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Biomedical subjects

T A Fleisher

Publications and source records attributed to T A Fleisher.

At least 19 recordsLinked to original sources
PubMed2003

Lymphocyte depletion with fludarabine in patients with psoriatic arthritis: clinical and immunological effects.

OBJECTIVE: To obtain preliminary information on the safety and efficacy of fludarabine in PsA and analyse its immunomodulatory effects in peripheral blood and synovial tissue. METHODS: 15 patients with active PsA who did not respond to DMARDs were randomly allocated to receive fludarabine every four weeks or placebo. Primary outcomes were the proportion of patients who met the ACR20 and the psoriatic arthritis response criteria (PsARC) at 16 weeks. Secondary outcomes were changes in tender or swollen joint counts and scores of the psoriasis area and severity index (PASI). Phenotypic analysis of peripheral blood mononuclear cells (PBMC), synovial immunohistochemistry, and functional analysis of PBMC were used to determine the immunomodulatory effects of fludarabine. RESULTS: At 16 weeks the ACR20 criteria were met by 3/7 (43%) fludarabine treated v 0/8 placebo treated patients (p=0.08); the PsARC was achieved by 4/7 (57%) fludarabine treated v 2/8 (25%) placebo treated patients; and 3/7 (43%) fludarabine treated v 0/7 placebo treated patients had > or =20% improvement in the PASI. Marked peripheral lymphopenia involving naive (CD4(+) CD45RA(+)) and memory (CD4(+) CD45RO(+)) T cells, CD8(+) T cells, and B cells was seen in fludarabine treated patients. CONCLUSIONS: In PsA fludarabine induces significant peripheral, but modest, synovial lymphopenia, and a trend towards improved clinical response.

Adult↗
PubMed2001

Immunophenotypic profiles in families with autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) type Ia is caused by inherited defects in apoptosis and is characterized by nonmalignant lymphoaccumulation, autoimmunity, and increased alpha/beta(+) double-negative T cells (alpha/beta(+)-DNT cells). This study reports immunophenotypic findings in 166 members of 31 families with ALPS type Ia, associated with genetic mutations in the TNFRSF6 gene encoding Fas. The ALPS type Ia probands (n = 31) and relatives having both a Fas mutation and clinically proven ALPS (n = 28) showed significant expansion of CD8(+) T cells, alpha/beta(+)-DNT cells, gamma/delta(+)-DNT cells, CD3(+)/ HLA-DR(+) T cells, CD8(+)/CD57(+) T cells, and CD5(+) B cells. Relatives with Fas mutations, but without all the required criteria for ALPS (n = 42), had expansions of CD8(+) T cells, alpha/beta(+)-DNT cells, and gamma/delta(+)-DNT cells. Interestingly, relatives without a Fas mutation and with no features of ALPS (n = 65) demonstrated a small but significant expansion of CD8(+) T cells, both DNT cell subsets, and CD5(+) B cells. As compared to unrelated healthy controls, lymphocyte subset alterations were greatest in the probands, followed by the relatives with mutations and ALPS. Probands and relatives with mutations and ALPS also showed a lower number of CD4(+)/CD25(+) T cells that, in combination with an independent increase in HLA-DR(+) T cells, provided a profile predictive of the presence of clinical ALPS. Because quantitative defects in apoptosis were similar in mutation-positive relatives regardless of the presence of clinical ALPS, factors, other than modifiers of the Fas apoptosis pathway, leading to these distinctive immunophenotypic profiles most likely contribute to disease penetrance in ALPS.

Adult↗
PubMed2001

Human T cell activation induces the expression of a novel CD45 isoform that is analogous to murine B220 and is associated with altered O-glycan synthesis and onset of apoptosis.

Activated T cells undergo changes during their transition to T cell blasts and, subsequently, via a phase of anergy, to apoptosis. For example, activated murine T cell blasts express the B-cell-specific CD45R isoform, B220, a marker also present on T cells in mice and humans with defective Fas-mediated apoptosis in vivo, suggesting a role for B220 up-regulation in the transition of activation to apoptosis. Human T cells, activated in vitro with superantigens and mitogens, also express B220 as they undergo blastogenesis and cell cycle progression. B220 expression peaks on T cells undergoing apoptosis. CD43-hexasaccharide glycoform expression and lectin binding profiles indicate that B220 expression is reflective of altered O-linked glycan biosynthesis found in specific T cell subsets transitioning through the phases of proliferation, anergy, and apoptosis. Potential implications of these alterations include changes in lymphocyte adhesion and trafficking and homeostasis through altered sensitivity to Fas-dependent and independent pathways of apoptosis.

