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PubMed1986

The T lymphocyte response to syngeneic lambda 2 light chain idiotopes. Significance of individual amino acids revealed by variant lambda 2 chains and idiotope-mimicking chemically synthesized peptides.

In the present study we have investigated the structure of the helper T cell (Th)-defined idiotope (Id) of myeloma protein 315 lambda 2 light chain (lambda 2(315] in BALB/c (H-2d) mice which carry a high-responder immune response gene for this Id. Three peptides were synthesized which spanned the third hypervariable region (HV3) of lambda 2(315): peptides 88-99, 94-108 and 91-108. Only peptide 91-108 was capable of eliciting carrier-specific Th that recognized M315 or free lambda 2(315). These Th did not recognize lambda 2(5-7) chain which differs from lambda 2(315) at 4 positions in this region; these are Tyr94, Ser95, Thr96, Tyr98 for lambda 2(5-7) and Phe94, Arg95, Asn96, Phe98 for lambda 2(315). Immunization with peptide analogues revealed that substitution of Tyr for Phe94 was compatible with Id-lambda 2(315) mimicry, but substitution of Ser for Arg95 or Thr for Asn96 destroyed the Th-recognized Id. Furthermore, Th primed with lambda 2(5-7) chain did not cross-react with lambda 2T952; these lambda 2 chains only differ from each other at positions 98 and 99 at the V lambda 2-J lambda 2 junction. The data indicate that individual amino acids of short peptide segments are critical for Th-recognized Id of the lambda 2 HV3 loop and V lambda 2-J lambda 2 junction. Furthermore, the immunogenicity of a small peptide suggests that the carrier (lambda 2)-specific Th recognize Id that have been processed by antigen-presenting cells (APC). This implies the existence of two categories of "internal images" of foreign or of self antigens: (a) serologically defined and (b) T lymphocyte defined. We propose that as a rule, Id processing by APC, including B cells, destroys the first and reveals the second category. The possible physiological function of these Id-specific T cells in network interactions with B cells is discussed.

Amino Acids↗
PubMed1986

Idiotope-specific T cell clones that recognize syngeneic immunoglobulin fragments in the context of class II molecules.

We have established idiotope (Id)-specific T cell lines and clones derived from at least 4 different BALB/c mice immunized with the light chain (lambda 2(315] of the BALB/c myeloma protein M315 (alpha, lambda 2). Independently derived clones were indistinguishable in that they reacted to V lambda 2(315), one or more of the amino acids corresponding to somatically mutated codons 94, 95 and 96 of the third hypervariable region being essential for expression of the Id. While the Id was efficiently expressed on V lambda 2(315), Fv315 and lambda 2(315) fragments, about a 100-1000-fold higher molar concentration of Fab315 and M315 was needed to induce equivalent responses. Thus, Ig quaternary structure heavily influenced the availability of the Id for T cells. The V lambda 2(315)-specific T cells were Thy-1.2+, L3T4+, Ly-2.2- and I-Ed restricted. Some of the T cell clones produced interleukin 2 (IL2), IL3 and B cell growth and differentiation factors upon activation. In addition, T cells were cytotoxic in long-term assays for Ed beta Ek alpha-, but not Ek beta Ek alpha- transfected L cells in the presence of Id. The cytotoxic effect was the basis for an L cell growth inhibition assay for T cell activation that was at least 10-fold more sensitive than ordinary proliferation assays.

Animals↗
PubMed1990

A new HLA-DRB1 allele within the DRw52 supertypic specificity (DRw13-DwHAG): sequencing and direct identification by oligonucleotide typing.

