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PubMed2002

[Relationship between type I autoimmune hepatitis and alleles of HLA-DRB1 in Chinese patients of Shanghai area].

OBJECTIVE: To analyze the association between alleles of HLA-DRB1 and type I autoimmune hepatitis (AIH) in patients from Shanghai, China. METHODS: In 32 Chinese patients with type I AIH and 48 healthy controls in Shanghai area, polymerase chain reaction amplification with sequence-specific primers (PCR-SSP) was performed to examine the association of the alleles of HLA-DRB1 and its subtypes with type I AIH. RESULTS: HLA-DRB1 typing by PCR-SSP showed that DR4 had a significantly increased frequency among patients with AIH versus healthy control (46.9% versus 20.8%; relative risk=3.35, chi(2)=5.99, P=0.014). No other alleles differed significantly between the two groups. In subtypes of DRB1*04, there was a trend for an increase in gene frequency of DRB1*0405 increased in patients with type I AIH versus healthy controls (21.9% vs 6.3%, chi(2)=4.23, P=0.04, but Pc=0.08). The frequency of arginine at position HLA-DR beta 71 of third hypervariable region significantly increased among patients with AIH versus healthy control (46.9% versus 18.8%, chi(2)=7.14, P=0.008). CONCLUSIONS: Type I AIH in Chinese patients of Shanghai area is associated with HLA-DR4 and arginine at position DR beta 71 of third hypervariable region

Alleles↗
PubMed1990

Shared molecular markers of genetic predisposition to seropositive rheumatoid arthritis.

Rheumatoid arthritis is associated with the human class II major histocompatibility complex antigens known as HLA-DR4. HLA-DR4 can be subdivided by cellular typing into five subtypes: Dw4, Dw10, Dw13, Dw14, and Dw15. By traditional serologic methods, 60-80% of rheumatoid arthritis patients type HLA-DR4 compared to approximately 20% of the general population. It has been demonstrated, using a panel of four alloreactive T-cell clones, each of which recognized HLA-DR4, Dw14 homozygous typing cells, that cells from all of a group of 23 rheumatoid arthritis patients could be recognized by one or more of these clones regardless of the patients' serologic typing. As the expressed polymorphism of the DR molecule is accounted for by the beta 1 gene, this gene was amplified, using the polymerase chain reaction, and sequenced. Seven patients whose cells were recognized by one of the DR4, DW14-specific T-cell clones, T431, were analyzed. All of these patients shared a common sequence in the third hypervariable region of the DR beta 1 chain gene. The sequence identified is the one normally associated with DR4, Dw14 and DR1. Patients and DR4-positive controls whose cells did not stimulate this clone did not share this sequence. These results suggest that this hypervariable region might be an important contribution to a restriction site for the putative causative agent(s) in rheumatoid arthritis.

Amino Acid Sequence↗
PubMed2005

Phylogeography of Barbary macaques (Macaca sylvanus) and the origin of the Gibraltar colony.

The Barbary macaque (Macaca sylvanus) is the earliest offshoot of the genus Macaca and the only extant African representative, all other species being Asiatic. Once distributed throughout North Africa, M. sylvanus is now restricted to isolated forest fragments in Algeria and Morocco. The species is threatened; the maximum total wild population size is estimated at 10,000 individuals. Relationships among surviving wild subpopulations in Algeria (96 samples) and Morocco (116 samples) were examined by using 468-bp sequences from hypervariable region I of the mitochondrial DNA control region. Twenty-four different haplotypes were identified, differing by 1-26 mutational steps (0.2-5.6%) and 1 insertion. With one exception (attributable to secondary introduction in coastal Morocco), Algerian and Moroccan haplotypes are clearly distinct. However, whereas Moroccan subpopulations show little divergence in hypervariable region I sequences and little correspondence with geographical distribution, there is a deep division between two main subpopulations in Algeria and one marked secondary division, with haplotypes generally matching geographical distribution. Accepting an origin of the genus Macaca of 5.5 million years ago, the Moroccan population and the two main Algerian subpopulations diverged approximately 1.6 million years ago. Distinction between Moroccan and Algerian haplotypes permitted analysis of the origin of the Gibraltar colony of Barbary macaques (68 samples; 30% of the population). It is generally held that the present Gibraltar population descended from a dozen individuals imported during World War II. However, the Gibraltar sample was found to include Algerian and Moroccan haplotypes separated by at least 16 mutational steps, revealing a dual origin of the founding females.

