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PubMed2003

Characterization of T-cell non-Hodgkin's lymphoma and its association with Epstein-Barr virus in Pakistani patients.

This study analyzes the prevalence of T-cell non-Hodgkin's lymphoma (T-NHL) in a major referral center of Pakistan and its association with Epstein-Barr virus (EBV). Ninety-two cases of T-NHL were characterized on the basis of morphology, immunohistochemistry and genetic features. The prevalence of T-NHL was 22.2% of the total NHLs diagnosed during the eight years period (1992-1999). Polymerase chain reaction (PCR) technique was used to assess T-cell clonality in paraffin-embedded tissues of known T-NHL. Amplifiable DNA was isolated from all the cases, which were further studied for T-cell receptor (TcR)-beta, gamma, delta, and IgH chain gene rearrangements. Out of 92 cases 51 cases showed clonal product and 33 demonstrated polyclonal smear for beta, gamma, or delta chain genes, respectively, whereas 8 cases exhibited IgH chain gene rearrangement for FR2 region. This study demonstrated frequent presence of EBV in T-NHL (55.4%) by PCR, which were further tested for the localization of the virus by in situ hybridization (ISH). The extent of polymorphism in EBV genome was studied by single stranded conformation polymorphism (SSCP) technique for Bam HI E, K, N and Z regions. Hypervariability in Bam HI K, and N regions was noticeably higher compared to E or Z regions. In conclusion, our study demonstrated that the prevalence of T-NHL in Pakistan is slightly higher to that reported for Western communities. In addition, the frequency of EBV genome in T-NHL is intermediate as compared to other studies. No association was established between EBV variants differentiated on the basis of sequence heterogeneity in Bam HI K, N, E and Z regions with the manifestation of different subsets of T-NHL.

Adolescent↗
PubMed2000

Human immune response to streptococcal inhibitor of complement, a serotype M1 group A Streptococcus extracellular protein involved in epidemics.

Streptococcal inhibitor of complement (Sic) is a highly polymorphic extracellular protein made by serotype M1 group A Streptococcus strains that contributes to bacterial persistence in the mammalian upper respiratory tract. New variants of the Sic protein arise very rapidly by positive selection in human populations during M1 epidemics. The human antibody response to Sic was analyzed. Of 636 persons living in diverse localities, 43% had anti-Sic serum antibodies, but only 16.4% had anti-M1 protein serum antibody. Anti-Sic antibody was also present in nasal wash specimens in high frequency. Linear B cell epitope mapping showed that serum antibodies recognized epitopes located in structurally variable regions of Sic and the amino terminal hypervariable region of the M1 protein. Phage display analyses confirmed that the polymorphic regions of Sic are primary targets of host antibodies. These results support the hypothesis that selection of Sic variants occurs on mucosal surfaces by a mechanism that involves acquired host antibody.

Adolescent↗
PubMed2001

Identification of conformational neutralizing epitopes on the capsid protein of canine calicivirus.

Two neutralizing monoclonal antibodies (MAbs) against canine calicivirus (CaCV), which has a distinct antigenicity from feline calicivirus (FCV), were obtained. Both MAbs recognized conformational epitopes on the capsid protein of CaCV and were used to identify these epitopes. Neutralization-resistant variants of CaCV were selected in the presence of individual MAbs in a cell culture. Cross-neutralization tests using the variants indicated that the MAbs recognized functionally independent epitopes on the capsid protein. Recombinantly expressed ORF2 products (capsid precursors) of the variants showed no reactivity to the MAbs used for the selection, suggesting that the resistance was induced by a failing in binding of the MAbs to the variant capsid proteins. Several nucleotide changes resulting in amino acid substitutions in the capsid protein were found by sequence analysis. Reactivities of the MAbs to the revertant ORF2 products produced from each variant ORF2 by site-directed mutagenesis identified a single amino acid substitution in each variant capsid protein responsible for the failure of MAb binding. The amino acid residues related to forming the conformational neutralizing epitopes were located in regions equivalent to the 5' and 3' hypervariable regions of the FCV capsid protein, where antigenic sites were demonstrated in previous studies. The recombinant ORF2 products expressed in bacteria failed to induce neutralizing antibody, suggesting that neutralizing antibodies were only generated when properly folded capsid protein was used as an antigen. In CaCV, the conformational epitopes may play a more important role in neutralization than do linear epitopes.

