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Biomedical subjects

C I Smith

Publications and source records attributed to C I Smith.

At least 91 records · Page 5Linked to original sources
PubMed1996

Reduction in serum levels of antimitochondrial (M2) antibodies following immunoglobulin therapy in severe combined immunodeficient (SCID) mice reconstituted with lymphocytes from patients with primary biliary cirrhosis (PBC).

The effect of gammaglobulin treatment on autoantibody production was investigated in SCID mice reconstituted with human peripheral blood mononuclear cells (PBMC) obtained from patients with PBC. All reconstituted mice displayed the presence of human antimitochondrial antibodies (alpha M2Ab) of both IgG and IgM types before treatment with human immunoglobulin. Two weeks after i.p. injection of 20 x 10(6) PBMC into SCID mice, i.p. treatment with various preparations of human immunoglobulin was initiated. In control animals treated with saline, serum levels of human alpha M2Ab of the IgG type increased with time, peaking around 4 weeks after reconstitution. In contrast, human IgG autoantibodies rapidly decreased in all animals treated with human IgG. Treatment with a human IgM preparation had no effect on serum levels of alpha M2Ab of the IgG type. The results may suggest that the pronounced reduction of specific IgG autoantibodies was due to an increased catabolism of human IgG, including the autoantibodies, in the gammaglobulin-treated mice. Although the production of human alpha M2Ab in reconstituted mice could be easily shown, PBC-specific liver lesions or bile duct destruction were not observed, irrespective of treatment protocol.

Animals↗
PubMed1996

Studies of the molecular basis of IgA production, subclass regulation and class-switch recombination in IgA nephropathy patients.

IgA nephropathy (IgAN), the most common form of glomerulonephritis, is characterized by normal to elevated levels of serum IgA. In order to understand the molecular mechanism(s) involved in the production of IgA in IgAN, peripheral blood mononuclear cells (PBMC) from these patients were analysed in this study. IL-10, transforming growth factor-beta 1 (TGF-beta 1) and CD40 have previously been shown to be involved in IgA production. We show here that CD40L expression was increased three-fold in these patients. However, expression of TGF-beta 1 in serum levels was comparable to controls. In vitro stimulation of PBMC with a polyclonal activator resulted in a three-fold increase in synthesis of both IgA subclasses, with a preference for IgA1 RNA. In situ hybridization studies also showed a three-fold increase in the numbers of IgA1- and IgA2-producing cells, but the subclass distribution was similar to the controls. Furthermore, using the nested primer polymerase chain reaction (PCR) for amplifying switch (S mu/S alpha) breakpoints we could demonstrate that in unstimulated PBMC the switch frequency did not differ from that of control donors. Sequence analysis of the amplified switch breakpoints and the I alpha regulatory region from patients showed no structural abnormality. Although we have previously demonstrated a correlation to in vivo germ-line RNA expression and class switching, no I alpha transcripts were detected in unstimulated PBMC from these patients. However, stimulation of PBMC with TGF-beta 1 resulted in I alpha production. Taken together, results from in vivo and in vitro studies suggest that increased cytokine production and hyperresponsiveness to polyclonal stimulation may play an important role in the increased synthesis of IgA. The preference for IgA1 is due to increased production of IgA1 per cell, and the absence of I alpha RNA indicates that additional defect(s) in immune regulation may play an important role in the pathogenesis of IgAN.

Adolescent↗
PubMed1996

X-linked agammaglobulinemia. A clinical and molecular analysis.