Molecular analysis of HLA class II polymorphism represents a crucial parameter for HLA matching in transplantation immunology, for the study of HLA-disease association and for the understanding of major histocompatibility complex (MHC)-restricted antigen presentation. We report here the DNA sequence and the deduced amino acid sequence of the polymorphic first domain exon of the DRB1 and DRB3 alleles of the homozygous cell line HAG (DRw13-DwHAG-DQw7). The DRB1 sequence represents a new DRB allele, which clearly shows a close relationship to other DRB1 genes from the DRw52 group and is now officially named DRB1* 1303. The DRB1* 1303 allele is very similar to the two DRw13 alleles we have described earlier, with only five amino acid differences at positions 32, 37, 47, 57 and 71. Furthermore, its sequence in the third hypervariable region is unique among all known DRB1 and DRB3 alleles. The sequence of the DRB3 gene of HAG shows that it corresponds to the previously described DRB3* 0101 (DRw52a) allele. In addition we present analyses of a panel of healthy blood donors and leukemic patients by oligonucleotide typing showing that this new HLA-DR specificity can now be unequivocally identified in routine oligotyping with an allele-specific oligonucleotide probe.

Alleles↗
PubMed1990

Association of a particular amino acid sequence of the HLA-DR beta 1 chain with HTLV-I-associated myelopathy.

Analyses of HLA-DRB1 gene using polymerase chain reaction and sequence-specific oligonucleotide probes reveal that the amino acid sequence Glu-Gln-Arg-Arg-Ala-Ala-Val at positions 69-75 of the third hypervariable region (HVR) of HLA-DR beta 1 chain is significantly associated with HTLV-I-associated myelopathy (HAM). Since the frequency of this sequence in HTLV-I carriers is almost the same as that in controls from Kagoshima (an endemic area of HTLV-I), this sequence may be related to susceptibility to HAM rather than to susceptibility to HTLV-I infection. Since this third HVR functions as putative antigen-binding sites and T cell recognition sites, the amino acid sequence of positions 68-73 was analyzed in detail. The analysis reveals that Gln70 and Arg71 are relevant to the occurrence of HAM.

Alleles↗
PubMed1992

T cell recognition of donor major histocompatibility complex class I peptides during allograft rejection.

LEW (RTI1) recipients of DA (RTIav1) skin and kidney allografts were tested for the capacity of their T lymphocytes to proliferate to three 22-24-amino acid peptides from the hypervariable regions of the RTI-Aav1 classical MHC class I molecule. Ten days after rejecting second-set DA kidney allografts, spleen cells (but interestingly not lymph node cells) from LEW recipients showed strong, LEW antigen-presenting cell-dependent, CD4+ T cell proliferation to peptide 1 (from the alpha helical region of the alpha 1 domain). CD8+ T cells showed no response to peptide 1. There was no response by the spleen cells to peptide 2 (from the beta sheet of the alpha 2 domain) or peptide 3 (from the alpha helical region of the alpha 2 domain). Immunization of LEW rats with pure RTI-Aav1 class I H chain in Freund's adjuvant gave responses identical to that seen after grafting, i.e. good CD4+ T cell proliferation to peptide 1, but none to peptides 2 and 3. However, immunization of LEW rats with peptides 1, 2 and 3 in Freund's adjuvant resulted in good CD4+ T cell proliferation responses to each of the peptides. These data demonstrate that indirect allorecognition can be stimulated by allograft rejection, and emphasize that the physiological processing of donor antigens will influence which peptides will be important in indirect allorecognition in transplantation.

Amino Acid Sequence↗
PubMed1996

Peripheral T cell tolerance as a tumor escape mechanism: deletion of CD4+ T cells specific for a monoclonal immunoglobulin idiotype secreted by a plasmacytoma.

Tumors could escape an immune attack by inducing peripheral T cell tolerance. To test this, T cell receptor (TCR)-transgenic mice were injected with plasmacytoma cells secreting a highly tumor-specific antigen, a monoclonal immunoglobulin (Ig), for which the transgene-encoded TCR is specific. The TCR recognizes a third hypervariable region idiotypic (Id) peptide of the Ig, presented by a class II molecule on host antigen-presenting cells. The TCR-transgenic mice have previously been shown to be protected against an Id+ plasmacytoma challenge. In the present experiments, the protection was deliberately overwhelmed by subcutaneous injection of large numbers of plasmacytoma cells. Such tumor mice, chronically exposed to increasing amounts of monoclonal Ig, delete Id-specific CD4+ T cells in their peripheral lymphoid organs and in the tumor. The residual CD4+ cells express endogenous, rather than transgene-encoded TCR alpha chains. Peripheral deletion, functional T cells unresponsiveness, and thymocyte deletion are all first detected at the same serum concentration of monoclonal Ig, approximately 50 micrograms/ml (0.3 microM), and become more and more profound as the tumor burden increases. The results suggest that peripheral T cell tolerance to Id could be a tumor escape mechanism in patients with B cell malignancies. In addition, the findings have implications for T cell tolerance to Ig V regions in normal individuals.

