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Biomedical subjects

K Weinberg

Publications and source records attributed to K Weinberg.

At least 37 records · Page 2Linked to original sources
PubMed1997

Differential activation of the tyrosine kinases ZAP-70 and Syk after Fc gamma RI stimulation.

Engagement of the high-affinity IgG Fc receptor (Fc gamma RI) activates a signal transduction pathway involving tyrosine phosphorylation of associated kinases. We compared the activation of the related protein tyrosine kinases (PTKs), Syk and ZAP-70, in Fc gamma RI-mediated signaling. Cross-linking of the Fc gamma RI multimeric receptor in monocytic cells results in tyrosine phosphorylation of the Fc epsilon RI gamma subunit and association of Syk with this complex. We stably introduced ZAP-70 via a retroviral vector into two monocytic cell lines, U937 and THP-1, which normally do not express ZAP-70. Neither Syk nor MAP kinase activation was affected by the presence of ZAP-70. Although transduced ZAP-70 had in vitro kinase activity and associated with Fc epsilon RI gamma after receptor aggregation, it was not tyrosine phosphorylated. In contrast, both ZAP-70 and Syk were phosphorylated in a T-cell line in which their respective levels of expression were similar to those detected in U937/ZAP-70 cells. Therefore, these results suggest that requirements for Syk and ZAP-70 phosphorylation are distinct in a monocytic cell context.

Cell Line↗
PubMed1996

Reconstitution of T cell receptor signaling in ZAP-70-deficient cells by retroviral transduction of the ZAP-70 gene.

A variant of severe combined immunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4+ cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I-transformed CD4+ T cell lines were established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropriately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SCID may be corrected by gene therapy.

CD4-Positive T-Lymphocytes↗
PubMed1996

Enhancement of thymopoiesis after bone marrow transplant by in vivo interleukin-7.

Bone marrow transplantation (BMT) is followed by a period of profound immune deficiency, during which new T lymphocytes are generated from either stem cells or immature thymic progenitors. Interleukin-7 (IL-7) induces proliferation and differentiation of immature thymocytes. We examined whether the in vivo administration of IL-7 to mice receiving BMT would alter thymic reconstitution. Lethally irradiated C57BL/6 mice received syngeneic BMT, followed by either IL-7 or placebo from days 5 to 18 post-BMT. At day 28, BMT recipients that had not received IL-7 had profound thymic hypoplasia (< 5% of normal), with relative increases in the numbers of immature thymocytes, decreased numbers of mature peripheral (splenic) T lymphocytes, and severely impaired T- and B-cell function. In contrast, transplanted mice treated with IL-7 had normalization of thymic cellularity, with normal proportions of thymic subsets and T-cell receptor beta variable gene (TCRV beta) usage, normal numbers of peripheral CD4+ T lymphocytes, and improved antigen-specific T- and B-cell function. In the BMT-IL-7 mice, there was an eightfold increase in the number of immature CD3-CD4-CD8- thymocytes in G2-M of the cell cycle, indicating that restoration of thymic cellularity was due to enhanced proliferation of immature thymic progenitors. Similar effects following IL-7 administration were also observed when donor bone marrow was depleted of mature T lymphocytes, indicating that IL-7 administration affected immature hematopoietic progenitors. IL-7 promotes thymic reconstitution after BMT, and may be useful in preventing post-BMT immune deficiency.

Animals↗
PubMed1996

Correction of interleukin-2 receptor function in X-SCID lymphoblastoid cells by retrovirally mediated transfer of the gamma-c gene.

X-SCID, the most common form of human SCID, is due to mutations in the common gamma chain gene (gamma-c) that encodes an essential component of the cytokine receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. Activation of the Janus family tyrosine kinases Jak1 and Jak3 is necessary for appropriate signalling through the IL-2 receptor (IL-2R). Neither Jak1 nor Jak3 was phosphorylated after IL-2 stimulation of an Epstein-Barr virus-transformed cell line (LCL) from an X-SCID patient with a gamma-c null mutation. However, we now show that appropriate IL-2R function can be restored in an X-SCID LCL by transduction of a wild-type gamma-c gene. A retroviral vector, G1gamma-cSvNa, was constructed and produced in the PG13 packaging line. Transduced X-SCID LCL expressed the G1gamma-cSvNa transcript. IL-2 stimulation of the transduced cell line resulted in appropriate tyrosine phosphorylation of both Jak1 and Jak3. Thus, retroviral-mediated transduction of normal gamma-c can reconstitute downstream signalling through the IL-2R in X-SCID cell lines, suggesting that gene therapy may be a treatment for this disease.

B-Lymphocytes↗
PubMed1995

Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not on thymocytes.