X-linked agammaglobulinemia (XLA), characterized by a profound deficiency of B lymphocytes due to an arrest in B lymphocyte development, is caused by mutations in the gene encoding Btk (Bruton tyrosine kinase). The BTK gene has been cloned and the genomic organization determined. BTK codes for 19 exons and is expressed in all hematopoietic cell lineages but is selectively down-regulated in T lymphocytes and plasma cells. The different Btk domains include PH, TH, SH3, SH2, and the kinase (SH1) domains. Btk, a cytoplasmic protein tyrosine kinase, is involved in cell signaling, although the precise pathway remains elusive. Mutation analysis has been performed in 236 families representing 282 patients. Mutations are scattered throughout the gene and consist of missense, nonsense, and splice site mutations as well as deletions and insertions. The major consequence of nonfunctional Btk appears to be a delay or block of the development of pro-B cells to pre-B cells and then to mature lymphocytes. Because IgG is actively transported across the placenta, affected newborns have normal levels of serum IgG at birth followed by gradually decreasing IgG levels and development of hypogammaglobulinemia and increased susceptibility to infections. Bacterial infections are the most common clinical manifestation. Resistance to viral infection is intact, except for an unusual susceptibility to infections with enteroviruses that may result in vaccine-related paralytic poliomyelitis or a dermatomyositis-meningoencephalitis syndrome. The diagnosis of XLA is based on the presence of lymphoid hypoplasia, markedly reduced serum levels of all 3 major classes of immunoglobulins, failure to make antibody to antigenic stimulation, and almost complete absence of B lymphocytes in the peripheral blood. Carrier detection and prenatal diagnosis are possible. The prophylactic infusion of high-dose intravenous immunoglobulin (IVIG) and the use of antibiotics have markedly improved the long-term prognosis of patients with XLA.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1996

Structural aspects of signal transduction in B-cells.

Signal transduction in hematopoietic cells is a highly specific process. The stimulation of B cell receptor following antigen binding triggers, as a first step, phosphorylation of the cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). Src family tyrosine kinases Blk, Fyn, Lck, and Lyn as well as spleen kinase, Syk, are activated to transmit the signal further. In this review the signaling events are discussed in structural terms. The factors related to B cell maturation and their targeted mutations are reviewed. During the last 2 years plenty of structural information concerning signaling molecules in B cells has been obtained by using X-ray crystallography, NMR spectroscopy, molecular modeling, and mutational analysis. The molecules discussed include Src family kinases, Syk, Grb2 adaptor protein, and Tec family kinases Bmx and Btk. The structure, function, and interactions of these signaling compounds are described in atomic detail.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Structural basis for pleckstrin homology domain mutations in X-linked agammaglobulinemia.

Deficiencies in a tyrosine kinase, designated Btk, cause X-linked agammaglobulinemia (XLA) in man, a hereditary defect of B-cell differentiation. Mutations in the newly found PH domain located at the N-terminus of Btk have been shown to be the direct cause of XLA, and here two new mutations, T33P and V64F, are presented. Btk is thus far the only protein in which mutations of the PH domain have been found to cause a disease. The three-dimensional structure of the Btk PH domain was modeled on the basis of the dynamin PH structure. Despite a relatively low sequence similarity the Btk PH domain seems to have the same two beta-sheet structure observed in the known structures. The model was used to interpret the structural basis for disease in five independent point mutations and in an insertion in patients with XLA. The mutated residues F25, V64, and V113, and possibly residue(s) around Q103, could form a binding site, since these amino acids are located close to each other on the surface of the molecule.

Agammaglobulinemia↗
PubMed1995

Molecular cloning, characterization, and chromosomal localization of a human lymphoid tyrosine kinase related to murine Blk.

Triggering of Ag receptors on lymphocytes induces rapid phosphorylation of several receptor-associated protein tyrosine kinases (PTKs), implicating their role in controlling cellular growth and differentiation. In this study, we report the cloning of a human cDNA encoding a nonreceptor PTK with a calculated M(r) of about 58 kDa. The kinase has an overall amino acid identity of approximately 87% with the murine Blk. However, in the unique domain there is only 58% homology and an insertion of six amino acids in the N-terminal region. The nature of this insertion suggests a functional role in membrane attachment. Northern blot analysis showed expression in all stages of B cell development and in T cell lines. The message was not observed in the nonlymphoid tissues examined. In contrast, expression of murine blk in plasma cells and T lymphocytes has not been reported. Importantly, transcripts were seen in human embryonic liver as early as 7.5 wk of gestation before the rearrangement of Ig H chain locus. Furthermore, transcripts were detected in human thymocytes and not in mature T cells. Southern blot analysis revealed polymorphism of this gene in a Caucasian population but not in a Gambian population, indicating a recent origin of this polymorphism. The gene was localized to chromosome 8p22-23. The homology at the protein level suggests that this kinase may be the human homologue of murine Blk. Expression of BLK in immature T cells suggests that BLK may play an important role in thymopoiesis.

