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Biomedical subjects

M L Markert

Publications and source records attributed to M L Markert.

At least 19 recordsLinked to original sources
PubMed1997

Successful formation of a chimeric human thymus allograft following transplantation of cultured postnatal human thymus.

Transplantation of cultured postnatal human thymus was performed in a patient with complete DiGeorge syndrome. Biopsy of the graft 3 mo after implantation revealed normal CD1+ thymocytes in thymic cortical epithelial regions and CD1- thymocytes in thymic medullary epithelial regions, respectively. HLA analysis of graft thymocyte and thymic microenvironment components demonstrated that developing thymocytes and thymic macrophages were recipient derived, while thymic epithelial components were of donor origin. The patient, who initially had no T cells and had profoundly defective T cell function, developed normal T cell responses to mitogens and Ags, tolerance to donor in a mixed lymphocyte reaction, and normal Ab titers after tetanus toxoid and pneumovax immunization. Thus, transplantation of cultured postnatal human thymic tissue in humans can form functional chimeric thymic tissue, and may provide a strategy to reconstitute the peripheral T cell pool in select congenital and acquired immune deficiency syndromes.

Chimera↗
PubMed1997

Mutations in purine nucleoside phosphorylase deficiency.

Purine nucleoside phosphorylase deficiency is an inherited disease of purine metabolism characterized clinically as combined immunodeficiency. The molecular defects have been published for 4 different alleles in 3 patients. We report four new mutations including two amino acid substitutions, A174P and G190V, a single codon deletion, delta I129, and a point mutation in intron 3 which leads to aberrant splicing and creation of a premature stop codon in exon 4 (286-18G-->A). Of the previously reported mutations, E89K was found in one additional patient, and R234P was found in 3 unrelated patients, making R234P the most common mutation reported to date in this disease.

Alleles↗
PubMed1997

The human thymic microenvironment during organ culture.

Cultured human thymic tissue has been transplanted into many patients with T cell dysfunction; however, little is known about the effect of in vitro culture on thymic tissue. Human postnatal thymic organ cultures were established in vitro to study the growth potential of the thymic epithelium and the expression of intracellular and surface antigens with time in culture. Marked depletion of bone marrow-derived cells was observed within 3 weeks of initiation of organ cultures although some viable CD3+ cells could still be detected. Thymic epithelial cells in in vitro explants continued to express MHC class I and class II antigens as well as cytokeratins. Thymic epithelial cells within cultured thymic organ slices maintained their postnatal growth potential, in that cytokeratin-positive epithelial monolayers could be established in vitro from these thymic slices up to 12 weeks after initiation of organ culture. Thus, thymic explants remained viable in culture and could potentially be used to reconstitute immunity in T cell deficient patients.

Antibodies, Monoclonal↗
PubMed1997

Human severe combined immunodeficiency: genetic, phenotypic, and functional diversity in one hundred eight infants.

OBJECTIVE: To determine the relative frequencies of the different genetic forms of severe combined immunodeficiency (SCID) and whether there are distinctive characteristics of the particular genotypes. STUDY DESIGN: The demographic, genetic, and immunologic features of 108 infants with SCID who were treated consecutively at Duke University Medical Center were analyzed. RESULTS: Eighty-nine subjects were boys and 19 were girls; there were 84 white infants, 16 black infants, and 8 Hispanic infants. Forty-nine had X-linked SCID with mutations of common cytokine receptor gamma chain (gamma c), 16 had adenosine deaminase (ADA) deficiency, 8 had Janus kinase 3 (Jak3) deficiency, 21 had unknown autosomal recessive mutations, 1 had reticular dysgenesis, 1 had cartilage hair hypoplasia, and 12 (all boys) had SCID of undetermined type. Deficiency of ADA caused the most profound lymphopenia; gamma c or Jak3 deficiency resulted in the most B cells and fewest natural killer (NK) cells; NK cells and function were highest in autosomal recessive and unknown types of SCID. CONCLUSIONS: Different SCID genotypes are associated with distinctive lymphocyte characteristics. The presence of NK function in ADA-deficient, autosomal recessive, and unknown type SCIDs, and low NK function in a majority of gamma c and Jak3 SCIDs indicates that some molecular lesions affect T, B, and NK cells (gamma c and Jak3), others primarily T cells (ADA deficiency), and others just T and B cells.