Recently, a severe combined immunodeficiency syndrome with a deficiency of CD8+ peripheral T cells and a TCR signal transduction defect in peripheral CD4+ T cells was associated with mutations in ZAP-70. Since TCR signaling is required in developmental decisions resulting in mature CD4 (and CD8) T cells, the presence of peripheral CD4+ T cells expressing TCRs incapable of signaling in these patients is paradoxical. Here, we show that the TCRs on thymocytes, but not peripheral T cells, from a ZAP-70-deficient patient are capable of signaling. Moreover, the TCR on a thymocyte line derived from this patient can signal, and the homologous kinase Syk is present at high levels and is tyrosine phosphorylated after TCR stimulation. Thus, Syk may compensate for the loss of ZAP-70 and account for the thymic selection of at least a subset of T cells (CD4+) in ZAP-70-deficient patients.

Cell Line↗
PubMed1995

Dysfunctional cytokine production by host-reactive T-cell clones isolated from a chimeric severe combined immunodeficiency patient transplanted with haploidentical bone marrow.

We have investigated the mechanism of tolerance in a patient with severe combined immunodeficiency (SCID) transplanted with HLA-haploidentical, T cell-depleted bone marrow cells obtained from the mother. At 4 years after transplantation, T cells, natural killer (NK) cells, and a small percentage (2%) of B cells were found to be of donor origin, whereas monocytes and the majority of B cells remained of host origin. In primary mixed lymphocyte cultures (MLC), the engrafted T cells of the donor did not proliferate in response to the host cells, whereas untransplanted donor T cells showed good proliferative responses. However, CD4+ and CD8+ T-cell clones of donor origin with specificity for class II and class I HLA determinants of the host were isolated. CD8+, host-reactive T-cell clones displayed normal cytotoxic activity after stimulation with the host cells, but proliferative responses of CD4+, host-reactive T-cell clones were considerably reduced. In addition, both CD8+ and CD4+, host-reactive T-cell clones produced very low to undetectable levels of interleukin-2 (IL-2), IL-4, IL-5, IL-10, interferon-gamma, and granulocyte-macrophage colony-stimulating factor after specific antigenic activation, which may be responsible for their nonresponsive state in vivo. Expression of the CD3 zeta subunit of the T-cell receptor (TcR) was normal, and after stimulation via CD3, Raf-1 and p42 mitogen activated protein (MAP) kinase were phosphorylated, indicating that this part of the signaling pathway after triggering of the TcR/CD3 complex is present. These results, together with our previous observation that dysfunctional, host-reactive T-cell clones can be isolated in SCID patients transplanted with fetal liver stem cells, demonstrate that lack of clonal deletion of host-reactive T cells is a general phenomenon after HLA-mismatched stem cell transplantation.

Bone Marrow Transplantation↗
PubMed1995

Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells.

Infection by murine retroviruses in embryonic carcinoma (EC) and embryonic stem cells is highly restricted. The transcriptional unit of the Moloney murine leukemic virus (MoMuLV) long terminal repeat (LTR) is inactive in EC and embryonic stem cells in association with increased proviral methylation. In this study, expression in F9 EC cells was achieved from novel retroviral vectors containing three modifications in the MoMuLV-based retroviral vector: presence of the myeloproliferative sarcoma virus LTR, substitution of the primer binding site, and either deletion of a negative control region at the 5' end of the LTR or insertion of a demethylating sequence. We conclude that inhibition of expression from the MoMuLV LTR in EC cells is mediated through the additive effects of multiple cis-acting elements affecting the state of methylation of the provirus.

3T3 Cells↗
PubMed1995

The effect of thymic function on immunocompetence following bone marrow transplantation.

We investigated the role of thymic function in the immunodeficiency that characterizes bone marrow transplantation (BMT) recipients. The capacity of histocompatible BMT recipients to generate new CD4+ T lymphocytes was determined by FACS analysis with antibodies to the two isoforms of CD45: CD45RA, which is expressed on newly generated CD4 T lymphocytes, and CD45RO, which is expressed on antigen-specific memory CD4 T lymphocytes. Immediately following BMT, all patients had low levels of CD45RA-expressing CD4 T lymphocytes, which increased during the first year and then stabilized. Since thymic function decreases with age in normal individuals, the impact of recipient age on the generation of new CD45RA,CD4 T lymphocytes was determined in BMT recipients with and without chronic graft-vs.-host disease (GVHD). In addition, antigen-specific immune function was determined by assessing in vitro blastogenic response to tetanus toxoid (TT). More than 1 year after transplantation, the results from BMT recipients without chronic GVHD were similar to those of normal individuals; there was an age-dependent decline in the number of CD45RA,CD4 T lymphocytes, and antigen-specific immune function was age-independent. On the other hand, recipients with chronic GVHD had an age-dependent decline in their immune function (r = 0.45 and p = 0.02) that correlated with the number of new CD45RA,CD4 T lymphocytes (p = 0.027) but not the number of memory CD45RO,CD4 T lymphocytes (p = 0.11). Thus recipients with chronic GVHD have decreased antigen-specific immune function that may be due to an acceleration of the normal thymic aging process induced by GVHD and its therapy.