Animals↗
PubMed1996

DNA sequencing using a four-color confocal fluorescence capillary array scanner.

The design, construction and operation of a four-color capillary array electrophoresis scanner are presented. The use of sensitive energy transfer primers facilitates four-color detection of the DNA sequencing fragments following excitation at a single laser wavelength (488 nm). This scanner collects fluorescence data from up to 25 capillaries in parallel. The resulting four-color image files are automatically reduced to four-color line plots, and a base-calling program (Sax) is used to call the sequence. The performance of this system for DNA sequencing is demonstrated by examining twelve different motifs of the hypervariable region I of human mitochondrial (mt) DNA obtained from a Sierra Leone population.

Base Sequence↗
PubMed1989

Increased risk for gestational diabetes mellitus associated with insulin receptor and insulin-like growth factor II restriction fragment length polymorphisms.

Gestational diabetes mellitus (GDM) is defined as glucose intolerance with onset or first recognition during pregnancy. We have examined restriction fragment length polymorphisms (RFLPs) near "candidate diabetogenic genes" as one approach to identify molecular markers for GDM genes. Genotypes for insulin hypervariable region (HVR), insulin-like growth factor II (IGF2), insulin receptor (INSR), and glucose transporter (GLUT1) RFLPs were studied in 96 GDM and 164 control subjects, matched to GDM for race, age, and gravidity. Logistic regression analysis was used to explore the relationship between genotypes at these candidate gene loci and GDM, while adjusting for the effects of potential confounding variables. Among black subjects, the INSR allele 1 (P = 0.001) and interactions between INSR allele 1 with body mass index (BMI) (P = 0.002) and history of DM in subject's mother (P = 0.004) contributed significantly to GDM risk. Among Caucasian subjects, a similar relationship between the INSR allele 1 (P = 0.007) and INSR allele 1-BMI interactions (P = 0.011) on GDM risk were observed. In Caucasians, an additional significant risk factor was determined by an INSR allele 1-IGF2 allele 2 interaction (P = 0.018). No risk factors were identified in Hispanic subjects. These data continue to support the hypothesis that GDM is a heterogeneous disorder with respect to phenotypic and genotypic features. Furthermore, our data suggest that risk for GDM in black and Caucasian subjects is not due to obesity perse but to interactions between obesity and INSR alleles. In Caucasian women, INSR and IGF2 alleles interact to confer additional risk for GDM. Thus genes underlying susceptibility to GDM in some women may be similar to genes conferring risk to NIDDM, while in others novel genes may contribute to GDM risk.

Chromosome Mapping↗
PubMed1995

Polymorphism at VNTR locus 3 to the apolipoprotein B gene in a Tunisian population: difference from other ethnic groups.

The Hypervariable region (HVR) detected at the 3' end of the apolipoprotein B (Apo B) locus has been the subject of numerous studies. As for many VNTR (variable number of tandem repeat), this locus is highly polymorphic and until now about 20 alleles have been described. The genotype distribution in all populations follows the Hardy-Weinberg predictions. A bimodal pattern of allele frequency distribution is apparent in all Caucasoid populations. We have analyzed the frequencies of different alleles in a Tunisian population (123 individuals) by the polymerase chain reaction technique and compared our results to those obtained in several ethnic groups. It appears that the distributions of the allele frequencies are very different: for Caucasoid populations, there are two peaks of frequencies for alleles with 36 and 48 repeats, but alleles of intermediate lengths are more frequent. Hixson et al. [(1993) Hum Genet 91:475-479] have shown a similar difference between black and white American populations. We found the same results in a black African group. Some of the repeat units of this HVR contain a Ssp I restriction site and digestion of the PCR products by this enzyme gives different patterns on gradient acrylamide gel [Desmarais et al., 1993, Nucleic Acids Res 21:2179-2184.] The DNA of African individuals (42) has been analyzed to discover the origin of this new allele. Preliminary results indicate that these particular alleles probably arose by introgression from the African population into the Tunisian one.

