Biomedical subjects

W G Wierda

Publications and source records attributed to W G Wierda.

15 recordsLinked to original sources

PubMed2005

Increased expression of CD152 (CTLA-4) by normal T lymphocytes in untreated patients with B-cell chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL) have defects in both cellular and humoral immunity. Since CD152 (CTLA-4) plays a critical role in downregulating T-cell responses, we studied the expression of surface and cytoplasmic CD152 (sCD152 and cCD152, respectively) in freshly isolated T cells from treatment-naïve patients with CLL. CD4+ and CD8+ T cells from these patients demonstrated significantly increased sCD152 and cCD152 compared to normal donors. Furthermore, these patients had an increased proportion of the regulatory CD4(+)/CD25(+)/CD152+ subset that correlated with advanced Rai stage, unfavorable cytogenetics and low serum IgG and IgA levels. The expression of sCD152 by T cells also correlated with ZAP-70 expression by CLL B cells. The proportion of CD4(+)/CD25+ cells was also correlated with unmutated immunoglobulin heavy chain variable gene status. Blockade of CD152 with monoclonal antibody (mAb) in proliferation assays was associated with potent T-cell proliferation in response to autologous and allogeneic CD40-activated CLL B cells. In summary, T cells from patients with CLL may be primed for anergy by expressing increased amounts of CD152; anti-CD152 mAb may represent a therapeutic opportunity to enhance an immune response against autologous leukemia cells.

Adult↗

PubMed2004

Increased mitochondrial biogenesis in primary leukemia cells: the role of endogenous nitric oxide and impact on sensitivity to fludarabine.

B cell chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in the Western hemisphere, yet many biological and molecular features of the disease remain undefined. CLL cells generate increased levels of radical species such as superoxide and nitric oxide (NO), which is associated with mitochondrial DNA mutations. Considering that NO levels can affect mitochondrial biogenesis, we hypothesized that the inherent nitrosative stress in CLL cells may lead to hyperactive mitochondrial biogenesis. Here we report that primary CLL cells contained significantly more mitochondria than normal lymphocytes and that their mitochondrial mass was significantly related to endogenous NO levels. Expression of the mitochondrial biogenesis factors nuclear respiratory factor-1 and mitochondrial transcription factor A was elevated in most CLL specimens examined and appeared to be related to cellular NO levels. Treatment of B cells with exogenous NO caused a substantial increase in mitochondrial mass. In vitro sensitivity of CLL cells to fludarabine was highly related to mitochondrial mass in that cells with greater mitochondrial mass were less sensitive to the drug. Taken together, our results suggest that NO is a key mediator of mitochondrial biogenesis in CLL and that modulation of mitochondrial biogenesis by NO may alter cellular sensitivity to fludarabine.

Antineoplastic Agents↗

PubMed2001

T cell activation following infection of primary follicle center lymphoma B cells with adenovirus encoding CD154.

Purified, high-titer adenovirus encoding murine CD154 (Ad-CD154) or human CD154 (Ad-hCD154) was used to infect lymph node cells isolated from patients with follicle center lymphoma. Infection of lymphoma B cells with Ad-CD154 at a multiplicity of infection (MOI) ratio of 100 or higher resulted in high-level transgene expression. Additionally, upon infection of lymphoma B cells, only Ad-CD154 resulted in surface expression of CD154, despite similar, high-level expression of either human or mouse CD154 by HeLa cells infected with Ad-hCD154 or Ad-CD154, respectively. Moreover, infection of lymphoma B cells with Ad-CD154, but not Ad-hCD154 or adenovirus encoding Eschericheria coli beta-galactosidase (Ad-LacZ), induced the neoplastic B cells to express higher levels of immune co-stimulatory molecules that are required for proficient presentation of antigen to T cells. Consistent with this, we found that Ad-CD154 infected lymphoma B cells could stimulate T cells to proliferate or produce interferon-gamma in allogeneic or autologous mixed lymphocyte interactions. We conclude that lymphoma B cells can be infected with Ad-CD154 and that this significantly enhances their recognition by allogeneic or autologous T cells. As such, Ad-CD154-transduced lymphoma B cells may have potential for the active immune therapy of patients with follicle center lymphoma.