Bone Marrow Transplantation↗
PubMed1994

Coverage gaps in child-restraint and seat-belt laws affecting children.

Although most U.S. states have enacted mandatory-seat-belt-use laws and all states have child-restraint laws requiring children under specified ages to be properly restrained in vehicles, some children are covered by neither law. Other children are covered only by belt-use laws that do not make any adult responsible for their compliance. This note analyzes the child-restraint and belt-use laws in each state and demonstrates the deficiencies in coverage as they apply to children younger than age 16.

Adolescent↗
PubMed1994

Immunophenotypic differences between putative hematopoietic stem cells and childhood B-cell precursor acute lymphoblastic leukemia cells.

Acute leukemia cells express myeloid, B-lymphoid and T-lymphoid lineage specific antigens. Many acute leukemias express the hematopoietic progenitor cell antigen CD34. Three proposed models of the normal human hematopoietic stem cell include CD34+ Thy-1low Lin-, CD34+ CD38-, and CD34+ HLA-DR-. The patterns of expression of CD34, Thy-1, CD38, HLA-DR, and multiple lineage-specific antigens on 49 consecutive pediatric B-cell precursor acute lymphoblastic leukemia (ALL) cases submitted for immunophenotyping (36 at first diagnosis, 13 at relapse) were analyzed. CD34+ expression was observed in 67% of the cases. CD34+ expression correlated with Thy-1low expression and expression of myeloid antigens (p < 0.001 and < 0.025, respectively). The CD34+ Thy-1low phenotype was observed in 65% of the cases; the CD34+ CD38- or CD34+ HLA-DR- phenotypes were observed in only three cases. Examples of heterogeneous expression of CD34 and Thy-1 were found in six cases, but CD38 expression was always bright and homogeneous in all positive cases. The data from this analysis indicates that the CD34+ CD38- or CD34+ HLA-DR- phenotypes would be more useful than the CD34+ Thy-1low phenotype for distinguishing normal hematopoietic stem cells from leukemic cells in childhood B-cell precursor ALL.

ADP-ribosyl Cyclase↗
PubMed1993

T lymphocyte ontogeny in adenosine deaminase-deficient severe combined immune deficiency after treatment with polyethylene glycol-modified adenosine deaminase.

Adenosine deaminase (ADA) deficiency causes severe combined immune deficiency (SCID) by interfering with the metabolism of deoxyadenosine, which is toxic to T lymphocytes at all stages of differentiation. Enzyme replacement with polyethylene glycol-modified ADA (PEG-ADA) has been previously shown to correct deoxyadenosine metabolism and improve mitogen-induced T lymphocyte proliferation. We studied the biochemical and immunologic effects of PEG-ADA in two infants with ADA-deficient SCID. While in a catabolic state, higher doses of PEG-ADA than previously described were required to normalize deoxyadenosine metabolism. After biochemical improvement, the patients recovered immune function in a pattern similar to that observed in normal thymic ontogeny and in patients with immunological reconstitution after bone marrow transplantation. Immune reconstitution was marked by the sequential appearance in the peripheral blood of phenotypic T lymphocytes corresponding to successive stages of thymic differentiation. Functional reconstitution was marked by the successive appearance of mitogen responses dependent on exogenous in vitro IL-2, mitogen responses not requiring exogenous IL-2, antigen-specific responses dependent on exogenous IL-2, and finally, antigen-specific responses not requiring exogenous IL-2. Natural killer function was tested in one patient and normalized with PEG-ADA therapy. Optimal PEG-ADA therapy appears to normalize thymic differentiation in ADA-deficient SCID, resulting in normal antigen-specific immune function.

Adenosine Deaminase↗
PubMed1993

Unrelated donor BMT for Wiskott-Aldrich syndrome.

The role of allogeneic sibling BMT for children with Wiskott-Aldrich syndrome is established. Mismatched T cell-depleted BMT has been successful, although significant problems with graft rejection, GVHD, and post-transplant lymphoproliferative disorders have been reported. We have performed four BMTs for children with Wiskott-Aldrich syndrome utilizing phenotypically HLA-identical unrelated donors. A non-TBI (total body irradiation) conditioning regimen was utilized, and BM was not T cell-depleted. All patients engrafted and developed significant, although manageable, GVHD. All patients are alive 3+ to 17+ months post-transplant. These results suggest that matched unrelated donor BMT has a definite role in the treatment of Wiskott-Aldrich syndrome.