Adult↗
PubMed2001

IL-10 improves skin disease and modulates endothelial activation and leukocyte effector function in patients with psoriatic arthritis.

Psoriatic arthritis (PsA) provides an ideal disease model in which to investigate the bioactivities of potentially therapeutic cytokines at multiple sites of tissue inflammation. We investigated the effects of IL-10, an antiinflammatory cytokine, given s.c. for 28 days in a double-blind, placebo-controlled study in PsA patients. Synovial/skin biopsies, peripheral blood leukocytes, articular magnetic resonance images, and clinical disease activity scores were obtained sequentially. Modest, but significant clinical improvement in skin, but not articular disease activity scores with only minor adverse effects was observed. Type 1, but not type 2 T cell cytokine production in vitro was suppressed in human rIL-10 compared with placebo recipients. Similarly, monokine production in vitro was reduced, whereas serum soluble TNFRII levels were elevated, indicating suppression of monocyte function. Decreased T cell and macrophage infiltration in synovial tissues was accompanied by reduced P-selectin expression. Moreover, suppressed synovial enhancement on magnetic resonance imaging and reduced alpha(v)beta(3) integrin expression on von Willebrand factor(+) vessels were observed. Together these data demonstrate that a short course of IL-10 modulates immune responses in vivo via diverse effects on endothelial activation, and leukocyte recruitment and effector function. Such biological changes may result in clinically meaningful improvement in disease activity.

Adult↗
PubMed2001

The development of lymphomas in families with autoimmune lymphoproliferative syndrome with germline Fas mutations and defective lymphocyte apoptosis.

Lymphomas were studied in kindreds with autoimmune lymphoproliferative syndrome (ALPS; Canale-Smith syndrome), a disorder of lymphocyte homeostasis usually associated with germline Fas mutations. Fas (CD95/APO-1) is a cell surface receptor that initiates programmed cell death, or apoptosis, of activated lymphocytes. Lymphoma phenotype was determined by immunohistochemistry, frequency of CD3(+)CD4(-)CD8(-) T-cell-receptor alpha/beta cells by flow cytometry, nucleotide sequences of the gene encoding Fas (APT1, TNFRSF6), and the percentage of lymphocytes undergoing apoptosis in vitro. Of 223 members of 39 families, 130 individuals possessed heterozygous germline Fas mutations. Eleven B-cell and T-cell lymphomas of diverse types developed in 10 individuals with mutations in 8 families, up to 48 years after lymphoproliferation was first documented. Their risk of non-Hodgkin and Hodgkin lymphomas, respectively, was 14 and 51 times greater than expected (each P <.001). Investigation of these 10 patients and their relatives with Fas mutations revealed that all had defective lymphocyte apoptosis and most had other features of ALPS. The tumor cells retained the heterozygous Fas mutations found in the peripheral blood and manifested defective Fas-mediated killing. These data implicate a role for Fas-mediated apoptosis in preventing B-cell and T-cell lymphomas. Inherited defects in receptor-mediated lymphocyte apoptosis represent a newly appreciated risk factor for lymphomas.

Adult↗
PubMed2001

Increases in circulating and lymphoid tissue interleukin-10 in autoimmune lymphoproliferative syndrome are associated with disease expression.

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder in which genetic defects in proteins that mediate lymphocyte apoptosis, most often Fas, are associated with enlargement of lymph nodes and the spleen and a variety of autoimmune manifestations. Some patients with ALPS have relatives with these same apoptotic defects, however, who are clinically well. This study showed that the circulating levels of interleukin 10 (IL-10) were significantly higher (P <.001) in 21 patients with ALPS than in healthy controls. Moreover, the peripheral blood mononuclear cells (PBMCs) and lymphoid tissues of these patients with ALPS contained significantly higher levels of IL-10 messenger RNA (mRNA; P <.001 and P <.01, respectively). By fractionating PBMC populations, disproportionately high concentrations of IL-10 mRNA were found in the CD4(-)CD8(-) T-cell population, expansion of which is virtually pathognomonic for ALPS. Immunohistochemical staining showed intense IL-10 protein signals in lymph node regions known to contain CD4(-)CD8(-) T cells. Nonetheless, in vitro studies showed no influence of IL-10 on the survival of CD4(-)CD8(-) T cells. Overexpression of IL-10 in patients with inherited apoptotic defects is strongly associated with the overt manifestations of ALPS.