Algeria↗
PubMed2000

Structural correlates of an anticarcinoma antibody: identification of specificity-determining residues (SDRs) and development of a minimally immunogenic antibody variant by retention of SDRs only.

Clinical utility of murine mAbs is limited because many elicit Abs to murine Ig constant and variable regions in patients. An Ab humanized by the current procedure of grafting all the complementarity determining regions (CDRs) of a murine Ab onto the human Ab frameworks is likely to be less immunogenic, except that its murine CDRs could still evoke an anti-variable region response. Previous studies with anticarcinoma mAb CC49 showed that light chain LCDR1 and LCDR2 of humanized CC49 could be replaced with the corresponding CDRs of a human Ab with minimal loss of Ag-binding activity. The studies reported in this paper were undertaken to dissect the CC49 Ag-binding site to identify 1) specificity determining residues (SDRs), the residues of the hypervariable region that are most critical in Ag-Ab interaction, and 2) those residues that contribute to the idiotopes that are potential targets of patients' immune responses. A panel of variants generated by genetic manipulation of the murine CC49 hypervariable regions were evaluated for their relative Ag-binding affinity and reactivity to sera from several patients who had been immunized with murine CC49. One variant, designated HuCC49V10, retained only the SDRs of CC49 and does not react with the anti-variable region Abs of the sera from the murine CC49-treated patients. These studies thus demonstrate that the genetic manipulation of Ab variable regions can be accomplished by grafting only the SDRs of a xenogeneic Ab onto human Ab frameworks. This approach may reduce the immunogenicity of Abs to a minimum.

Amino Acid Sequence↗
PubMed1975

Structural, functional, and idiotypic characteristics of a phosphorylcholine-binding IgA myeloma protein of C57BL/ka allotype.

An IgA phosphorylcholine (PC)-binding myeloma protein with IgCH allotypic determinants different from those of BALB/c mice is characterized. The myeloma, CBPC 2, was induced in the CB-20 strain of mice which is congenic to BALB/c but differs from it by carrying the A15 allotypic determinant of C57BL/ka mice. Sequence analysis of the CBPC 2 light chain through the first hypervariable region, as well as isoelectric point analysis, show that this chain is indistinguishable from that of T15, a PC-binding myeloma protein of BALB/c origin. The heavy chains of CBPC 2 and T15 differ by only two amino acids (positions 14 and 16) through the first hypervariable region. As measured by inhibition of precipitation, both CBPC 2 and T15 have the same specificity for PC, glycerophosphorylcholine, acetylcholine, and choline. In addition, CBPC 2 possesses the binding site-associated idiotypic determinant which is present on T15. However, like normal or induced C57BL/6 anti-PC antibody, it does not possess the nonbinding site idiotypic determinant.

Amino Acid Sequence↗
PubMed1989

Identification of a doubly spliced viral transcript joining the separated domains for putative protease and reverse transcriptase of hepatitis B virus.

Hepatitis B virus (HBV), like retroviruses, replicates through reverse transcription. However, the identity and mechanism for the synthesis of HBV reverse transcriptase remain unknown. The open reading frame (ORF) for HBV putative reverse transcriptase (pol), as a consequence of overlapping with the whole ORF of envelope proteins (hepatitis B surface antigens), includes a hypervariable region at the N terminus. Thus, compared with retroviruses, it is unlikely that HBV reverse transcriptase is translated from complete pol ORF in the full-length pregenomic RNA. We have now detected in infected human livers a novel doubly spliced RNA in which one splicing event removed the hypervariable region of the pol gene but retained the conserved region homologous to retroviral reverse transcriptase. The other splicing event deleted the central region of hepatitis B core antigen and thus brought the protease domain which is important for maturation of reverse transcriptase close to that of pol. For this sequence organization, the spliced RNA as the possible template for the synthesis of HBV reverse transcriptase is discussed.