Adenoviridae↗

PubMed2001

Immunotherapy of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is typically an indolent B-cell malignancy, primarily affecting the aging population. Standard cytotoxic treatment with alkylating agents or purine analogs is very effective at inducing remission. However, curative treatment is not yet available. Immunotherapy is emerging as an exciting modality with significant potential to advance the treatment of this disease. This review discusses the different modalities of immunotherapy under investigation for the treatment of CLL. These modalities include passive immunotherapy with monoclonal antibodies against antigens on CLL B-cells including CD52 and CD20. Active immunotherapy by vaccination with genetically modified autologous leukemia cells is being evaluated in clinical trials. Allogeneic stem cell transplant for adoptive immunotherapy of CLL is yet another modality being investigated. While this modality may have limited application due to morbidity in older patients, it may result in improved survival and possibly cure. The use of immunotherapy in CLL is in the early stages of development. It is likely that this approach will significantly improve the treatment of CLL and possibly contribute to the cure of this disease.

Antibodies, Monoclonal↗

PubMed2000

CD40-ligand (CD154) gene therapy for chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) cells can be made to express recombinant CD40-ligand (CD154) by transduction with a replication-defective adenovirus vector (Ad-CD154). Ad-CD154-transduced and bystander leukemia cells become highly effective antigen-presenting cells that can induce CLL-specific autologous cytotoxic T lymphocytes in vitro. This study investigated the immunologic and clinical responses to infusion of autologous Ad-CD154-CLL cells in patients with CLL. After a one-time bolus infusion of autologous Ad-CD154-transduced leukemia cells, there was increased or de novo expression of immune accessory molecules on bystander, noninfected CLL cells in vivo. Treated patients also developed high plasma levels of interleukin-12 and interferon-gamma, the magnitudes of which corresponded to absolute blood CD4(+) T-cell counts before therapy. On average, patients experienced a greater than 240% increase in absolute blood T-cell counts within 1 to 4 weeks of treatment. Moreover, treatment increased the numbers of leukemia-specific T cells, demonstrated by autologous ELISPOT assay and mixed lymphocyte reactions. These biologic effects were associated with reductions in leukemia cell counts and lymph node size. Treatment did not induce autoimmune thrombocytopenia or hemolytic anemia and no dose-limiting toxicity was observed. This approach may provide a novel and effective form of gene therapy for patients with this disease.

Adenoviridae↗

PubMed2000

CD40 activation does not protect chronic lymphocytic leukemia B cells from apoptosis induced by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) can kill target cells by the granule/exocytosis pathway or the Fas-mediated apoptosis pathway. The sensitivity of chronic lymphocytic leukemia (CLL) B cells to CTL-mediated apoptosis before and after CD40 activation was examined. Resting or CD40-activated CLL cells were found to be equally sensitive to class I-restricted CTL-mediated killing. Despite expressing CD95, the CD40-activated CLL target cells were found to be resistant to apoptosis induced by CH11, an IgM CD95 monoclonal antibody (mAb). Consistent with this, inhibitors of caspases, which are involved in the Fas-induced apoptotic pathway (eg, N-carbobenzoxy-Val-Ala-Asp fluoromethyl ketone [z-VAD-fmk]), were unable to block destruction of CLL target cells by CTL. In addition, preincubation of the effector T cells with the anti-Fas ligand mAb NOK-2 failed to inhibit their subsequent ability to kill CLL target cells. On the other hand, CTL activity was blocked by inhibitors of the granule exocytosis pathway such as ethylene-glyco-tetra-acetic acid or concanamycin A. These results indicate that CD40 activation does not impair the sensitivity of CLL cells to Fas-independent CTL-mediated apoptosis. (Blood. 2000;95:3853-3858)

Anti-Bacterial Agents↗

PubMed2000

Gene therapy of hematologic malignancies.

