pauciarticular-onset juvenile rheumatoid arthritis.

The immunogenetic basis of pauciarticular-onset juvenile rheumatoid arthritis is unclear. We therefore analyzed the HLA and T cell receptor genes present in a clinically well-defined group of patients. We found that the DR8 haplotype contributes most of the HLA-associated risk, although alleles at other loci contribute independently. A candidate disease-associated T cell receptor polymorphism, in contrast, was not identified in this population. Mechanistic implications of these findings are discussed.

HLA-DQ alpha polymorphisms: oligonucleotide probes characterize the contribution of first and second domains to electrophoretic variants.

Polymorphism is known to exist within the HLA-DQ alpha locus in the human major histocompatibility complex, although such polymorphism may be "silent" in standard HLA typing. However, DQ alpha polymorphism may be functionally significant, either through DQ alpha epitopes functioning directly in the immune response or by affecting tertiary conformation of Ia molecules through differential alpha/beta pairing. We have previously defined a particular DQ alpha polymorphism through reactivity with a monoclonal antibody and restriction fragment length polymorphism pattern. We now characterize this DQ alpha polymorphism through two-dimensional gel electrophoretic analysis and identify a subset of DQ alpha molecules with unique characteristics. Investigation of these allelic variants using synthetic oligonucleotide probe analysis of genomic DNA suggests a localization of the DNA region encoding the DQ alpha 5 epitope and suggests possible evolutionary mechanisms accounting for these unique patterns.