Base Sequence↗
PubMed2002

Antibodies highly effective in SCID mice during infection by the intracellular bacterium Ehrlichia chaffeensis are of picomolar affinity and exhibit preferential epitope and isotype utilization.

Although often considered to be ineffective against intracellular bacteria, Abs, in the absence of lymphocytes, have been shown previously to protect SCID mice from lethal infection by the obligate intracellular bacterium Ehrlichia chaffeensis, even when administered well after infection has been established. To identify characteristics of Abs that are critical for host defense during this intracellular infection, a panel of Ehrlichia-specific mAbs was generated and analyzed. Among 100 Abs recovered, 39 recognized an amino-terminal hypervariable region of an outer membrane protein (OMP), demonstrating that the OMPs are both antigenically variable and immunodominant. A subset of 16 representative OMP-specific Abs was further examined to identify characteristics that were essential for in vivo efficacy. The highly effective Abs recognized a linear epitope within the first hypervariable region of OMP-1g. Only IgG were found to be effective, and among the effective IgG, the following hierarchy was observed: IgG2a > IgG3 = IgG2b. The most striking characteristics of the highly effective Abs were their picomolar binding affinities and long binding t(1/2). Thus, although epitope recognition and isotype use may contribute to efficacy, high affinity may be a critical characteristic of Abs that can act effectively during this intracellular bacterial infection.

Animals↗
PubMed1991

T-cell clones identify three distinct epitopes associated with HLA-Dw14.

Alloreactive T-cell clones were used to study allodeterminants associated with the HLA-DR1, -DR4, and -DRw14 allelic families. Three clones derived by priming against the DR4,Dw14 alloantigen were tested against a panel of HLA-D homozygous B-cell lines and homozygous and heterozygous peripheral blood lymphocytes. Each clone was blocked by monoclonal antibodies specific for HLA-DR, but not HLA-DQ or -DP, molecules, and each showed a unique pattern of allorecognition when tested against the cell panel. Clone 14B appeared to recognize a specific sequence, termed L67-A74, comprised of amino acids in the third hypervariable region of the alpha-helix of the DR beta 1 molecule, and expressed on certain DR1-, DR4-, and DRw14-positive cells. Clone EMO25 recognized the same L67-A74 sequence, but only when expressed on DR4-positive cells, suggesting a role for residues in the first and second hypervariable regions of DR4-positive DR beta 1 molecules in T-cell recognition. Clone EM036 also recognized the L67-A74 sequence, but only when expressed on DR4,Dw14.1-positive cells, implicating residues at positions 57 and 86 of the alpha-helix in T-cell recognition. These results demonstrate the range of specific T-cell responses that are possible against alloepitopes expressed by a single class II allele (Dw14), and are an indication of the diverse regions of the class II molecule that can contribute to allorecognition sites.

Amino Acid Sequence↗
PubMed1998

Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample.

We constructed a full-length complementary DNA (cDNA) clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier. The blood from the carrier was eventually transfused to a patient who later developed typical posttransfusion hepatitis C. It was also shown to be infectious to chimpanzees. We obtained 12 overlapping cDNA fragments altogether, covering the entire HCV genome. By subcloning and sequencing, clones considered to constitute the major population were selected. We could also detect 98 base pairs of extra sequences at the 3' end of the genome. After confirming the overlapping sequences, we combined the fragments to make a full-length cDNA. The HCV population in the donor was heterogeneous, as determined by their nucleotide sequences of the hypervariable region in envelope protein, but a few virus clones were selected in the recipient after transmission. The similar convergence of the virus population was previously observed when the same blood sample was injected into a chimpanzee. Interestingly, virus clones isolated during the acute phase in the recipient and the chimpanzee had sequences in the hypervariable region identical to that of the full-length cDNA clone. The full-length cDNA clone of HCV constructed in this study may originate from infectious virus clones.

