Delayed-type hypersensitivity to allogeneic cells in mice. III. Sensitivity to cell-surface antigens coded by the major histocompatibility complex and by other genes.

DTH could be induced to cell-surface antigens coded by either H-2 or non-H-2 genes. Sensitivity was more readily induced across I region than across K- or D-region differences. The presence of an I-region difference during sensitization did not significantly increase the DTH response to K- or D-region-coded antigens. Macrophage processing appeared to be the major route of sensitization to background antigens. Thus, high levels of sensitivity were achieved equally well using viable or disrupted cells, the response was independent of the H-2 haplotype of the allogeneic cells, and transfer was restricted to the K end of the host H-2 complex. Although sensitization to H-2 antigens was obtained with disrupted cells, transfer of sensitivity against viable cells was unrestricted. This suggests a minor role for macrophage processing in sensitization to H-2 antigens.

Suppression of T cells specific for the nonthymic parental H-2 haplotype in thymus-grafted chimeras.

The mechanism of restriction of T-cell specificity by the genotype of the thymus in allogeneic and semiallogeneic chimeras was investigated. Lack of induction of delayed-type hypersensitivity (DTH) directed against antigen in association with the nonthymic parental haplotype in naive cells adoptively transferred into chimeras suggests the existence of an in vivo suppressive mechanism. However, it was not possible to suppress the expression of DTH in sensitized cells transferred into chimeras, or to transfer this suppression to normal naive recipients.

Contact sensitivity to azobenzenearsonate and its inhibition after interaction of sensitized cells with antigen-conjugated cells.

Painting mice on the skin with the diazonium salt of p-arsanilic acid elicited two types of T cell activity. One was restricted by the I region of the major histocompatibility complex and was responsible for the transfer of azobenzenearsonate (ABA) sensitivity to naive mice. The other was H-2K restricted and could be demonstrated by its ability to interact specifically with ABA-coupled cells in vitro and to inhibit nonspecifically the transfer of sensitivity by cells sensitized either to ABA or to another antigen. Free antigen, or antibody directed against the cross-reactive idiotype on the anti-ABA antibodies of A/J mice, could inhibit the H-2K-restricted suppressive activity induced in the ABA immune A/J cells.

N-glycosylation site occupancy of rat alpha-1,3-fucosyltransferase IV and the effect of glycosylation on enzymatic activity.

All mammalian alpha-1,3-fucosyltransferases (Fuc-Ts) so far characterized have potential N-glycosylation sites, but the role of these sites in enzymatic activity or localization has not been investigated. When one member of this family, rFuc-TIV, is expressed in bacteria, the unglycosylated form of rFuc-TIV has no detectable enzymatic activity. The two potential N-glycosylation sites of rFuc-TIV were mutated to determine site occupancy and the effect of site occupancy on enzyme activity and targeting of this enzyme. Results obtained with singly mutated forms of rFuc-TIV indicate that both sites are occupied in mammalian cells. Lack of glycosylation at sites 117-119, 218-220, or both of these sites, decreased enzyme activity to approximately 64%, 5% or 1%, respectively, of that seen in the unmutated enzyme. These results show that N-glycosylation is necessary for optimal enzyme activity, with glycosylation at site 218-220 playing the major role. However, N-glycosylation does not appear to affect the major intracellular location of the enzyme, as immunocytochemistry reveals the same perinuclear pattern of staining for the unglycosylated mutants as is seen for the wild-type rFuc-TIV in transfected cells.

Regulation of the human acid beta-glucosidase promoter in multiple cell types.

Acid beta-glucosidase (beta Glc) is a housekeeping enzyme whose expression is ubiquitous, but differs greatly according to tissue of origin. Expression of a reporter gene under the control of a 622 bp fragment of the beta Glc promoter correlated roughly with the relative amount of beta Glc mRNA detected in five different cell lines, suggesting that elements within this region play a role in determining differential expression of the beta Glc gene. Experiments using deletion mutants revealed that differential expression of beta Glc is not due to the presence of promoter elements that are active in only certain cell types, but rather due to subtle changes in the magnitude of the effect of the different elements. Strikingly, regulatory elements located upstream of the TATA box are dispensible in several cell types, whereas elements located within exon 1 of the beta Glc gene are essential for reporter gene expression in cultured cells. At least two exon 1 elements regulate mRNA levels, and one double stranded probe containing exon 1 sequences binds a factor present in extracts from HeLa and glioblastoma cells. Additionally, at least two of the exon 1 elements act in an orientation-independent fashion. Thus, it is likely that at least a subset of the exon 1 elements act as transcriptional enhancers.