Adenosine Deaminase↗
PubMed1997

Normalization of the peripheral blood T cell receptor V beta repertoire after cultured postnatal human thymic transplantation in DiGeorge syndrome.

Complete DiGeorge syndrome is an immunodeficiency disease characterized by thymic aplasia and the absence of functioning peripheral T cells. A patient with this syndrome was transplanted with cultured postnatal human thymic tissue. Within 5 weeks of transplantation, flow cytometry, T cell receptor V beta sequence analysis, and cell function studies showed the presence of oligoclonal populations of nonfunctional clonally expanded peripheral T cells that were derived from pretransplantation T cells present in the skin. However, at 3 months posttransplantation, a biopsy of the transplanted thymus showed normal intrathymic T cell maturation of host T cells with normal TCR V beta expression on thymocytes. By 9 months postransplantation, peripheral T cell function was restored and the TCR V beta repertoire became polyclonal, coincident with the appearance of normal T cell function. These data suggest that the transplanted thymus was responsible for the establishment of a new T cell repertoire via thymopoiesis in the chimeric thymic graft.

Base Sequence↗
PubMed1996

Antibodies against retroviral proteins and nuclear antigens in a subset of idiopathic CD4+ T lymphocytopenia patients.

Idiopathic CD4+ T lymphocytopenia (ICL) is an immunodeficiency syndrome characterized by severe depletion of CD4+ T lymphocytes, but in which human immunodeficiency virus cannot be detected. Peripheral blood mononuclear cells (BPMCs) from an ICL patient were cocultured with HUT78 T-lymphoblastoid cells, and an acute cytopathic effect and formation of multinucleated cells were observed. A human intracisternal A-type retroviral particle designated HIAP-II was detected in cells surviving the acute cytopathic effect. Eight of 13 ICL patients in a blinded screen of a serological panel provided by the National Centers for Disease Control and Prevention (CDC) had serum antibodies that specifically reacted with HIAP-II associated proteins by Western immunoblotting. None of 19 control sera in the panel that were unreactive with HIV Gag proteins produced a positive result on HIAP-II immunoblots. Comparable results were obtained in a blinded screen of a second CDC serological panel. Sera from 8 of 14 ICL patients in the second serological panel were positive for antinuclear autoantibodies (ANAs) commonly observed in patients with systemic autoimmune diseases. These results suggest the possible involvement of an A-type retrovirus or autoimmunity in development of ICL in a subset of patients.

Adult↗
PubMed1995

Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development.

Males with X-linked severe combined immunodeficiency (XSCID) have defects in the common cytokine receptor gamma chain (gamma c) gene that encodes a shared, essential component of the receptors of interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. The Janus family tyrosine kinase Jak3 is the only signaling molecule known to be associated with gamma c, so it was hypothesized that defects in Jak3 might cause an XSCID-like phenotype. A girl with immunological features indistinguishable from those of XSCID was therefore selected for analysis. An Epstein-Barr virus (EBV)-transformed cell line derived from her lymphocytes had normal gamma c expression but lacked Jak3 protein and had greatly diminished Jak3 messenger RNA. Sequencing revealed a different mutation on each allele: a single nucleotide insertion resulting in a frame shift and premature termination in the Jak3 JH4 domain and a nonsense mutation in the Jak3 JH2 domain. The lack of Jak3 expression correlated with impaired B cell signaling, as demonstrated by the inability of IL-4 to activate Stat6 in the EBV-transformed cell line from the patient. These observations indicate that the functions of gamma c are dependent on Jak3 and that Jak3 is essential for lymphoid development and signaling.

Amino Acid Sequence↗
PubMed1995

Correction of proliferative responses in purine nucleoside phosphorylase (PNP)-deficient T lymphocytes by retroviral-mediated PNP gene transfer and expression.

Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficiency is associated with a fatal T cell immunodeficiency in children, a candidate condition for gene therapy by introduction of functional PNP sequences into either T lymphocytes or more primitive progenitor cells in the bone marrow. To test the effectiveness of PNP gene transfer in T lymphocytes, a retroviral vector (LmPSN-2) was designed and constructed to express the murine PNP cDNA under transcriptional regulation of the Moloney murine leukemia virus long terminal repeat. LmPSN-2 was first used to mediate gene transfer and expression of electrophoretically distinct murine PNP in normal (PNP-positive) human PBL. Peripheral blood leukocytes were then obtained from a PNP deficient patient and characterized phenotypically. Despite their paucity and general mitogenic unresponsiveness, T lymphocytes from this patient were successfully grown in culture by using anti-CD3 with rIL-2 and then transduced with LmPSN-2. Elevated PNP enzyme activity was observed in the transduced cell population. Mitogenic and allogeneic responses, normally depressed in PNP-deficient patients' cells, were partially corrected in the transduced cell population relative to nontransduced cells. These results suggest the possibility of effecting improved immunologic function in PNP-deficient T lymphoid cells by retroviral-mediated gene transfer as therapy for PNP deficiency.