Gene therapy offers many new and exciting treatment strategies for patients with hematologic malignancies. Through the transfer of genes into hematopoietic stem cells, one can reduce the sensitivity of myeloid cells to chemotherapy. Donor T cells can be modified to become sensitive to otherwise nontoxic prodrugs, allowing for their safer use as effectors in graft-versus-leukemia immune reactions following allogeneic transplantation. Neoplastic cells also may be modified to enhance their sensitivity to various drugs. Finally, neoplastic cells can be modified to enhance their immunogenicity using genes that encode immune stimulatory cytokines or cell surface proteins. Recent studies, for example, indicate that the stealth-like phenotype of leukemia cells can be reversed through transfer of genes such as the one encoding CD154, the ligand for CD40. A phase I clinical trial using autologous CD154-transduced leukemia cells as a cellular vaccine has provided encouraging results. Indeed, we may soon enter an era of effective gene therapy for hematologic malignancies.

Apoptosis↗

PubMed2000

Immunogenetic therapy for B-cell malignancies.

Neoplastic B cells are stealthlike in their ability to evade immune detection, even by allogeneic T cells of normal healthy donors. This stealthlike phenotype can be reversed by activating neoplastic B cells through ligation of CD40, a cell surface molecule that can interact with a ligand expressed on activated T cells. The gene encoding this ligand, CD154, can be transferred into neoplastic B cells ex vivo through infection with a modified adenovirus vector called Ad-CD154. This results in a dramatic change in the phenotype and function of the neoplastic B cells. Infected malignant B cells can stimulate T cells reactive with potential tumor antigens and induce autologous cytotoxic T cells capable of destroying the neoplastic B cells in vitro. This formed the basis for an immune gene therapy protocol in which patients were infused with Ad-CD154-transduced leukemic B cells. Treatment was well tolerated, without apparent long-term toxicity, and without a maximum tolerated dose. Biologic and clinical responses were observed, including significant reductions in leukemia cell counts and lymph node sizes after a single one-time infusion. Furthermore, preliminary data suggest that this approach can enhance antibody-dependent cellular cytotoxicity and thereby augment the activity of antitumor monoclonal antibody therapy. Development of such strategies may allow for effective immunogenetic therapy for B-cell malignancies.

Antibodies, Monoclonal↗

PubMed1999

Chronic lymphocytic leukemia.

Significant strides have been made in our understanding of the biology and treatment of B cell chronic lymphocytic leukemia. Recent studies have defined cytogenetic and molecular lesions that may be responsible for leukemogenesis or disease progression. Molecular analyses of immunoglobulin genes have delineated two or more subgroups of chronic lymphocytic leukemia that may differ in their clinical behavior. Research in the biochemistry of chronic lymphocytic leukemia has provided insight into the noted resistance of leukemia cells to cytotoxic drugs. Investigations into the immunology has revealed mechanisms whereby chronic lymphocytic leukemia cells can contribute to the immune deficiency that commonly develops in patients with this disease. Clinical studies have delineated factors that are helpful in predicting prognosis and have provided data on promising new therapies for patients with this disease, including stem cell transplantation, monoclonal antibodies, and gene therapy.

Antibodies, Monoclonal↗

PubMed1993

Induction of porcine granulocyte-mediated tumor cytotoxicity by two distinct monoclonal antibodies against lytic trigger molecules (PNK-E/G7).

PNK-E and G7 mAb bind to distinct porcine granulocyte function-associated molecules and induce granulocyte-mediated cytotoxicity against tumor targets. PNK-E mAb binds to a 205-kDa molecule that reduces to a dispersed band of 50 kDa on SDS-PAGE analysis. Previous work demonstrates that G7 mAb immunoprecipitates a molecule that appears as a heterodispersed 40-kDa band under both reducing and nonreducing conditions on SDS-PAGE analysis. Whole but not F(ab')2 PNK-E and G7 mAb induce dose-dependent porcine granulocyte-mediated lysis of FcR+ but not FcR- targets, suggesting a redirected cytotoxicity mechanism of granulocyte-mediated killing. Fresh porcine granulocytes also mediate significant levels of antibody-dependent cellular cytotoxicity (ADCC) against nucleated (SB) target cells. Neither whole nor F(ab')2 PNK-E mAb affects granulocyte-mediated ADCC against SB target cells. However, both whole and F(ab')2 G7 mAb inhibit granulocyte-mediated ADCC against SB targets by approximately 50%. Bound F(ab')2 G7 mAb inhibits PNK-E mAb-induced granulocyte-mediated cytotoxicity against K562 targets, but bound F(ab')2 PNK-E mAb does not inhibit G7 mAb-induced granulocyte-mediated cytotoxicity, suggesting a physical association between the PNK-E and G7 molecules on the surface of porcine granulocytes. PNK-E and G7 hybridoma cells are readily lysed by granulocyte effectors, further supporting that the PNK-E and G7 molecules are cytolytic trigger molecules on granulocytes. These data demonstrate that PNK-E and G7 mAb bind to distinct granulocyte lytic trigger molecules and induce potent granulocyte-mediated cytotoxicity against nucleated tumor targets through a mechanism of redirected cytotoxicity.