Alleles↗
PubMed2004

Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes.

Differences in the composition of the hepatitis C virus (HCV) quasispecies between plasma and blood mononuclear cells (BMC) strongly suggest that BMCs support viral replication. We examined the frequency of such compartmentalization, the cell types involved, the constraints exerted on the different variants, and the role of immunoglobulin-complexed variants. We screened the hypervariable region (HVR1) of HCV isolates from 14 HBsAg- and HIV-seronegative patients with chronic HCV infection. HCV RNA was amplified and cloned from plasma, the immunoglobulin G (IgG)-bound fraction, and total and sorted BMCs (CD19+, CD8+, CD4+, and CD14+ cells). Compartmentalization was estimated using a matrix correlation test. The ratio of nonsynonymous/synonymous substitutions (d(N)/d(S) ratio) was calculated for each compartment. HCV RNA was detected in 3/3 BMC, 11/11 CD19+, 10/11 CD14+, 4/11 CD8+ and 0/11 CD4+ cell samples. HVR1 sequences were significantly different between plasma and at least one cellular compartment in all nine cases analyzed, and between B cells (CD19+) and monocytes (CD14+) in all five available cases. IgG-bound variants were distinct from cellular variants. D(N)/d(S) ratios were similar (n = 3) or lower (n = 6) in cellular compartments compared with plasma and the IgG-bound fraction. In conclusion, HCV compartmentalization is a common phenomenon. B cells and monocytes harbor HCV variants showing a low rate of nonsynonymous mutations, a feature that might contribute to the persistence of HCV infection.

Adult↗
PubMed1997

A specific antibody response to HCV E2 elicited in mice by intramuscular inoculation of plasmid DNA containing coding sequences for E2.

As the chimpanzee, the only reliable animal model for hepatitis C virus (HCV) infection, is impractical for early stage testing of HCV vaccine candidates, we have evaluated the immune response in mice to an experimental plasmid based HCV vaccine. We used this system because DNA vaccines can be rapidly constructed without the necessity of large scale protein production and purification. In this preliminary study we tested the immune response in mice to HCV envelope glycoprotein, E2, induced by a eukaryotic expression plasmid. Protein expression was monitored by immunofluorescence in transfected tissue culture cells. Each mouse was inoculated intramuscular with 100 microg plasmid DNA and some mice were boosted after 5 weeks. Among 12 BALB/C mice inoculated, 10 developed antibody to E2 by the second week. The antibody levels increased steadily before reaching a plateau in mice receiving the booster, but in the nonboosted mice the antibody declined over time. The serum from one mouse was tested against a series of overlapping peptides covering most of E2. This serum contained antibodies recognizing two distinct epitopes beginning at amino acid 57 and amino acid 113 but no antibody was directed against peptides representing the hypervariable region of E2, antibody to which is thought to be important in HCV neutralization. We have shown that the use of plasmid based vaccines can induce a specific immune response in mice against HCV antigens. This system should be useful as the first step in vaccine development.

Animals↗
PubMed1997

Hepatitis C virus genome complexity correlates with response to interferon therapy: a study in French patients with chronic hepatitis C.