3T3 Cells↗
PubMed1994

Molecular basis of adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency is an autosomal recessive disorder resulting in immunodeficiency. Since the cDNA for ADA was cloned approximately 10 years ago, investigators have determined the molecular basis for disease in many patients with ADA deficiency. Mutations that have been identified include point mutations causing amino acid substitutions, premature stop codons, RNA splicing errors, and deletion mutations. Approximately one third of patients are homozygous for their mutation; in some of these cases the parents are known to be related. One mutation, Ala329-Val, is the most common, being present in 8 of the 21 ADA-deficient SCID patients whose mutations have been reported.

Adenosine Deaminase↗
PubMed1993

Haploidentical bone marrow stem cell transplantation in human severe combined immunodeficiency.

From May 1992 to March 1993, 50 infants with severe combined immunodeficiency (SCID) were given bone marrow transplants at Duke University Medical Center. None received chemotherapy for conditioning or for graft-versus-host disease (GVHD) prophylaxis. Forty-one received haploidentical parental marrow depleted of T cells by soybean lectin and sheep red blood cell resetting, and nine received HLA-identical marrow. Forty (80%) survived from 1 week to almost 11 years posttransplantation, including nine of nine (100%) HLA-identical marrow recipients and 31 of 41 haploidentical recipients. T-cell function was present within 2 weeks after transplantation of unfractionated HLA-identical marrow, but not until 3 to 4 months after T-cell-depleted haploidentical marrow stem cells. All 37 patients who are more than 4 months posttransplantation have good T-cell function, and all but one have 100% donor T cells. B-cell function developed slowly or not at all in some recipients of haploidentical marrow. Fourteen (four HLA-identical and 10 haploidentical recipients) have some donor B cells; 19 patients are receiving intravenous immune globulin (IVIG) therapy.

B-Lymphocytes↗
PubMed1992

Exon skipping in purine nucleoside phosphorylase mRNA processing leading to severe immunodeficiency.

We report a defect in splicing of precursor messenger RNA (pre-mRNA) resulting from a naturally occurring mutation of the gene encoding purine nucleoside phosphorylase (PNP) in a patient with PNP-deficient severe combined immunodeficiency. This defects results from a G to T transversion at the terminal nucleotide of exon 2 within the 5' splice site of intron 2 and causes skipping of exon 2 during processing of PNP pre-mRNA. Translation of the misspliced mRNA results in a reading frameshift at the exon 1-exon 3 junction. The predicted polypeptide encoded by the aberrant mRNA is severely truncated, terminating at 31 amino acids. Only 4 residues at the NH2 terminus of the polypeptide correspond to PNP amino acids. Otherwise the translation product of the misspliced mRNA differs completely from PNP in amino acid sequence and has no PNP activity. The finding of exon skipping in PNP is the first report of a splicing defect resulting in PNP-deficient severe combined immunodeficiency. Analysis of the genomic context of the G-1 to T mutation of the 5' splice site lends support for the exon definition model of pre-mRNA splicing and contributes to the understanding of splice site selection.

Amino Acid Sequence↗
PubMed1992

Molecular analysis of mutations in a patient with purine nucleoside phosphorylase deficiency.

Purine nucleoside phosphorylase (PNP) deficiency is an inherited autosomal recessive disorder resulting in severe combined immunodeficiency. The purpose of this study was to determine the molecular defects responsible for PNP deficiency in one such patient. The patient's PNP cDNA was amplified by PCR and sequenced. Point mutations leading to amino acid substitutions were found in both alleles. One point mutation led to a Ser-to-Gly substitution at amino acid 51 and was common to both alleles. In addition, an Asp-to-Gly substitution at amino acid 128 and an Arg-to-Pro substitution at amino acid 234 were found in the maternal and paternal alleles, respectively. In order to prove that these mutations were responsible for the disease state, each of the three mutations was constructed separately by site-directed mutagenesis of the normal PNP cDNA, and each was transiently expressed in COS cells. Lysates from cells transfected with the allele carrying the substitution at amino acid 51 retained both function and immunoreactivity. Lysates from cells transfected with PNP alleles carrying a substitution at either amino acid 128 or amino acid 234 contained immunoreactive material but had no detectable human PNP activity. In summary, molecular analysis of this patient identified point mutations within the PNP gene which are responsible for the enzyme deficiency.