Amino Acid Sequence↗
PubMed2003

The C-terminal end of R-Ras contains a focal adhesion targeting signal.

R-Ras promotes cell adhesion and activation of integrins through a process that is yet unknown. We show here that active R-Ras (38V) promotes the formation of focal adhesions and a spread cell shape. By contrast, the dominant-negative mutant of R-Ras (43N) reduces the number of focal adhesions, leading to the formation of refractile cells. In adherent cells wild-type R-Ras, activated (38V) R-Ras and endogeous R-Ras were preferentially targeted to focal adhesions, whereas the dominant-negative mutant (43N) of R-Ras was excluded from these structures. Activated mutants of H-Ras and K-Ras were not found in focal adhesions. We dissected R-Ras to find out the determinants that are important for the targeting process. The outermost region in the N-terminus of R-Ras, as well as the intact proline-rich sequence in the C-terminus of RRas that mediates binding to Nck, were not essential. Mutating the potential palmitoylation site (C213A) of RRas results in depalmitoylation and accumulation of R-Ras in the Golgi. Using H-Ras/R-Ras, R-Ras/H-Ras and RRas/K-Ras hybrid molecules we showed that the C-termini (175-218 amino acids) of R-Ras contains the signal for focal adhesions targeting. Exchanging the hypervariable region of H-Ras to R-Ras inhibited the targeting of R-Ras to focal adhesions, whereas H-Ras obtained the ability to localize to focal adhesions after receiving the hypervariable region of R-Ras. This indicates that R-Ras targeting is mediated both by the nucleotide binding status as well as through a specific region in the C-terminus of R-Ras. These results indicate that targeting and activation of R-Ras are linked processes in the formation of focal adhesions.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Genetic characterization of chicken anemia virus from commercial broiler chickens in Alabama.

Chicken anemia virus (CAV) isolates show extremely limited genetic variability worldwide. We determined the nucleotide sequence of an 823-nucleotide portion of the 2.3-kb CAV genome found in 10 liver and/or spleen specimens of Alabama 29-to-49-day-old commercial broiler chickens exhibiting lymphocyte depletion of the thymus submitted to the state diagnostic laboratory because of problems unrelated to anemia. We determined the nucleotide sequence directly from DNA isolated from tissues, without isolation of virus in culture. This procedure enabled us to characterize CAV that might not have replicated in culture and avoided the potential for changes during passage. Results confirmed the limited genetic variability of CAV. All sequences were identical in 93% of nucleotide positions. The sequences encoded only two distinct VP1 hypervariable regions, and both had been found previously in other CAV isolates. A novel amino acid, glutamine, was found at VP1 position 22 in half the sequences, replacing the histidine residue encoded by most previously characterized CAV genomes. We were able to distinguish among CAV genomes with different codons at VP1 amino acid 22 and different hypervariable regions by restriction endonuclease analysis of polymerase chain reaction products.

Alabama↗
PubMed2002

Characterisation of an Indonesian very virulent strain of infectious bursal disease virus.

An Indonesian very virulent (vv) strain of infectious bursal disease virus (IBDV), designated Tasik94, was characterised both in vivo and at the molecular level. Inoculation of Tasik94 into 5-week-old specific-pathogen-free (SPF) chickens resulted in 100% morbidity and 45% mortality. The complete nucleotide and predicted amino acid sequences of genomic segments A and B were determined. Across each of the three deduced open reading frames (ORFs), Tasik94 shared the greatest nucleotide homology to Dutch vv strain D6948. Phylogenetic analyses were performed using 15 full-length polyprotein sequences and a total of 105 VP2 hypervariable region sequences from geographically and pathogenically diverse strains. In each case, Tasik94 grouped closely with vv strains, particularly those from Europe. The deduced VP1, VP2, VP3, VP4 and VP5 protein sequences of Tasik94 were aligned with those from published strains and putative virulence determinants were identified in VP2, VP3 and VP4. Alignment of additional protein sequences across the VP2 hypervariable region confirmed that residues Ile[242], Ile[256] and Ile[294] were highly-conserved amongst vv strains, and may account for their enhanced virulence.