Apoptosis↗
PubMed2001

TcR-alpha/beta(+) CD4(-)CD8(-) T cells in humans with the autoimmune lymphoproliferative syndrome express a novel CD45 isoform that is analogous to murine B220 and represents a marker of altered O-glycan biosynthesis.

Autoimmune lymphoproliferative syndrome (ALPS), caused by inherited defects in apoptosis secondary to mutations in genes encoding Fas/CD95/APO-1 and Fas ligand (Fasl)/CD95L, is characterized by nonmalignant lymphadenopathy and splenomegaly, increased T cell receptor alpha/beta(+) CD4(-)CD8(-) T cells (alpha/beta(+) double-negative T cells [alpha/beta(+)-DNT cells]), autoimmunity, hypergammaglobulinemia, and cytokine abnormalities. The alpha/beta(+)-DNT cells are immunophenotypically and functionally similar to alpha/beta(+)-DNT cells that accumulate in lpr and gld mice, which bear genetic mutations in Fas and FasL. In these mice, alpha/beta(+)-DNT cells express the B-cell-specific CD45R isoform B220. We show that alpha/beta(+)-DNT cells of ALPS patients, with either Fas or FasL mutations, also express B220. In addition, also similar to LPR/gLD mice, they have an unusual population of B220-positive CD4(+) T cells. B220 expression, together with our finding of characteristic lectin binding profiles, demonstrates that cell surface O-linked glycoproteins have undergone specific modifications, which may have consequences for lymphocyte trafficking, cell-cell interactions, and access to alternative apoptosis pathways.

Autoimmune Diseases↗
PubMed2001

Detection of intracellular phosphorylated STAT-4 by flow cytometry.

The convergence of innate and adaptive immunity is critical for host defense, allowing for early protection and the generation of specific responses. STAT-4 is at that point of convergence, unifying the IFNalpha and IL-12 pathways. Activation of STAT-4 is crucial to T cell polarization, B cell and NK cell activation, and the control of intracellular pathogens. However, techniques to detect phosphorylated STAT-4 are cumbersome and require many cells. We have developed a flow cytometric detection technique to investigate IL-12 signaling in human peripheral blood mononuclear cells. Using different polyclonal antibodies that recognize either total STAT-4 protein or tyrosine-phosphorylated STAT-4, we can easily detect IL-12 and IFNalpha signaling in PHA/IL-2 blasts derived from peripheral blood lymphocytes. This technique not only allows us to evaluate IL-12 signaling, but it is also less time consuming and labor intensive than alternative methods. Using this flow cytometry-based method, we should be able to detect patients with defects in IL-12 receptor signal transduction, who typically present with disseminated nontuberculous mycobacterial infections.

Animals↗
PubMed2001

A genetic disorder of lymphocyte apoptosis involving the fas pathway: the autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a recently characterized human disorder that typically presents with lymphocyte accumulation in the first few years of life. This is often associated with the development of autoimmunity, most commonly affecting the hematopoietic system. A key laboratory feature is the marked expansion of double-negative (CD4- and CD8-) T cells that express the alpha/beta T-cell receptor. ALPS is associated with defective Fas-mediated lymphocyte apoptosis, and in most patients, this results from a heterozygous mutation in the TNFRSF6 gene encoding Fas. The clinical features of ALPS reveal the importance of the Fas apoptotic pathway in maintaining lymphocyte homeostasis and protecting against autoimmunity and lymphoid malignancy.

Apoptosis↗
PubMed2001

Immunophenotyping.

Immunophenotyping of leukocytes for nonmalignant conditions uses the power of multiparameter flow cytometry to count specific lymphocyte populations and evaluate the presence or absence of particular cell surface markers. Its main clinical indications, and the focus of this chapter, are enumeration of CD4 T-cell counts and activation markers on T cells in human immunodeficiency virus (HIV) infection, immunophenotypic characterization of primary immunodeficiency disorders and immune-mediated diseases, and the study of immune reconstitution following stem cell transplantation (SCT). Immunophenotyping has advanced our understanding of many immunologic disorders, which in turn have stimulated new developments in the field of flow cytometry, such as quantitative flow cytometry and single-platform technology. Semin Hematol 38:100-110. This is a US government work. There are no restrictions on its use.