Amino Acid Sequence↗
PubMed1991

Purine nucleoside phosphorylase deficiency.

Purine nucleoside phosphorylase (PNP) deficiency is a rare inherited disease accounting for approximately 4% of patients with severe combined immunodeficiency. Thirty-three patients have been reported. PNP-deficient patients suffer from recurrent infections, usually beginning in the first year of life. Two thirds of patients have evidence of neurologic disorders. Findings range from spasticity to developmental delay, to mental retardation. One third of patients develop autoimmune disease. The most common manifestation of this is autoimmune hemolytic anemia. Idiopathic thrombocytopenic purpura and systemic lupus erythematosis have also been reported. Patients usually present with infections but approximately one fourth have come to medical care initially for neurological problems. In PNP deficiency, T- and B-cell immunity are affected. T-cell function may be profoundly deficient, may be normal at birth and then decrease with time, or may fluctuate repeatedly between low and normal. B-cell function can be normal but is deficient in approximately one third of patients. PNP protein is a trimer of approximately 90,000 daltons. It is found in most tissues of the body but is at highest levels in lymphoid tissues. This tissue distribution explains why the lymphoid system is predominantly affected in PNP deficiency. Many mechanisms have been proposed to explain the metabolic toxicity in PNP deficiency. The elevated dGTP found in PNP deficiency is thought to inhibit ribonucleotide reductase and, thus, impede cell division. Depressed GTP levels may correlate with neurologic dysfunction. The gene for PNP has been cloned; it is located on the long arm of chromosome 14. Studies of a mutant PNP gene isolated from one patient showed that a point mutation resulting in an amino acid substitution was responsible for PNP deficiency. PNP deficiency has a grave prognosis. No patient has reached the third decade of life. Twenty-nine of the 33 reported patients have died from their disease. Prenatal diagnosis is currently available. Many different therapies have been utilized for PNP deficiency including bone marrow transplantation, red cell transfusions, and supplementation of the diet with purines and pyrimidines. None of these therapies has been consistently successful. In light of the poor prognosis for PNP deficiency, bone marrow transplantation should be considered for all patients. In the future, improved forms of therapy such as gene therapy may become available.

B-Lymphocytes↗
PubMed1989

A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution.

In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation in exon 11. This resulted in the amino acid substitution of a valine for an alanine at position 329 of the ADA protein. Sequence analysis revealed that this mutation created a new BalI restriction site. Using Southern analyses, we were able to directly screen individuals to determine the frequency of this mutation. By combining data on eight families followed at our institution with data on five other families reported in the literature, we established that five of 13 patients (seven of 22 alleles) with known or suspected point mutations have this defect. This mutation was found to be associated with three different ADA haplotypes. This argues against a founder effect and suggests that the mutation is very old. In summary, a conservative amino acid substitution is found in a high proportion of patients with ADA deficiency; this can easily be detected by Southern analysis.

Adenosine Deaminase↗
PubMed1988

Adenosine deaminase (ADA) deficiency due to deletion of the ADA gene promoter and first exon by homologous recombination between two Alu elements.

In 15-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The goal of this study was to determine the precise molecular defect in a patient with ADA-deficient SCID whom we previously have shown to have a total absence of ADA mRNA and a structural alteration of the ADA gene. By detailed Southern analysis, we now have determined that the structural alteration is a deletion of approximately 3.3 kb, which included exon 1 and the promoter region of the ADA gene. DNA sequence analysis demonstrates that the deletion created a novel, complete Alu repeat by homologous recombination between two existing Alu repeats that flanked the deletion. The 26-bp recombination joint in the Alu sequence includes the 10-bp "B" sequence homologous to the RNA polymerase III promoter. This is the first example of homologous recombination involving the B sequence in Alu repeats. Similar recombination events have been identified involving Alu repeats in which the recombination joint was located between the A and B sequences of the polymerase III split promoter. The nonrandom location of these events suggests that these segments may be hot spots for recombination.

Adenosine Deaminase↗