Low affinity of kappa chain bearing (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific antibodies in the primary antibody repertoire of C57BL/6 mice may explain lambda chain dominance in primary anti-NP responses.

The primary anti-(4-hydroxy-3-nitrophenyl)acetyl (anti-NP) antibody response of C57BL/6 mice was analyzed by several methods. Serum analyses by solid-phase radioimmunoassay (RIA) showed that stimulation with the thymus-independent (TI) type 1 antigen NP-LPS results in an anti-NP antibody response dominated by kappa (kappa) light chain bearing antibodies, whilst responses to NP-Ficoll (a TI-2 antigen) and NP-KLH (a thymus dependent antigen) are dominated by lambda (lambda) 1 light chain bearing antibodies. However, in all these responses NP-specific plaque-forming cells (PFCs) were predominantly heteroclitic, and inhibitable by anti-lambda antiserum. In addition, kappa-bearing IgM-producing hybridomas obtained by fusion of spleen cells from NP-LPS-immunized mice, although producing NP-specific antibodies detectable by RIA, were unable to produce NP-specific plaques. Direct determination of the affinity of 5 of those hybridomas by fluorometric titrations showed that their affinity is indeed lower than 10(-5) M. These results suggest that most NP-specific antibodies stimulated by NP-LPS are of too low affinity to be detected in a direct PFC assay, with the exception of a population of lambda-bearing antibodies. Therefore, the differential expression of kappa- or lambda-bearing antibodies in primary responses to the hapten NP presented on different carriers may be due to different affinity requirements for B-cell triggering via different activation pathways.

HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells.

Novel hybrid vectors, which incorporate critical elements of both herpes simplex virus type 1 (HSV-1) amplicon vectors and adeno-associated virus (AAV) vectors, are able to sustain transgene expression in dividing glioma cells for over 2 weeks. These vectors combine the high infectibility and large transgene capacity of HSV-1 vectors with the potential for episomal amplification and chromosomal integration of AAV vectors. The hybrid vectors contain the HSV-1 origin of DNA replication, oriS, and the DNA cleavage/packaging signal, pac, which allow amplicon replication and packaging in HSV-1 virions. The lacZ reporter gene under control of the CMV IE1 promoter is flanked by AAV inverted terminal repeat (ITR) sequences, which facilitate replication and genomic integration of this cassette in the host cell nucleus. Constructs were generated with or without the AAV rep gene (rep+ and rep-) to assess its importance in extending transgene expression. Expression of Rep proteins was confirmed by Western blot analysis. An HSV-1 amplicon construct containing the reporter gene, but no AAV sequences, was used as a control. Constructs were packaged into HSV-1 virions with or without helper virus and these vector stocks were used to infect human U87 glioma cells in culture. The hybrid vectors supported transgene retention and expression for over 2 weeks, whereas the control amplicon vector lost the transgene after 10 days. Expression was somewhat longer for the rep+ as compared to the rep- hybrid vectors. Toxicity due to the HSV-1 helper virus was eliminated using helper virus-free amplicon vector stocks. Transgene constructs could also be packaged in AAV virions, using AAV and adenovirus or HSV-1 helper functions. These HSV/AAV hybrid vectors should allow long-term, nontoxic gene delivery of DNA constructs to both dividing and nondividing cells.

Regulation of expression of the gene encoding human acid beta-glucosidase in different cell types.

Acid beta-glucosidase (beta Glc) activity and mRNA levels were measured in several human cell lines, and found to vary over 50-fold. A comparison between relative levels of beta Glc enzyme and mRNA levels revealed three patterns. The first group, including epithelial, lymphoblast, histiocyte, glioblastoma and astrocytoma cell lines, showed a direct relationship between relative levels of mRNA and enzyme activity, indicating that mRNA levels play an important role in determining enzyme activity. The second group, including fibroblast, promyelocyte and neuroglioma cell lines, also showed a direct relationship between beta Glc enzyme and mRNA levels within this group, but had enzyme activities that were approximately sixfold higher than expected, when compared with enzymes within the first group. The third pattern was exhibited by a single monocyte cell line, which showed high levels of beta Glc mRNA, but only intermediate levels of enzyme activity. These results suggest that although beta Glc mRNA levels play a major role in regulating beta Glc activity, other mechanisms also influence enzyme levels in certain cell lines. These results also demonstrate the importance of examining several different cell types when considering mechanisms of housekeeping gene regulation. Additionally, culturing cells in the presence of the beta Glc-specific inhibitor, conduritol-B-epoxide, did not affect beta Glc mRNA levels, and cells derived from normals had levels of beta Glc mRNA comparable to those from Gaucher disease patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of myelin proteolipid protein in oligodendrocytes and transfected cells.