3' Untranslated Regions↗
PubMed2007

Epidemic dynamics of two coexisting hepatitis C virus subtypes.

Hepatitis C virus (HCV) infection affects about 3% of the human population. Phylogenetic analyses have grouped its variants into six major genotypes, which have a star-like distribution and several minor subtypes. The most abundant genotype in Europe is the so-called genotype 1, with two prevalent subtypes, 1a and 1b. In order to explain the higher prevalence of subtype 1b over 1a, a large-scale sequence analysis (100 virus clones) has been carried out over 25 patients of both subtypes in two regions of the HCV genome: one comprising hypervariable region 1 and another including the interferon sensitivity-determining region. Neither polymorphism analysis nor molecular variance analysis (attending to intra- and intersubtype differences, age, sex and previous history of antiviral treatment) was able to show any particular difference between subtypes that might account for their different prevalence. Only the demographic history of the populations carrying both subtypes and analysis of molecular variance (AMOVA) for risk practice suggested that the route of transmission may be the most important factor to explain the observed difference.

Analysis of Variance↗
PubMed2004

Mutations in the putative HCV-E2 CD81 binding regions and correlation with cell surface CD81 expression.

The hepatitis C virus (HCV) envelope (E)2 protein interacts with the cellular receptor CD81 leading to modulation of B and T cell function. Recently, a higher binding affinity of subtype 1a in comparison with 1b derived E2 proteins for CD81 in vitro was described. The importance of mutations within the putative CD81 binding regions of different HCV geno-/subtypes in correlation with CD81 expression is unknown. In the present study, CD81 expression on blood lymphocytes of patients with chronic hepatitis C infected with different HCV geno-/subtypes were analysed by fluorescence activated cell sorter analyses. In addition, the putative CD81 binding regions on the E2 gene comprising the hypervariable region (HVR)2 were analysed by direct sequencing. CD81 expression on CD8(+) T-lymphocytes from patients infected with subtype 1a (n = 6) was significantly higher in comparison with subtype 1b (n = 12) and 3 (n = 5) infected patients before and during antiviral therapy (P = 0.006; P = 0.021, respectively). Sequencing of the putative CD81 binding regions in the E2 protein comprising the HVR2 (codon 474-495 and 522-552 according to the HCV-1a prototype HCV-H) showed a highly conserved motif within HVR2 for subtype 1a isolates and an overall low number of mutations within the putative CD81 binding regions, whereas numerous mutations were detected for subtype 1b isolates (12.0 vs 23.6%). HCV-3 isolates showed an intermediate number of mutations within the putative binding sites (19.2%; P = 0.022). In conclusion, the highly conserved sequence within HVR2 and putative CD81 binding sites of subtype 1a isolates previously associated with a high CD81 binding affinity in vitro is correlated with high CD81 expression on CD8(+) T-lymphocytes in vivo.

Adult↗
PubMed1987

Guinea pig MSEL-neurophysin. Sequence comparison of eight mammalian MSEL-neurophysins.

The amino acid sequence of guinea pig MSEL-neurophysin has been determined using tryptic peptides derived from the performic acid-oxidized protein and staphylococcal proteinase peptides obtained from the reduced-carboxamidomethylated neurophysin. Guinea pig MSEL-neurophysin consists of a 93-residue polypeptide chain that shows 12 substitutions and 2 deletions when compared to bovine MSEL-neurophysin. It displays the highest number of variations among known mammalian MSEL-neurophysins. These variations are mainly found in the C-terminal region (residues 88-93). Moreover guinea pig MSEL-neurophysin, like rat homologous protein, exhibits substitutions in positions 2, 5, 29 and 81 and lacks an arginine in the penultimate position. Comparison between eight mammalian MSEL-neurophysins reveals a highly conserved region (residues 1 to 88) and a hypervariable region (residues 89 to 93/95). On the other hand the eight species examined are endowed with arginine vasopressin except pig, which has a lysine vasopressin. In the vasopressin-MSEL-neurophysin precursor, the hormonal moiety and the MSEL region of neurophysin (residues 1-9) are encoded by a common exon in ox, rat and man; it can be concluded that this exon is evolutionarily conservative in contrast to the one encoding the C-terminal region of MSEL-neurophysin.

Amino Acid Sequence↗
PubMed1995

V3-independent determinants of macrophage tropism in a primary human immunodeficiency virus type 1 isolate.