Amino Acid Sequence↗
PubMed1995

Family and linkage study of selective IgA deficiency and common variable immunodeficiency.

Screening of close relatives of Swedish patients with selective immunoglobulin A deficiency (IgAD) and common variable immunodeficiency (CVID) for serum immunoglobulin levels has identified the positive family history of IgAD/CVID as the most significant risk factor for developing the disease. The relative risk for siblings of patients with IgAD was estimated to be approximately 50. In 12 of 34 Swedish multiplex families identified in the study, both IgAD and CVID occurred, usually CVID in the parental generation and IgAD in the subsequent generation. This proportion was much higher than expected by chance and strongly suggests that the two clinically discernible disorders represent an allelic condition, reflecting a variable expressivity of a common defect. In 27 multiplex families the disorders segregated as an autosomal dominant trait, affecting at least two generations. A high relative risk for siblings, permanent phenotype, low number of phenocopies, and common population prevalence, which makes it possible to obtain a sufficient sample size, make these immunoglobin deficiencies amenable to genetic linkage analysis. In a pilot multicenter linkage study involving 16 multiplex families with dominant transmission of IgAD/CVID, we have attempted to confirm previously reported genetic linkage of the disease susceptibility to the major histocompatibility complex (MHC) region. Using both parametric and nonparametric linkage analyses with a set of microsatellite markers at and flanking the MHC region, no evidence for linkage was found. In accordance with these results, no evidence for linkage to the MHC region was obtained by analyzing previously published segregation data at the MHC region in multiplex families with IgAD/CVID in more than one generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Common Variable Immunodeficiency↗
PubMed1995

Structural basis for X-linked agammaglobulinemia (XLA): mutations at interacting Btk residues R562, W563, and A582.

It has been suggested that tryptophan 563 is sandwiched between residues R562 and A582 in Bruton's agammaglobulinemia tyrosine kinase (Btk). Mutations of the surrounding residues have been shown to cause X-linked agammaglobulinemia. Substitutions R562P and A582V were noticed to have impaired kinase activity. However, based on Western blot analysis, the mutant proteins were expressed at normal levels. Molecular modeling of the kinase domain has previously indicated that these residues presumably govern the position of the W563 side chain, which is thought to interact with the catalytic loop. W563 is inside the molecule and too far away from the catalytic center to interact directly with the substrate or cofactors. To prove these model-based conclusions, a conservative substitution with phenylalanine for W563 was made, and the resultant mutant lacked kinase activity. These results confirm our previous assumption that the side chain of W563, invariant in protein tyrosine kinases, is crucial for Btk kinase activity. Mutations in the surrounding residues seem to inactivate Btk by affecting the location of W563.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1995

The influence of gene deletions and duplications within the IGHC locus on serum immunoglobulin subclass levels.

To study the influence of heterozygous gene deletions and duplications in the human IGHC region on serum concentrations of antibodies, we have analyzed the IgG subclass levels in a large number of individuals from three different populations. Our results show that heterozygous gene deletions of the C gamma 1, C gamma 2, or C gamma 4 genes are all associated with a lower mean serum level of the affected subclass whereas duplication of the C gamma 2 or C gamma 4 genes result in either no change (IgG2) or a slightly elevated (IgG4) level of serum immunoglobulins.

Gene Deletion↗
PubMed1995

DNA-based mutation analysis of Bruton's tyrosine kinase gene in patients with X-linked agammaglobulinaemia.