The data presented in this paper show that the appropriate tools are now available to study the behavior of PLP and DM20 transcripts engineered with either point mutations or deletion of specific domains. Such studies should begin to provide new insights into the functions of PLP and DM20 and their role in relation to the optimal functioning of the nervous system.

Heterogeneity of mutations in the acid beta-glucosidase gene of Gaucher disease patients.

Gaucher disease is inherited in an autosomal recessive manner and is the most prevalent lysosomal storage disease. Gaucher disease has marked phenotypic variation and molecular heterogeneity, and several simple and complex alleles of the acid beta-glucosidase gene have been identified as causal to this disease. Certain combinations of alleles have been shown to correlate well with the severity of the disease, but many Gaucher disease patients exist whose disease is not explained by any of the published mutations. This study was undertaken to identify mutant alleles in such incompletely characterized Gaucher disease, in an attempt to find further correlations between clinical phenotype and the presence of acid beta-glucosidase alleles. RNA was isolated from Gaucher cell lines and converted to cDNA, the cDNA was amplified by PCR and cloned, and several clones for each allele were sequenced. Several new singly mutated and multiply mutated alleles were identified, and sequence-specific oligonucleotide hybridization was used to verify the presence of these mutations in the genome of these patients. All newly identified mutations occurred only rarely in the Gaucher disease population, making it difficult to determine whether inheritance of a particular combination of alleles always correlates with the clinical manifestations seen in the test patients. Three of the newly described alleles were single missense mutations in exon 8, one was a single missense mutation in exon 5, and the fifth was a complex allele, comprising a series of different point mutations scattered throughout exons 5 and 6.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of home health care in patients with peripheral vascular disease.

This investigation was designed to study the effects of home health care (HHC) on patients who have been hospitalized with peripheral vascular disease. For a patient to have HHC, the patient had to have a defined wound, educational needs, or both. Sixty patients, 30 with HHC and 30 without, were contacted approximately 30 days after their last hospital discharge. The 30 patients with HHC were deemed to be at increased risk because of multisystem disease with multiple medications, infirmity, early senility, and often complex wounds. In a prospective fashion, each patient was interviewed by either a registered nurse or medical student using a standardized data collection form. The following issues were assessed: incidence of postoperative complications, knowledge of the patient of his or her disease, compliance with medication (knowledge of, regular use), incidence of readmission, and unscheduled clinic or emergency department visits. Upon statistical analysis using the two-sample t-test and Pearson chi-square test, no significant differences were found between the two groups in terms of complications, compliance, or patient education. HHC, therefore, was found to be helpful to patients with peripheral vascular disease. In our study, the use of HHC made the risk of complications in a group of patients with defined teaching needs and wound care needs equal to that in a group with no such defined needs on discharge from the hospital.

Detection of single-base mutations in DNA molecules using the solution melting method.

RNA molecules that differ by a single base pair in their highest melting domain can be separated by the solution melting method (Smith et al., 1988) due to sequence-specific melting properties of double-stranded (ds) RNA heteroduplexes. We now show that this method can be used to reliably detect single base pair differences in DNA molecules, and we define the upper limit of the high melting domain length that can be analyzed by this method for dsRNA and RNA-DNA molecules as about 250 bp and 130 bp, respectively. The usefulness of this method to detect point mutations in human genomic DNA is evaluated. X-linked genes are most amenable to study, because results are most easily interpretable when only a single allele is present. Using the human factor VIII gene as an example, we show this method is capable of detecting polymorphisms present in genomic DNA after amplification by the polymerase chain reaction. This technique should prove useful in the simultaneous rapid screening of multiple exons of X-linked genes for single-base mutations.

Determination of the mutation rate of a retrovirus.

The mutation rate of Rous sarcoma virus (RSV) was measured. Progeny descended from a single virion were collected after one replication cycle, and seven regions of the genome were analyzed for mutations by denaturing-gradient gel electrophoresis. In all, 65,250 nucleotides were screened, yielding nine mutations, and the RSV mutation rate was calculated as 1.4 x 10(-4) mutations per nucleotide per replication cycle. These results indicate that RSV is an extremely mutable virus. We speculate that the mutation rate of a virus may correlate inversely with the effectiveness of vaccination against a given virus and suggest that prevention of retrovirus-mediated disease via vaccination may prove difficult.