Human immunodeficiency virus type 1 isolates differ in their ability to productively infect macrophages, and several groups have mapped the genetic basis for macrophage tropism to regions of env that include the third hypervariable region (V3 loop). We recently described a primary isolate (89.6) which is highly macrophage tropic and yet differs from other macrophage-tropic strains studied in that it is cytopathic in T cells. Genetic mapping of macrophage tropism determinants in this virus was done by using chimeras generated with the prototypic non-macrophage-tropic strain HXB2. Replacement of a 2.7-kb env-containing region of HXB with corresponding sequences from 89.6 conferred the macrophage-tropic phenotype, but insertion of the 89.6 V3 loop along with V4/V5 sequences did not. Conversely, placement of HXB sequences that included V3 into 89.6 did not impair this strain's ability to replicate in macrophages. Sequence analysis of V3 shows that 89.6 differs markedly from previously described macrophage-tropic consensus sequences and that it is more similar to highly charged non-macrophage-tropic strains. This suggests either that macrophage tropism is defined by structural determinants resulting from complex interactions among multiple env regions rather than V3 sequence-specific requirements or that there are multiple mechanisms by which different strains may establish productive macrophage infection. In addition, because the HXB V3 loop supports productive macrophage infection in the background of 89.6, phenotypic characterization of V3 sequences should be considered specific to the viral context in which they are placed.

Amino Acid Sequence↗
PubMed1994

Epidemiological studies on hepatitis C virus infection: detection, prevalence, exposure and prevention.

Highly sensitive and specific tests for HCV infection are now available for epidemiological studies. These include: (1) the second-generation ELISA or agglutination technique with supplemental testing; (2) serotyping of antibody; (3) detection of HCV-RNA by RT-nested double PCR with the 5' noncoding region for the highest sensitivity; (4) quantitation of HCV-RNA; (5) genotyping using specific primers in the core region, and (6) sequence analysis of E2/NS1 hypervariable region 1 to identify homology among HCV isolates. Transmission of HCV is commonly done by percutaneous routes, although the route in a significant proportion of sporadic infections is unknown. Sexual transmission and perinatal transmission appear to be relatively infrequent.

Africa↗
PubMed1994

Isolated VH4 heavy chain variable regions bind DNA characterization of a recombinant antibody heavy chain library derived from patient(s) with active SLE.

In many autoimmune diseases autoantibodies are intimately involved in disease manifestations. Molecular characterization of these autoantibodies should provide insights into the pathogenesis of these diseases, as well as suggest novel avenues for development of therapeutics. While some prior studies suggest that DNA binding may be a characteristic of individual heavy chain variable regions, the ability of these V regions to bind DNA in isolation has not been investigated. We have utilized a bacterial vector for cloning and expressing isolated antibody heavy chain variable regions. RNA was extracted from peripheral blood mononuclear cells of patients with active SLE, cDNA synthesized and heavy chain V regions amplified with VH specific oligonucleotide primers. The VH fragments were cloned into a bacterial expression plasmid including the pelB leader peptide to direct appropriate expression. Recombinant antibodies were screened for binding to 32P-labeled double-stranded plasmid DNA and later also characterized for binding to single-stranded DNA. Binding was confirmed by standard ELISA methodology. Sequence analysis of seven DNA binding VH fragments revealed that they utilized the VH gene family previously described to be associated with autoimmune responses, with a JH6 segment. On VH sequence analysis only one residue substitution in the consensus sequence is needed to form a VH4 germline gene. Potential contact residues with DNA were delineated by three-dimensional structure analysis. We concluded that the DNA binding characteristics of VH regions can be examined in the absence of light chain. DNA binding specificity appears to be a property of the germline VH4 gene. Analysis of such V regions can aid in the identification of hypervariable region contact residues important for DNA binding.

Amino Acid Sequence↗
PubMed1992

Analysis of DNA structure in the human insulin gene-linked polymorphic region in vivo.

An altered DNA structure exists within the hypervariable region located 360 bp upstream of the human insulin gene. The aim of the present study was to determine whether this structure exists in the insulin gene in vivo, and whether its presence is related to the expression of the insulin gene. However, since there were no clonal human beta-cell lines available for such studies, the human insulin gene was transfected into a rat insulinoma-derived beta-cell line and several human insulin-expressing clones were selected. One such cell line was treated in vivo with the DNA structural probe bromoacetaldehyde and the chromosomal DNA was extracted. Following digestion with TaqI and subsequent digestion with S1-nuclease to cleave at the bromoacetaldehyde-reactive sites, the DNA was subjected to agarose gel electrophoresis, and insulin gene fragments were detected by Southern blot analysis. Bromoacetaldehyde generated subfragments of 2500, 1700 and 800 bp in the human insulin gene isolated from the rat beta-cell line, while the human insulin gene in the non-expressing HeLa cell line was unreactive to bromoacetaldehyde. These results suggest that an altered structure might exist in the insulin gene-linked polymorphic region of the human insulin gene in vivo, and that this structure may play a role in the expression of the insulin gene.