Recent studies performed in Japan have suggested that hepatitis C virus (HCV) genome heterogeneity might be taken as a predictive virological parameter of response to interferon alfa (IFN-alpha) treatment. However, there is presently no information on the impact of this virological parameter in patients from Western countries infected by different HCV genotypes. We have investigated this issue by using amplification of HCV E2 hypervariable region 1, followed by single-strand conformation polymorphism assay (PCR-SSCP). We have studied 95 French patients infected with various genotypes and treated with IFN-alpha-2b. We analyzed the impact of the following parameters by univariate and multivariate analyses: HCV heterogeneity, HCV genotype, viral load, and liver histology in response to therapy. Age >40 years (P < .01), viral load >35 x 10(5) Eq/mL (P < .01), genotype 1 (P < .01), and a number of SSCP bands >3 (P < .001) were significantly associated with nonresponse or relapse; cirrhosis was associated with nonresponse. In multivariate analysis, three variables were independently associated with the absence of long-term response: SSCP bands >3, genotype 1, and viral load >35 x 10(5) with odds ratios of 19, 7.5, and 11.8, respectively. Our data establish the major importance of HCV genome heterogeneity in patients infected with both HCV types associated with low (genotype 1b) or high (genotype 3a) response to IFN-alpha.

Adult↗
PubMed1997

Virological response to interferon treatment in hepatitis C virus carriers with normal aminotransferase levels and chronic hepatitis.

Hepatitis C virus (HCV) carriers with normal aminotransferase levels often show histological chronic hepatitis. This study was undertaken to determine the effect of interferon (IFN) in such patients. Nineteen HCV carriers with normal aminotransferase activities and chronic hepatitis were randomized to receive IFN-alpha2b (3 million units 3 times weekly for 12 months) or no treatment. Therapy was monitored by qualitative and quantitative determination of viral RNA. Patients who did not clear HCV RNA after 6 months discontinued therapy. In all, 9 patients constituted the control group, while 10 patients were treated. Five of these patients, still viremic after 6 months, stopped IFN. The remaining 5 patients, who cleared the viral RNA within 6 months, completed the 12-month course. Three of these patients relapsed off treatment, and 2 were still free of viremia 12 months after stopping therapy. A transient flare-up of aminotransferase activities was detected in 2 patients during treatment and in 3 patients after. None of the 9 control patients cleared the viral RNA during follow-up. A variable degree of sequence heterogeneity was detected in the hypervariable region before therapy, and IFN treatment decreased sequence diversity in all patients. These results indicate that IFN therapy can be effective in chronic HCV carriers with normal aminotransferase activities, inducing short-term virological response in 3 of 10 patients and sustained response in 2. The effects of treatment on viral load and quasispecies complexity were similar to those reported previously in patients with increased aminotransferase activities.

Adult↗
PubMed1999

Evolution of hepatitis C virus quasispecies in patients with severe cholestatic hepatitis after liver transplantation.

Evolution of hepatitis C quasispecies may be one mechanism by which fibrosing cholestatic hepatitis develops after liver transplantation. In this study, we compared changes in quasispecies complexity and/or divergence in (1) hepatitis C-infected immunosuppressed transplant recipients and in immunocompetent controls; (2) transplant recipients with mild recurrence, and in those with the most severe form of posttransplantation recurrence. Quasispecies were measured in 12 hepatitis C-infected patients pretransplantation and posttransplantation (6 with mild and 6 with severe recurrence), and in 5 immunocompetent patients with similar follow-up, and characterized by heteroduplex mobility and sequence analysis of the hypervariable region. Although the number of variants (complexity) did not change with time in either group, there was a qualitative change in the variants with time (divergence) in immunocompromised, but not in immunocompetent patients. These changes were most marked with severe recurrence, and preceded the development of severe disease. Phylogenetic analysis confirmed that most posttransplantation variants were unrelated to those detected pretransplantation. These observations suggest that in the absence of immune suppression, there is minor evolution of quasispecies. With immune suppression, divergence of quasispecies is enhanced, resulting in selection/emergence of many new variants, particularly in those with fibrosing cholestatic hepatitis. Thus, quasispecies may influence disease progression in immune suppressed populations.

Cholestasis, Intrahepatic↗
PubMed1983

Amino acid sequence data on glial fibrillary acidic protein (GFA); implications for the subdivision of intermediate filaments into epithelial and non-epithelial members.