Amino Acid Sequence↗
PubMed1996

Coupling protein design and in vitro selection strategies: improving specificity and affinity of a designed beta-protein IL-6 antagonist.

The minibody is a designed small beta-protein conceived to enable the construction of large libraries of minimal discontinuous epitopes displayed on the surface of filamentous phage. The 61 residue molecule consists of three strands from each of the two beta-sheets of the variable domain of immunoglobulins packed face to face, along with the exposed H1 and H2 hypervariable regions. We have previously shown that from a minibody repertoire of more than 50 million molecules displayed on phage, we were able to select a minibody with micromolar affinity for human interleukin-6 that behaves as a selective cytokine antagonist. The minibody exposes a surface composed of two constrained loops, which provides the possibility of improving IL-6 binding and specificity by swapping the hypervariable regions, followed by further selection. We established experimental conditions for "stringent" selection such as monovalent phage display, competitive selection and epitope masking. Here, we show that by virtue of the optimization/selection process, we have isolated a minibody with improved antagonistic potency and greater specificity. Furthermore, using hIL-6 mutants carrying amino acid substitutions in distinct surface sites it was possible to carefully define the cytokine region that binds the minibody.

Amino Acid Sequence↗
PubMed1997

Insertions, duplications and substitutions in restricted gp90 regions of equine infectious anaemia virus during febrile episodes in an experimentally infected horse.

We have studied a horse which exhibited typical clinical signs of disease when experimentally infected with a non-adapted virulent strain of equine infectious anaemia virus (EIAV), designated V70. Five viruses (F1V, F2V, F3V, F4V and F5V) were recovered during periodic febrile episodes. Cross-neutralization tests revealed that all of these variants and the parental V70 were antigenically distinct. Sequencing of their full-length env gp90 genes and gp45 5' sequences revealed novel mutations at a limited number of nucleotide positions, consisting of insertions and duplications in the gp90 principal neutralizing domain (PND) in F1V, F3V and F5V. Parts or all of small units (6, 9 and 12 nucleotides) located just before the insertion site were used for the duplications. Furthermore, amino acid substitutions in the env PND and hypervariable region were also observed in all five viruses. These mutations may contribute to the generation of serial variants. Consequently, the full-length gp90 sequences showed close relationships between V70, F2V and F4V, and between F1V, F3V and F5V. In addition to the two domains (PND and hypervariable region), a comparison of these viruses with the reported env gp90 sequences revealed four additional variable domains, although these four domains were highly conserved among the five variants.

Amino Acid Sequence↗
PubMed1998

Comparison of serum and liver hepatitis C virus quasispecies in HCV-related hepatocellular carcinoma.

BACKGROUND/AIMS: The hepatitis C virus (HCV) genome consists of quasispecies populations of heterogeneous variants, especially in the hypervariable region. To assess the profiles of viral quasispecies in HCV-related hepatocellular carcinoma, we studied the viral population patterns in serum and liver tissues of 13 HCV-positive patients with hepatocellular carcinoma developed on cirrhotic and non-cirrhotic livers (5 and 8 cases, respectively). METHODS: HCV genome heterogeneity was analyzed by polymerase chain reaction-mediated single-strand conformation polymorphism analysis, which showed multiple DNA bands representing different hypervariable region sequences. RESULTS: The HCV populations were different between tumorous and nontumorous tissues in 3/5 hepatocellular carcinomas with cirrhosis and in 6/8 without cirrhosis. At least one or more than one common band was detected in both compartments in all but one case. No significant differences in the complexity of HCV quasispecies were found in hepatocellular carcinoma with or without underlying cirrhosis. Comparison of the HCV quasispecies profiles in serum and liver tissues showed a different distribution of HCV variants between these two compartments in 6/7 patients. In four cases, both common and compartmentalized sequences were detected, whereas in two cases, both without cirrhosis, the HCV population in serum was completely different from that found in the liver. CONCLUSIONS: These results suggest that the complexity of HCV populations is influenced by the presence of hepatocellular carcinoma rather than by the severity of the underlying chronic liver disease. The different quasispecies patterns found in serum and liver may reflect different biological properties of circulating and intrahepatic HCV particles or the existence of extrahepatic sites of replication.