Animals↗
PubMed1990

[Production of antibodies with the aid of genetic technology].

During the past decade we have witnessed a rapid development of methods for the production of monoclonal antibodies with predefined specificity. These antibodies are now regularly being used both for diagnostic and therapeutic purposes. The first antibodies thus produced were mainly from cell lines generated by fusion of a mouse B lymphocyte with a given specificity with a mouse myeloma line. Efforts have lately been made also to make cell lines producing human antibodies either by immortalizing human cells or by "humanizing" mouse antibodies. The latter has recently been accomplished by genetic engineering. In these experiments the entire variable region of a mouse antibody or the hypervariable regions only (complementarity determining regions) have been grafted onto human constant region genes thus creating chimeric antibodies. Fragments of antibodies with retained binding properties have also been generated and some of these have been effectively produced in bacteria, allowing large scale production. Bifunctional antibodies have also been created with success and have been applied in different systems. The ease with which antibodies with desired specificities can now be designed and produced will form the beginning of a new era in immunology with a potential that we can not as yet foresee.

Animals↗
PubMed1996

Differential binding avidities of human IgM for staphylococcal protein A derive from specific germ-line VH3 gene usage.

Human IgM that express the variable region genes of the VH3 family bind staphylococcal protein A (SPA). We previously reported that the SPA-binding IgM can be divided into two groups based on the differential binding avidities for solid-phase SPA. To study the molecular basis for these differences, we cloned B cells from human blood by EBV transformation. The nucleotide sequences of the expressed Ig heavy chain genes were determined on 20 B cell clones that produce SPA-binding IgM. The germ-line VH3 gene usage in IgM with high avidities for SPA were distinct from the germ-line VH3 genes used in IgM with low avidities for SPA. There was no correlation in the usage of D or JH genes or in the usage of light chains in IgM according to the SPA binding avidity. These results suggest that the differential binding avidities for SPA are at least partly due to specific germ-line VH3 gene usage. An investigation of direct binding of SPA to the synthetic peptides corresponding to the portions of the variable regions of SPA-binding and non-SPA-binding IgM showed that the peptides corresponding to the VH3 family specific framework region 3 sequences had significant SPA binding capacities, while the peptides corresponding to the other subdomains and those corresponding to framework region 3 of the reported VH3 sequences from non-SPA-binding IgM showed little or no binding. It is of interest that the Ig-framework region 3 subdomain corresponds to the fourth hypervariable region, which in the TCR-beta chain has been implicated as a critical site for T cell superantigen binding.

Adult↗
PubMed1987

The use of defined peptides in characterizing idiotypes.

The structural correlates of idiotypes have been sought in several antibody systems. The cumulative results suggest that the hypervariable regions (or complementarity-determining regions) of the heavy and light chains are the structural basis of idiotypes. However, in most cases, it is exceedingly difficult to associate a particular idiotypic determinant with a specific amino acid sequence. Recently, synthetic peptides were used to induce antibodies specific for predefined determinants in intact proteins. These findings led us and others to use synthetic peptides corresponding to the hypervariable regions/complementarity-determining regions to induce anti-idiotypes. These novel anti-idiotypic antibodies are easy to prepare, and are ideal reagents for structural and genetic studies of antibody responses.

Amino Acid Sequence↗
PubMed1998

Heteroplasmic substitutions in the mitochondrial DNA control region in mother and child samples.

The sequences of the two hypervariable regions of the mitochondrial DNA control region (HV1 and HV2) from close maternal relatives (mother-child pairs) were compared to determine the frequency of mutations between two generations. A total of 68 blood samples were sequenced only in HVI and 86 were analysed for HV1 and HV2. The intergenerational comparison led to the identification of six heteroplasmic point mutations affecting the children only. In one case a heteroplasmy of the maternal sequence was resolved to homoplasmy in the corresponding sequence of the child.