Animals↗

PubMed1993

Two distinct porcine natural killer lytic trigger molecules as PNK-E/G7 molecular complex.

PNK-E and G7 mAbs regulate porcine NK and ADCC activities by binding to distinct NK function-associated trigger molecules on porcine NK cells. Previous work demonstrates that PNK-E mAb binds to a 205-kDa tetrameric molecule composed of two 47-kDa peptides and two 50-kDa peptides and G7 mAb binds to a distinct 40-kDa heterodispersed monomeric peptide on porcine NK cells. The data presented herein demonstrate that all PNK-E+ PBLs are G7+ and all G7+ PBLs are PNK-E+ indicating that the PNK-E and G7 molecules are coexpressed by porcine NK cells. Bound G7 mAb blocks subsequent binding of PNK-E mAb but not the converse. Bound F(ab')2 G7 mAb abrogates the ability of whole PNK-E mAb to enhance NK activity but bound F(ab')2 PNK-E mAb has no affect on G7 mAb enhancement of NK activity. PNK-E mAb enhanced NK activity is inhibited by binding of F(ab')2 G7 mAb even though whole PNK-E mAb remains bound. However, bound F(ab')2 PNK-E mAb has no affect on G7 mAb-enhanced NK activity. When PNK-E and G7 mAbs were tested alone and together in NK assays, comparable levels of enhancement were observed. PNK-E and G7 hybridomas express surface mAb through which NK cells bind and specifically lyse these hybridomas. Lysis of PNK-E and G7 hybridomas is inhibited by pretreatment of PBLs with F(ab')2 G7 mAb. These data indicate a physical association between the PNK-E and G7 molecules on NK cells and suggest that the G7 molecule is external to the PNK-E molecule.

Animals↗

PubMed1992

A triggering structure recognized by G7 monoclonal antibody on porcine lymphocytes and granulocytes.

Monoclonal antibody (mAb) G7 has been developed and appears to recognize a triggering structure on porcine natural killer (NK) cells and granulocytes. G7 mAb binds to approximately 13% of lymphocytes, 70% of monocytes, and greater than 95% of granulocytes. G7 mAb does not react with B cells. G7 mAb immunoprecipitates a heterodispersed molecule of approximately 40 kDa. Functionally, whole but not F(ab')2 fragments of G7 mAb enhance NK killing of Fc receptor positive K562, U937, and MOLT-4 targets but not Fc receptor negative CEM, WEHI-164, or YAC-1 targets. Both whole and F(ab')2 fragments of G7 mAb inhibit lymphocyte-mediated antibody-dependent cellular cytotoxicity. Interestingly, G7 mAb induces dramatic levels of granulocyte killing against nucleated K562 targets. These results suggest that G7 mAb recognizes a trigger molecule involved in porcine cellular cytotoxicity.

Animals↗

PubMed1991

Further characterization of PNK-E: a monoclonal antibody enhancing porcine natural killer cell activity.

Monoclonal antibody PNK-E binds to approximately 15% of porcine peripheral blood lymphocytes (PBL) which are PT4 negative and PT8 positive. When cells from tissues of adult pigs are treated with PNK-E, enhancement of natural killer (NK) cell activity is observed from PBL and spleen cells, and a dramatic induction of NK activity is observed from bone marrow cells. With cells derived from tissues of neonatal piglets, PNK-E induces NK activity from PBL and bone marrow cells. To investigate the mechanism of PNK-E-mediated enhancement of NK, proliferation assays, calcium-pulse assays, single-cell assays, and kinetic analyses were performed. PNK-E did not induce proliferation of PBL. PNK-E could be added as late as 30 min prior to termination of Ca(2+)-pulse assays and still enhance NK activity. Using kinetic analysis PNK-E was found to increase the rate of NK lysis (Vmax) and rate of lytic programming per NK cell (k2). In addition, results from single-cell assays indicate that PNK-E activates a population of normally inactive effector cells. These results indicate that PNK-E enhances the lytic capacity of mature NK cells and induces a population of nonlytic cells to become highly cytolytic cells. Furthermore, the enhancing effects are immediate and do not require an induction period. Thus, PNK-E recognizes and activates a unique triggering molecule that is present on NK cells.