Aged↗
PubMed1989

The cell attachment site on foot-and-mouth disease virus includes the amino acid sequence RGD (arginine-glycine-aspartic acid).

The amino acid sequence RGD (arginine-glycine-aspartic acid) is highly conserved in the VP1 protein of foot-and-mouth disease virus (FMDV), despite being situated in the immunodominant hypervariable region between amino acids 135 and 160. RGD-containing proteins are known to be important in promoting cell attachment in several different systems, and we report here that synthetic peptides containing this sequence are able to inhibit attachment of the virus to baby hamster kidney (BHK) cells. Inhibition was dose-dependent and could be reversed on removal of the peptide. A synthetic peptide corresponding to a portion of the same hypervariable region but not containing the RGD sequence did not inhibit virus attachment under the same conditions. Antibody against the RGD region of VP1 blocked attachment of the virus to BHK cells, and neutralizing monoclonal antibodies, which neutralize virus by preventing cell attachment, were blocked by RGD-containing peptides from binding virus in an ELISA test. Cleavage of the C-terminal region of virus VP1 in situ with proteolytic enzymes reduced cell attachment, and antiserum against a peptide corresponding to this region was also able to inhibit attachment of virus to BHK cells. These results indicate that the amino acid sequence RGD at positions 145 to 147 and amino acids from the C-terminal region of VP1 (positions 203 to 213) contribute to the cell attachment site on FMDV for BHK cells.

Amino Acid Sequence↗
PubMed2002

Possible association of non-binding of HSP70 to HLA-DRB1 peptide sequences and protection from rheumatoid arthritis.

The beta-chains of HLA-DR molecules associated with susceptibility to rheumatoid arthritis (RA) share a common amino acid sequence in their third hypervariable region at position 70-74. This shared epitope could either contribute to preferential binding of a given disease-associated peptide, be involved in disease-induction by molecular mimicry or, by binding to heat shock proteins, influence antigen presentation. It is known that the Escherichia coli M(r)70,000 heat shock protein DnaK can bind peptides from the shared epitope. Using a highly sensitive method, we show that peptides covering the third hypervariable region of associated, but also most of the non-associated HLA-DR alleles, bind to DnaK. Similar binding specificities could be found for the constitutively expressed mammalian M(r)70,000 heat shock protein Hsc73 and the inducible mammalian Hsp72. However, peptides containing the amino acid sequence DERAA, found in HLA-DR alleles and strongly associated with protection from RA, did not bind any HSP70. Thus, our results suggest a possible association of non-binding of HSP70 to HLA-DR molecules or its 70-74 fragments and protection from RA.

Alleles↗
PubMed1991

Discontinuous sequence change of human immunodeficiency virus (HIV) type 1 env sequences in plasma viral and lymphocyte-associated proviral populations in vivo: implications for models of HIV pathogenesis.

Sequence change in different hypervariable regions of the external membrane glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1) was studied. Viral RNA associated with cell-free virus particles circulating in plasma and proviral DNA present in HIV-infected peripheral blood mononuclear cells (PBMCs) were extracted from blood samples of two currently asymptomatic hemophiliac patients over a 5-year period. HIV sequences were amplified by polymerase chain reaction to allow analysis in the V3, V4, and V5 hypervariable regions of gp120. Rapid sequence change, consisting of regular replacements by a succession of distinct viral populations, was found in both plasma virus and PBMC provirus populations. Significant differences between the frequencies of sequence variants in DNA and RNA populations within the same sample were observed, indicating that at any one time point, the predominant plasma virus variants were antigenically distinct from viruses encoded by HIV DNA sequences in PBMCs. How these findings contribute to current models of HIV pathogenesis is discussed.

Acquired Immunodeficiency Syndrome↗