I-region-coded products expressed on both macrophages and thymus epithelium influence T-cell activities.

I-region-coded products expressed on the surface of both macrophages (Mph) and thymus epithelium were shown to restrict T-cell activities at the level of antigen recognition and determination of the T-cell repertoire. Thus keyhole limpet haemocyanin (KLH) -pulsed Mph could elicit a delayed-type hypersensitivity response in a KLH-sensitized mouse only when the Mph and the sensitized mouse were compatible at the I-A region of the major histocompatibility complex. Also, experiments with thymus-grafted chimaeras showed that I-region compatibility between the thymus of the cell donor and the reticuloendothelial system of the recipient was essential for successful transfer of the DTH reaction. Thus, F1 T cells derived from stem cells differentiating in a P1 thymus graft, where F1 parents (P1 and P2) differed only at defined regions of the H-2 complex, could always transfer sensitivity to P1 mice. However, successful transfer to P2 mice was possible only if P1 and P2 were H-21-compatible.

Rapid RNA sequencing using double-stranded template DNA, SP6 polymerase, and 3'-deoxynucleotide triphosphates.

A simple and efficient nucleic acid sequencing method is described in which RNA transcription by the SP6 polymerase is specifically terminated using 3'-deoxynucleotide triphosphates. Initial difficulties in resolving the RNA ladder were overcome by replacing guanosine triphosphate by inosine triphosphate in the reaction mixture and electrophoresing gels at high temperature (50 degrees C). This method presents advantages over current sequencing techniques: Unprocessed plasmid DNA is the template and preparation of inserts and/or single-stranded templates is unnecessary. Use of the specific promoter for SP6 polymerase removes the need for a primer in sequencing reactions.

Heterogeneous and monoclonal helper T cells induce similar anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody populations in the primary adoptive response. II. Lambda light chain dominance and idiotope expression.

When the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) is presented on different carrier molecules, different anti-NP antibody responses are stimulated. On stimulation with NP-lipopolysaccharide (LPS) [T-independent type 1 (TI-1) antigen] kappa + antibodies are the major population, whereas on stimulation with NP-Ficoll [T-independent type 2 (TI-2) antigen], NP-keyhole limpet hemocyanin (KLH) or NP-chicken gamma globulin (CG) [T-dependent (TD) antigens], lambda 1+ antibodies dominate. The relative contribution of idiotopes Ac38 or Ac146 to the lambda 1+ anti-NP response was also different on comparison of TI-1 with TI-2 or TD anti-NP responses. We investigated whether light chain- or idiotype-specific T cells are responsible for these differences. Analysis of the anti-NP response of nude mice after immunization with NP-Ficoll showed lambda 1 dominance. Likewise primary adoptive transfer experiments using carrier-specific T cell lines to reconstitute the TD anti-NP response to NP-KLH or NP-CG, showed that help from carrier-specific T cells alone is capable of stimulating the characteristic lambda 1 dominant response. No significant difference could be found in the levels of Ac38 and Ac146 idiotope expression between mice reconstituted with splenic T cells and those reconstituted with T cell lines. These results suggest that light chain- or idiotype-specific T cells are required neither for the production of lambda 1 light chain dominance, nor for the appearance of idiotopes characteristic of the primary anti-NP response. The possible reasons for differences seen in both light chain and idiotope expression between primary anti-NP responses to the TI-1 antigen NP-LPS and those to TD or TI-2 antigens are discussed.

Molecular cloning of rat alpha1,3-fucosyltransferase IX (Fuc-TIX) and comparison of the expression of Fuc-TIV and Fuc-TIX genes during rat postnatal cerebellum development.