Adult↗
PubMed1991

DNA length polymorphism of the apo B 3' region: frequency distribution of the alleles in the German population.

A hypervariable region has been described 3' to the human apolipoprotein B (apo B) gene. Using the polymerase chain reaction amplification followed by agarose gel electrophoresis, at least 16 different alleles can be distinguished. In order to introduce this system into forensic DNA analysis detailed knowledge of the allele frequency is one of the most important prerequisites. For this reason we studied the allele distribution of 340 unrelated individuals originating predominantly from Southern Germany.

Alleles↗
PubMed1984

[Genetic bases of antibody diversity].

Repertoire of immunoglobulin paratopes is estimated as at least 10(7)-10(8) per individual. This repertoire is created by the variability of paratope coding VH- and VL-genes. Three events contribute to the necessary diversity of VH- and VL-genes: 1. Sets of germline DNA segments: VH, DH, JH and VL, JL containing genetic information for different parts of V-domains amino acid sequence. 2. Ontogenic rearrangements of these segments resulting in generation and expression of complete VH- and VL-genes. These rearrangements create the third hypervariable region diversity. 3. Succeeding hypermutational process leading to numerous substitutions of single amino acids along the V-domain localized essentially in hypervariable (complementary determining) regions. This process possesses the greatest possibilities for generation of somatic diversity of V-genes. Final VH- and VL-genes diversity reaches the necessary paratope repertoire, due to epigenetic mechanism of heavy and light chains combination in immunoglobulin molecules. Mechanisms of somatic generation of V-genes diversity are interpreted to spring up and be maintained in the course of evolution because of the fact that micro-parasites (viruses and bacteria) have much higher changeability rate than their hosts--highest vertebrates. Since future evolution of micro-parasites cannot be foreseen with the past events as a basis, natural selection of many thousands of germline V-genes fails to bring adaptation of the immune system to changeability of infection agents. Optimal evolution strategy of immunoglobulin gene complex of host species is expected to ensure developing somatic mechanisms. These mechanisms would generate de novo broad and random V-gene variability which is able, through structure diversity of corresponding paratopes, to foresee not only arbitrary micro-parasite, but also any arbitrary antigen not known in phylogenesis.

Animals↗
PubMed1986

The predicted structure of immunoglobulin D1.3 and its comparison with the crystal structure.

Predictions of the structures of the antigen-binding domains of an antibody, recorded before its experimental structure determination and tested subsequently, were based on comparative analysis of known antibody structures or on conformational energy calculations. The framework, the relative positions of the hypervariable regions, and the folds of four of the hypervariable loops were predicted correctly. This portion includes all residues in contact with the antigen, in this case hen egg white lysozyme, implying that the main chain conformation of the antibody combining site does not change upon ligation. The conformations of three residues in each of the other two hypervariable loops are different in the predicted models and the experimental structure.

Amino Acid Sequence↗
PubMed1998

Structural determinants in the sequences of immunoglobulin variable domain.

To determine the general relation between the sequence and structure of variable domains of immunoglobulins we have carried out an analysis of their atomic structures, some 5300 different expressed sequences and the human germline gene segments. Variable domains are formed by two beta-sheets, packed face to face, and the inter-strand turns. Comparison of the different known structures shows that they have a core of 76 residues which has the same main-chain conformation in all structures. This common core contains almost all of the beta-sheet structure and three inter-strand turns. The regions that differ in conformation are the three hypervariable regions, three other inter-strand turns and a few adjacent residues. The 5300 expressed sequences currently known for variable domains were examined to determine the residues that occur at the 76 sites. Ignoring site conservations that occur for functional reasons, there are eight sites that have the same residue in almost all sequences; 12 that have one of a small group of very similar residues, and 52 where the chemical character of the residues is strongly conserved but not their volume. The role of residues at each site in the core was determined from the examination of their accessible surface areas, contacts, packing and buried side-chain hydrogen bonds. The most strongly conserved sites form the "deep" structure of the domain at the centre of the interface between the beta-sheets. It includes eight invariant sites and 11 sites that have one of a set of very similar residues. Around the deep structure there are buried hydrophobic residues that, in different variable domains, can differ greatly in volume. These differences in volume are accommodated by conformational changes in turn regions that are outside the common core. On the surface nearly all residues not involved in function or turn conformations strongly conserve hydrophilic or neutral residues. The implications of these results for the general relations between the sequence and structure of proteins are discussed.

CD2 Antigens↗