Acute Disease↗
PubMed1993

Recombination activating gene-1 (RAG-1) expression in all differentiation stages of B-lineage precursor acute lymphoblastic leukemia.

The recombination activating gene-1 (RAG-1), which is required for immunoglobulin (Ig) gene rearrangement, is expressed in murine B-lymphoid precursors but not in mature B lymphocytes. In order to characterize the temporal relationship of RAG-1 expression to other markers of human B-lymphoid differentiation [cell surface antigens, terminal deoxynucleotidyl transferase (TdT), Ig gene rearrangements], RAG-1 expression was studied in a group of B lineage childhood acute lymphoblastic leukemia (ALL). ALL cells from 21 patients were grouped into three developmentally related phenotypes based on the expression of the differentiation antigens CD19, CD10, and CD20. All 21 leukemias were surface Ig (slg) negative. There were leukemias representing each developmental stage of Ig gene rearrangement. RAG-1 was expressed in 20 of 21 B-lineage ALL, including leukemic cells from each stage of differentiation, as defined by immunophenotype and IgH and IgL gene rearrangement status. RAG-1 was expressed in slg- ALL, regardless of the Ig heavy chain (IgH) or Ig light chain (IgL) gene configuration. RAG-1 was not expressed in two Burkitt lymphomas and Burkitt lymphoma cell lines with slg+ mature B-lymphocyte phenotype. In two cases, RAG-1 was expressed in TdT-negative ALL; conversely TdT was expressed in the one RAG-1 negative ALL. These results suggest that RAG-1 in B-lineage ALL is expressed at all phenotypic and genotypic developmental stages preceding surface immunoglobulin expression, and that TdT and RAG-1 may be regulated by different mechanisms.

Antigens, Neoplasm↗
PubMed1992

Transcription of IL-2 and IL-4 genes is not inhibited by cyclosporin A in competent T cells.

Cyclosporin A (CsA) inhibits T-cell proliferation primarily by blocking the transcription of several early activation genes, especially those of the important T-cell growth factors IL-2 and IL-4. This effect seems to be mediated through inhibition of the activity of the transcription factor NF-AT which is essential for IL-2 and probably for IL-4 gene transcription. However, once T cells are rendered "competent" to proliferate following a brief exposure to the phorbol ester, phorbol 12,13-dibutyrate (PDBu), and the calcium ionophore, ionomycin, CsA no longer inhibits cell cycle progression supported by the presence of PDBu alone. Here it is shown that transcription of the IL-2 and IL-4 genes occurs normally throughout this "progression" phase, even in the presence of CsA. However, further production of functional NF-AT, which began during the competence phase of the cell cycle, is inhibited. These data indicate that, although the primary initiation of transcription of IL-2 and IL-4 mRNA during induction of competence may be NF-AT-dependent and CsA-sensitive, the augmentation in the progression phase is both NF-AT-independent and CsA-resistant.

Base Sequence↗
PubMed1991

Defining protective responses to pathogens: cytokine profiles in leprosy lesions.

The immunological mechanisms required to engender resistance have been defined in few infectious diseases of man, and the role of specific cytokines is unclear. Leprosy presents clinically as a spectrum in which resistance correlates with cell-mediated immunity to the pathogen. To assess in situ cytokine patterns, messenger RNA extracted from leprosy skin biopsy specimens was amplified by the polymerase chain reaction with 14 cytokine-specific primers. In lesions of the resistant form of the disease, messenger RNAs coding for interleukin-2 and interferon-gamma were most evident. In contrast, messenger RNAs for interleukin-4, interleukin-5, and interleukin-10 predominated in the multibacillary form. Thus, resistance and susceptibility were correlated with distinct patterns of cytokine production.

Base Sequence↗
PubMed1991

Hepatic veno-occlusive disease post-bone marrow transplantation in children conditioned with busulfan and cyclophosphamide: incidence, risk factors, and clinical outcome.

We performed a retrospective analysis of the incidence, risk factors, and clinical outcome of hepatic veno-occlusive disease (VOD) in 50 children prepared for bone marrow transplantation with busulfan (16 mg/kg) and cyclophosphamide (200 mg/kg). The overall incidence of VOD was 28% (14/50). The incidence of VOD among patients transplanted for leukemia was 36% (14/39). In contrast, no patient transplanted for a genetic disease developed VOD. Neither patient age, sex, remission status, type of graft (i.e. allogeneic or autologous), past history of liver disease nor pretransplant liver function tests were associated with an increased risk of VOD. In addition, 23 of 50 patients had pretransplant samples available for antihepatitis C virus (HCV) testing; 3/23 were reactive (two of nine patients with VOD and one of 14 patients without VOD were positive for anti-HCV). We found a high incidence of pleural effusion in patients with VOD (7/14), an association that has previously not been described. VOD was manageable and resolved in all patients.

Adolescent↗