Fucosylated glycoconjugates play an essential role in central nervous system development, but the regulation of expression of these molecules is not well understood. The final biosynthetic step for a major group of cerebellar fucosylated glycoconjugates (those bearing the developmentally regulated epitope 3-fucosyl-N-acetyllactosamine, CD15, and related fucosylated epitopes) is catalyzed by an alpha-1,3-fucosyltransferase (FucT). The major FucT activity in postnatal rat cerebellum has a specificity consistent with that encoded by either a Fuc-TIV- or Fuc-TIX-like gene, and thus the expression of these genes was investigated during postnatal rat cerebellar development. A rFuc-TIX cDNA was cloned and a comparison of its enzymatic activity with rFuc-TIV revealed similar results on oligosaccharides, but strikingly higher activity on lipid acceptors, suggesting a greater role for rFuc-TIX than rFuc-TIV in the synthesis of CD15 glycolipids. rFuc-TIX mRNA levels were much higher than those of rFuc-TIV in neonatal cerebellum. Whereas rFuc-TIX mRNA levels remained relatively constant, rFuc-TIV mRNA levels declined during postnatal cerebellar development. In situ hybridization of postnatal rat cerebella also revealed different patterns of expression for these two genes. The rFuc-TIV gene was expressed predominantly in Purkinje cells and the deep cerebellar nuclei throughout postnatal development, but was expressed in granule neurons only in the neonatal, and not the adult, rat. In contrast, the rFuc-TIX gene was expressed in cells in the granule cell layers in both neonatal and in the adult rat. The potential implications of the different enzymatic activities and cell localization of rFuc-TIV and rFuc-TIX expression for the regulation of fucosylated glycoconjugates during cerebellar development are discussed.

Detection of single base substitutions in influenza virus RNA molecules by denaturing gradient gel electrophoresis of RNA-RNA or DNA-RNA heteroduplexes.

Single point mutations in the NS gene of influenza virus were detected by electrophoresis of double-stranded RNA heteroduplexes in denaturing gradient gels. The heteroduplex RNAs were made by hybridization of virion RNA with SP6-derived RNA probes of varying length. Mutations located at different positions along the NS gene (890 nucleotides long) were all detected in a predictable fashion. The method of heteroduplex analysis was also successfully used in detecting single point mismatches in DNA-RNA hybrids.

Gaucher disease type 1: cloning and characterization of a cDNA encoding acid beta-glucosidase from an Ashkenazi Jewish patient.

Gaucher disease (GD) type 1 is the most prevalent lysosomal storage disease and the most prevalent genetic disease among the Ashkenazi Jews. The defective activity of acid beta-glucosidase is the enzymatic basis of GD and is inherited as an autosomal recessive trait. To investigate the genetic basis of Ashkenazi Jewish GD type 1, a cDNA encoding acid beta-glucosidase was isolated from a cDNA library constructed using splenic poly(A)+RNA from a patient. The cDNA was sequenced to identify mutations, and the presence of a single missense mutation in the patients' genome was confirmed by selective oligonucleotide hybridization and by restriction endonuclease digestion analyses of amplified genomic sequences. This G----A transition (Arg-119 to Gln-119) was present heterozygously in the index patient and his affected third cousin but was not present in normal non-Jewish individuals. This mutation is the second single base mutation found in Ashkenazi Jewish GD type 1 patients. Furthermore, results obtained with the affected third cousin suggest that at least three mutant alleles may be present in this GD subpopulation.

Gaucher disease: molecular heterogeneity and phenotype-genotype correlations.

Gaucher disease (GD) is the most prevalent lysosomal storage disease. This autosomal recessive trait results from the defective activity of acid beta-glucosidase (beta-Glc). Four different exonic point mutations have been identified as causal alleles for GD. To facilitate screening for these alleles, assays were developed using allele-specific oligonucleotide hybridization to amplified genomic DNA sequences. Specifically, intron bases flanking exons 5, 9, and 10 were determined, and conditions for PCR amplification of these exons were obtained. Two different procedures were developed to distinguish signals obtained from the structural beta-Glc gene exons and those from the pseudogene. These procedures were used to determine the distribution of all known GD alleles in a population of 44 affected patients of varying phenotypes and ethnicity. The high frequency of one of the exon 9 mutations in Ashkenazi Jewish GD type 1 patients was confirmed, and, in addition, this mutation was present in ethnically diverse non-Jewish type 1 GD patients. Homozygotes (N = 5) for this allele were midly affected older individuals, and this mutant allele was not found in any patient with neuronopathic disease. The exon 10 mutation was confirmed as the predominant allele in types 2 and 3 GD. However, several type 1 GD patients, including one of Ashkenazi-Jewish heritage, also were heterozygous for this allele. The presence of this allele in type 1 patients did not correlate with the severity of clinical symptoms. The second exon 9 mutation and the exon 5 mutation were rare, since they occurred only heterozygously either in one type 2 GD patient or in two related Ashkenazi-Jewish GD patients, respectively. Although most GD patients (38 of 44) had at least one of the known mutant alleles, 57% were heterozygotes for only one of these mutations. Fourteen percent of patients were negative for all mutations. A total of 73% of GD patients had at least one unknown allele. The varying clinical phenotypes and ethnic origins of these incompletely characterized patients suggest that multiple other GD alleles exist.