Animals↗
PubMed2001

Cell function-based flow cytometry.

Flow cytometry is increasingly used to assess the functional status of leukocytes, and practically all aspects of their life (and death) are accessible to flow cytometric study. Together with familiar features of flow cytometry, such as multiparameter immunophenotyping, cell function-based flow cytometry has provided many new insights into the relationships among lymphocyte cell surface features; intracellular processes, such as cytokine production and protein phosphorylation; and the functional status of lymphocytes in a variety of human diseases. Direct visualization and quantification of antigen-specific T cells using major histocompatibility complex (MHC)-peptide tetramer technology, in combination with functional assays, has provided the means to study specific T-cell subsets of interest. Even early in its development, this technology already has offered a better understanding of basic and clinical immunology and has invited reassessment of several long-standing immunologic concepts. Semin Hematol 38:169-178. This is a US government work. There are no restrictions on its use.

Animals↗
PubMed2001

Unexpected and variable phenotypes in a family with JAK3 deficiency.

Mutations of the Janus kinase 3 (JAK3) have been previously described to cause an autosomal recessive variant of severe combined immunodeficiency (SCID) usually characterized by the near absence of T and NK cells, but preserved numbers of B lymphocytes (T-B+SCID). We now report a family whose JAK3 mutations are associated with the persistence of circulating T cells, resulting in previously undescribed clinical presentations, ranging from a nearly unaffected 18-year-old subject to an 8-year-old sibling with a severe lymphoproliferative disorder. Both siblings were found to be compound heterozygotes for the same deleterious JAK3 mutations: an A96G initiation start site mutation, resulting in a dysfunctional, truncated protein product and a G2775(+3)C mutation in the splice donor site sequence of intron 18, resulting in a splicing defect and a predicted premature stop. These mutations were compatible with minimal amounts of functional JAK3 expression, leading to defective cytokine-dependent signaling. Activated T cells in these patients failed to express Fas ligand (FasL) in response to IL-2, which may explain the accumulation of T cells with an activated phenotype and a skewed T cell receptor (TcR) Vbeta family distribution. We speculate that residual JAK3 activity accounted for the maturation of thymocytes, but was insufficient to sustain IL-2-mediated homeostasis of peripheral T cells via Fas/FasL interactions. These data demonstrate that the clinical spectrum of JAK3 deficiency is quite broad and includes immunodeficient patients with accumulation of activated T cells, and indicate an essential role for JAK3 in the homeostasis of peripheral T cells in humans.

Adolescent↗
PubMed2001

The combination of zidovudine and interferon alpha-2B in the treatment of adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is frequently a very aggressive malignancy with a poor survival despite aggressive multiagent chemotherapy. The combination of the antiretroviral drug zidovudine (AZT) and interferon alpha (IFNalpha) has been reported to induce remissions in patients with ATL. The purpose of this study was to evaluate the clinical response and toxicity following administration of a combination of IFNalpha-2b and AZT in patients with human T-cell lymphotropic virus type I (HTLV-I)-associated ATL. Eighteen patients with ATL (chronic. crisis, acute or lymphoma type) were treated with the combination of AZT (50 - 200 mg orally 5 times a day) and IFNalpha-2b (2.5 - 10 million units subcutaneously daily). Three patients had objective responses lasting more than one month. One patient had a clinical complete remission, lasting 21.6 months and two patients had partial remissions lasting 3.7 and 26.5 months. Six patients were not considered evaluable for response due to short and/or interrupted periods of treatment. Seventeen patients have died with a median survival time after initiation of therapy of 6 months. Neutropenia and thrombocytopenia were the dose limiting toxicities. In conclusion, the response rate in this study was lower than noted in the two previous published series. This may be due to the amount and type of prior treatment our patients had received.

Adult↗
PubMed2000

Prolonged CD4 depletion after sequential autologous peripheral blood progenitor cell infusions in children and young adults.