The identification of the BTK (Bruton's tyrosine kinase) gene defective in human immunoglobulin deficiency X-linked agammaglobulinaemia (XLA) and characterisation of BTK exon-intron boundaries has now allowed the analysis of mutations and polymorphisms at the level of genomic DNA. Using Southern blot analysis and the polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) assay, amplifying all 19 exons and the putative promoter region with a single annealling temperature, mutations have been identified in 19 out of 24 unrelated patients diagnosed as having XLA. Apart from a large deletion involving exon 19, nine missense (F25S, R288W, 1370M, M509V, R525P, N526K, R562W, A582V and G594R), two nonsense (E277X and R525X), five frameshift and two splice site mutations have been found affecting most coding exons and all major enzyme domains. No mutations or polymorphisms were detected in the putative promoter region. A single nucleotide deletion located in the last exon, resulting in a truncation of the eight C-terminal residues of Btk and a typical XLA phenotype, indicates structural and/or functional importance of Btk helix I in the catalytic domain. Although allelic heterogeneity at the BTK locus may partly explain clinical variability in families with XLA, compensatory and redundant mechanisms involved in B-cell development must play a role in the phenotypic diversity of the disease.

Adolescent↗
PubMed1995

Identification of Btk mutations in 20 unrelated patients with X-linked agammaglobulinaemia (XLA).

X-linked agammaglobulinaemia (XLA) is an inherited immunodeficiency resulting from mutations in the gene for a cytoplasmic protein tyrosine kinase (Btk). We have utilised reverse-transcription-based PCR in combination with the chemical cleavage and mismatch technique (CCM) to screen for Btk mutations in 42 unrelated patients having classical XLA or 'leaky' XLA-like phenotypes. A variety of mutations, including point mutations, large deletions and splicing defects were detected using this strategy. In total, 20 mutations were found in these patients. All the mutations were different with the exception of three unrelated patients who all showed the same Arg-->His amino acid substitution (R641H) at a highly-conserved residue in the kinase domain. We have also used structural modelling of the Btk kinase domain to predict how two different amino acid substitution mutations at highly-conserved residues are likely to affect the Btk kinase activity.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1995

Retroviral-mediated gene transfer into human bone marrow stromal cells: studies of efficiency and in vivo survival in SCID mice.

Retroviral-mediated gene transfer into bone marrow cells is extensively used in gene therapy protocols. Cytokines are needed for stimulation, to achieve a high rate of gene transfer. However, the stromal cell compartment of bone marrow is characterized by rapid proliferation even without cytokines. In this study, human bone marrow stromal cells were isolated and subsequently infected with recombinant retrovirus in a cell-free supernatant. The LN retroviral vector used in this study carries the bacterial neomycin phosphotransferase gene (neoR). Transduction efficiency was significantly enhanced by repeated cycles of infection, with a maximum of 91% transduced cells by four rounds of infection. Presence of the neoR-gene was detected by PCR from all stromal cells selected by G418. After culture in vitro for 3 months, cells were still positive for PCR amplification of the neoR-gene. Transduced stromal cells were also injected into SCID mice to study their homing and survival ability in vivo.

Animals↗
PubMed1995

Prevalence, genetics and clinical presentation of chronic granulomatous disease in Sweden.

To estimate the prevalence of chronic granulomatous disease (CGD) in Sweden, an inquiry asking for known and possible CGD cases was mailed to paediatric, internal medicine and infectious disease departments all over Sweden. The detected patients were characterized as to genetics and the clinical presentation. Twenty-one patients (belonging to 16 different families) were found, corresponding to a prevalence of approximately 1/450,000 individuals. The patients with X-linked disease, lacking a functional gp91phox protein (n = 12), comprised 57% and 43% of the patients had an autosomal recessive (AR) disease lacking p47phox (n = 7) or p67phox (n = 1), respectively. All unrelated patients with X-linked disease displayed different gene abnormalities such as point mutations predicting nonsense (n = 3), missense (n = 1) or splice site mutations (n = 2), but also a total deletion and a unique 40 base pair duplicature insertion. The patients with p47phox-deficiency showed a GT deletion at a GTGT tandem repeat, and the p67phox-deficient patient displayed a heterozygous in-frame deletion of AAG combined with a large deletion in the other allele. Three patients died during the study period, two from pseudomonas cepacia infections. Patients with X-linked disease had more frequent infections (mean of 1.7 per year), than the patients with AR inheritance (0.5 infections per year). The most common infections were dermal abscesses (n = 111), followed by lymphadenitis (n = 82) and pneumonias (n = 73). Inflammatory bowel disease-like symptoms, mimicking Crohn's disease of the colon, was seen in three CGD patients.

Adolescent↗