Determination of 50% of the sequence of the astrocyte-specific intermediate filament (IF) protein documents the hypervariable regions as well as parts of the coiled-coil array of glial fibrillary acidic protein (GFA). The results show that the four non-epithelial IF proteins (myogenic desmin, mesenchymal vimentin, GFA and neurofilament 68 K protein) known to form homopolymers are much more closely related than the epithelial keratins, which seem to form heteropolymers only. Of the four non-epithelial proteins, desmin and vimentin are the most closely related, since GFA has a shorter non-alpha-helical array at the amino terminus. We discuss the possibility that the non-alpha-helical terminal arrays, because of their sequence and length variability, are responsible for differences of distinct IF with respect to physical-chemical properties such as the low ionic strength-induced depolymerization into protofilaments.

Amino Acid Sequence↗
PubMed1986

T cell receptor genes in an alloreactive CTL clone: implications for rearrangement and germline diversity of variable gene segments.

Both cDNA and genomic clones of the T cell receptor (TCR) alpha- and beta-chain genes of the alloreactive cytotoxic T lymphocyte (CTL) clone F3 were examined. Two distinct rearrangement events, one functional and one non-functional, were found for both the alpha and beta loci. Thus only a single functional TCR alpha beta heterodimer could be defined, consistent with allelic exclusion in the TCR genes. The V alpha gene employed by F3 is part of a six-member V alpha subfamily. Genomic clones containing each member of this subfamily were isolated and the V alpha nucleotide sequences determined. Five of these six genes are functional; these genes differ from each other by 7-14% at the amino acid level. A single dominant hypervariable region was defined within this subfamily, in contrast to the pattern of variability seen between V alpha genes in general.

Alleles↗
PubMed1989

Intragenic recombination in a flagellin gene: characterization of the H1-j gene of Salmonella typhi.

Salmonella typhi, the etiologic agent of typhoid fever, typically has only a phase-1 flagellar antigen, d, but some isolates, found only in Indonesia, have antigen j instead, and may have a second flagellar antigen, z66. It appears that intragenic recombination involving a directly repeated 11 bp sequence in the H1-d flagellin gene changed the flagellar antigen to j, by deleting 261 bp in its central, antigenically determinant, part. Sequencing of the hypervariable regions of genes H1-d and H1-j, and hybridization of such genes, after amplification by the polymerase chain reaction, with oligonucleotide probes specific for the deleted segment or for the sequence produced by the recombination confirmed that all the j alleles have the postulated deletion. By applying the polymerase chain reaction to study S. typhi isolates from Jakarta, not previously tested in respect to flagellar antigen, we showed that gene H1-j was nearly as common as H1-d in these isolates.

Antigens, Bacterial↗
PubMed2002

Molecular analysis of measles virus genome derived from SSPE and acute measles patients in Papua, New Guinea.

A very high annual incidence of 56 per million population below the age of 20 years for subacute sclerosing panencephalitis (SSPE) has been reported from Papua New Guinea (PNG). In a more recent study, we have confirmed this unusual high incidence for Eastern Highlands Province (EHP) of PNG. In the study, it was observed that the vaccination rate among SSPE patients registered at Goroka Base General Hospital (GBGH) in EHP was higher than that of other infants in the province in recent years. To identify the measles virus (MV) responsible for SSPE in EHP, sequence analysis of hypervariable region of the N gene was performed from 13 MV genomes: 2 amplified from clinical specimens of SSPE patients and 11 from acute measles patients. In 2 cases among the 11 with acute measles, nucleotide sequence of the entire H gene derived from isolated viruses was determined. Both nucleotide sequence and phylogenetic tree analyses showed that the amplified MV cDNAs were closely related to one another and belonged to the D3 genotype though they were different from any previously reported MV sequences. No genome sequences of vaccine strains were detected. These findings suggest that the MV strains prevailing in the highlands of PNG belong to genotype D3 of the MV and this wild-type MV rather than the vaccine strains was likely to be responsible for SSPE in these patients.

Acute Disease↗