Administration of mobilized peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy rapidly restores multilineage hematopoiesis, but the ability of such products to restore lymphocyte populations remains unclear. In this report, we evaluated immune reconstitution in a series of patients treated with sequential cycles of high-dose chemotherapy, followed by autologous PBPC infusions (median CD34(+) cell dose 7.2 x 10(6) cells/kg [range 2-29.3]). Although patients experienced rapid reconstitution of B cells and CD8(+) T cells, we observed CD4 depletion and diminished immune responsiveness in all patients for several months after completion of therapy. Mature CD4(+) T cells contained within the grafts did not appear to contribute substantially to immune reconstitution because CD4 counts did not differ between recipients of unmanipulated T-cell replete infusions versus CD34 selected, T-cell-depleted infusions. Rather, at 12 months after therapy, total CD4 count was inversely proportional to age (rho = -0.78, P =.04), but showed no relationship to CD34 cell dose (rho = -0.42, P =.26), suggesting that age-related changes within the host are largely responsible for the limited immune reconstitution observed. These results demonstrate that in the autologous setting, the infusion of large numbers of PBPCs is not sufficient to restore T-cell immune competence and emphasize that specific approaches to enhance immune reconstitution are necessary if immune-based therapy is to be used to eradicate minimal residual disease after autologous PBPC transplantation. (Blood. 2000;96:754-762)

Adolescent↗
PubMed2000

Efficient detection of thirty-seven new IL2RG mutations in human X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency (XSCID) is a rare and potentially fatal disease caused by mutations of IL2RG, the gene encoding the interleukin-2 receptor gamma chain, a component of multiple cytokine receptors that are essential for lymphocyte development and function. To date, over 100 different mutations of IL2RG resulting in XSCID have been published. Using nonradioactive, direct DNA sequencing of a single PCR amplicon containing the whole IL2RG gene, we found IL2RG mutations in 78 previously unpublished unrelated cases of XSCID. We report 37 newly identified mutations of IL2RG, including 23 point mutations, 10 small deletions, 3 instances of the same single nucleotide insertion, 1 large deletion, and 2 complex mutations. More than half of the mutations (22 of 37) were predicted to result in unstable IL2RG mRNA. The remaining 14 mutations disrupted conserved functional motifs common to all cytokine receptor family members; changed protein conformation, charge, or hydrophobicity; or altered the intracellular portion of the protein, which is critical for proper interaction with signal-transducing molecules including Janus family tyrosine kinase 3.

Base Sequence↗
PubMed2000

Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection.

Coupling laser capture microdissection (LCM) with sensitive quantitative chemiluminescent immunoassays has broad applicability in the field of proteomics applied to normal, diseased, or genetically modified tissue. Quantitation of the number of prostate-specific antigen (PSA) molecules/cell was conducted on human prostate tissue cells procured by LCM from fixed and stained frozen sections. Under direct microscopic visualization, laser shots 30 microm in diameter captured specific cells from the heterogeneous tissue section onto a polymer transfer surface. The cellular macromolecules from the captured cells were solubilized in a microvolume of extraction buffer and directly assayed using an automated (1.5 hour) sandwich chemiluminescent immunoassay. Calibration of the chemiluminescent assay was conducted by developing a standard curve using known concentrations of PSA. After the sensitivity, precision, and linearity of the chemiluminescent assay was verified for known numbers of solubilized microdissected tissue cells, it was then possible to calculate the number of PSA molecules per microdissected tissue cell for case samples. In a study set of 20 cases, using 10 replicate samples of 100 laser shots per sample, the within-run (intraassay) SD was approximately 10% of the mean or less for all cases. In this series the number of PSA molecules per microdissected tissue cell ranged from 2 x 10(4) to 6. 3 x 10(6) in normal epithelium, prostate intraepithelial neoplasia (PIN), and invasive carcinoma. Immunohistochemical staining of human prostate for PSA was compared with the results of the soluble immunoassay for the same prostate tissue section. Independent qualitative scoring of anti-PSA immunohistochemical staining intensity paralleled the LCM quantitative immunoassay for each tissue subpopulation and verified the heterogeneity of PSA content between tissue subpopulations in the same case. Extraction buffers were successfully adapted for both secreted and membrane-bound proteins. This technology has broad applicability for the quantitation of protein molecules in pure populations of tissue cells.

Calibration↗