Age Factors↗

PubMed1991

Immobilized IgG immune complex induces secretion of tumor necrosis factor-alpha by porcine alveolar macrophages.

Tumor necrosis factor-alpha (TNF-alpha) is an important inflammatory mediator produced by activated monocytes and macrophages. We have previously shown that porcine alveolar macrophages (PAM) mediate bystander cytotoxicity through hydrogen peroxide production following activation with immobilized IgG immune complex (IIC) (J. Immunol. 1983; 131:1438-1442). In this report, we have investigated whether IIC induces TNF-alpha secretion by PAM. Isolated PAM from Minnesota miniature swine were cultured for 18 h with and without recombinant human interferon-gamma (rhIFN-gamma). Cultured PAM were then incubated with IIC or IgG immune complex in suspension (SIC). The supernatants generated were assessed for cytotoxic activity using a TNF-alpha-sensitive WEHI-164 cell line. Anti-recombinant human TNF-alpha (rhTNF-alpha) monoclonal antibody neutralized the observed cytotoxicity of IIC-activated PAM supernatant completely, indicating that this cytotoxicity is mediated by TNF-alpha. IIC induced TNF-alpha secretion by PAM after 3 h of incubation, reaching a plateau from 6 to 12 h and decreasing thereafter. TNF-alpha release was enhanced by pretreatment of PAM with rhIFN-gamma. SIC did not induce significant levels of TNF-alpha secretion by PAM; however, SIC with cytochalasin B-pretreated PAM induced equivalent levels of TNF-alpha secretion as IIC-activated PAM. We conclude that IIC or SIC with cytochalasin B pretreatment, both of which prevent internalization of IgG immune complex-bound Fc receptor (FcR), provide a signal for PAM to generate TNF-alpha through FcR modulation. This suggests that in vivo, deposited (immobilized) IgG immune complexes-bound FcR may be a stimulus for activation of PAM to generate TNF-alpha rather than circulating (mobilized) immune complexes, which may contribute to the pathogenesis of diffuse interstitial fibrosis of the lung, especially in idiopathic pulmonary fibrosis.

Animals↗

PubMed1989

Comparison of fluorochrome-labeled and 51Cr-labeled targets for natural killer cytotoxicity assay.

An alternative method for measuring in vitro cellular cytotoxicity has been developed utilizing the carboxyfluorescein derivative 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) as the target cell label. Target cells labeled with the fluorescent dye are incubated with effector cells, if killing of targets occurs, BCEDF is released analogous to 51Cr release. Measurement of specific lysis in this assay is based on the direct measurement of dye retained by the remaining viable target cells using the Pandex FCA. In paired experiments we have compared the fluorochrome assay to the standard 51Cr release assay in measuring porcine natural killer cytotoxicity. The target labeling time with BCECF is 30 min as opposed to 1 h with 51Cr; and there is no significant dye reincorporation after release. The optimal target number per incubation well for the BCECF assay is 5 X 10(3) cells. In both the BCECF and 51Cr release assays, maximum percent specific lysis is reached after 3-4 h incubation. By 2 h incubation, the BCECF assay reaches the maximum seen with 51Cr and in a 4 h assay the maximum NK activity measured with BCECF labeled targets is always higher than that measured with 51Cr-labeled targets. In paired experiments, we have shown the reproducibility of the BCECF assay and that the BCECF assay measures NK enhancement by NK enhancing monoclonal antibody and inhibition by NK inhibiting monoclonal antibody as good as the 51Cr release assay, if not better. In conclusion, the BCECF assay is a reliable and reproducible measure of in vitro cellular cytotoxicity, eliminates the use of radioisotopes and is cost efficient.

Cell Line↗