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Biomedical subjects

D W Hunt

Publications and source records attributed to D W Hunt.

At least 37 records · Page 2Linked to original sources
PubMed1998

Rapid cytochrome c release, activation of caspases 3, 6, 7 and 8 followed by Bap31 cleavage in HeLa cells treated with photodynamic therapy.

Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. The initiating events of PDT-induced apoptosis are poorly defined. It has been shown for other proapoptotic stimuli that the integral endoplasmic reticulum protein Bap31 is cleaved by caspases 1 and 8, but not by caspase-3. Further, a 20 kDa Bap31 cleavage fragment is generated which can induce apoptosis. In the current report, we sought to determine whether Bap31 cleavage and generation of p20 is an early event in PDT-induced apoptosis. The mitochondrial release of cytochrome c, involvement of caspases 1, 2, 3, 4, 6, 7, 8, and 10 and the status of several known caspase substrates, including Bap31, were evaluated in PDT-treated HeLa cells. Cytochrome c appeared in the cytosol immediately following light activation of the photosensitizer benzoporphyrin derivative monoacid ring A. Activation of caspases 3, 6, 7, and 8 was evident within 1-2 h post PDT. Processing of caspases 1, 2, 4, and 10 was not observed. Cleavage of Bap31 was observed at 2-3 h post PDT. The caspase-3 inhibitor DEVD-fmk blocked caspase-8 and Bap31 cleavage suggesting that caspase-8 and Bap31 processing occur downstream of caspase-3 activation in PDT-induced apoptosis. These results demonstrate that release of mitochondrial cytochrome c into the cytoplasm is a primary event following PDT, preceding caspase activation and cleavage of Bap31. To our knowledge, this is the first example of a chemotherapeutic agent inducing caspase-8 activation and demonstrates that caspase-8 activation can occur after cytochrome c release.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Overexpression of Bcl-X(L) prevents caspase-3-mediated activation of DNA fragmentation factor (DFF) produced by treatment with the photochemotherapeutic agent BPD-MA.

Photodynamic therapy (PDT) is a clinically effective cancer treatment. For human promyelocytic leukemia HL-60 cells, cleavage of pro-caspase-3 (CPP32/Yama/apopain) into its proteolytically active subunits rapidly follows the photodynamic treatment of these cells with cytotoxic levels of the photosensitizer benzoporphyrin derivative monoacid ring A and visible light. Cleavage of a recently identified cytosolic 45 kDa protein, DNA fragmentation factor (DFF), is required for endonuclease activation leading to DNA fragmentation. In the present study, DFF was rapidly processed following PDT. Overexpression of the anti-apoptotic Bcl-X(L) gene in HL-60 cells prevented PDT-induced caspase activation, DFF cleavage and DNA fragmentation. These results demonstrate for the first time an example of chemotherapeutic drug-induced activation of DFF and its regulation by Bcl-X(L).

Apoptosis↗
PubMed1998

Photofrin increases murine spleen cell transferrin receptor expression and responsiveness to recombinant myeloid and erythroid growth factors.

When given to normal male mice, the photochemotherapeutic agent Photofrin (porfimer sodium), a complex mixture of monomeric and oligomeric non-metallic porphyrins, significantly increased relative spleen weight, spleen cell numbers, DNA replication, levels of granulocyte-macrophage and erythroid progenitors and cellular responsiveness to different recombinant (r) myeloid growth factors. In contrast, monomeric hematoporphyrin had no effect on spleen weight, cellularity, erythroid progenitor levels or spleen cell cycle status. Photofrin significantly increased spleen cell expression of the receptor (CD71) for the iron transport protein transferrin by 72 h post-injection but did not affect levels of a receptor (CD25) for the T-cell growth factor interleukin-2 (IL-2) or spleen cell responsiveness to rIL-2. Further evidence of increased splenic erythropoietic activity in Photofrin-treated mice was provided by double color flow cytometric studies which indicated that the treatment elevated the number of nucleated spleen cells recognized by the erythroid lineage-specific monoclonal antibody TER-119. A majority of TER-119+ cells also expressed CD71 and the heat stable antigen, a marker of developing hematopoietic cells as well as mature erythrocytes. The capacity of Photofrin to stimulate murine hematopoietic activity appears to be a property not shared by other porphyrin photosensitizers.

Animals↗
PubMed1998

Interaction of viral proteins with host cell death machinery.

In recent years, intense research has been directed towards understanding molecular mechanisms involved in viral pathogenesis. It is now known that many viruses manipulate host defense mechanisms to prevent apoptosis in order to maximize viral replication. Towards the end of their replication cycle, certain viruses direct the synthesis of proteins that induce apoptosis or cell lysis thereby facilitating viral release from the cell. The present review summarizes the current understanding of interactions between viral proteins and the host cell death machinery.

Animals↗
PubMed1998

Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells.

Coxsackievirus B3 (CVB3), an enterovirus in the family Picornaviridae, induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Immunomodulatory aspects of photodynamic therapy.

In its conventional form, photodynamic therapy (PDT) is a clinically effective technique with which to treat tumours accessible to visible light. PDT utilises light absorbing compounds which catalyse the generation of toxic oxygen species, to produce localised antitumour effects. It has become apparent over the past decade that PDT also exhibits immunomodulatory attributes. Experimental animals may possess heightened antitumour immunity after tumour ablation with PDT. In contrast, at sub-phototoxic levels of photosensitiser, in combination with whole body light irradiation, PDT lessened disease severity when applied in different models of autoimmunity. Although the behaviour of lymphocytes may be affected by treatment, the ability of PDT to down-regulate autoimmune processes appears to be related to its capacity to influence the immunostimulatory attributes of antigen presenting cells.

Journal Article↗
PubMed1998

The porphyrin photosensitizer Photofrin elevates murine splenic erythropoiesis.

Changes occurring within the spleens of the genetically distinct DBA/1 and DBA/2 mouse strains produced by the photosensitizer Photofrin in the absence of direct light exposure were analyzed. Photofrin significantly increased spleen weight, cellularity and erythroid progenitor levels in both mouse strains tested. The expression of heat stable antigen (HSA), a marker present upon different immature leukocytes as well as certain fully-differentiated cell types including erythrocytes, was increased in the spleens of mice given Photofrin. It was shown for Photofrin-injected DBA/1 mice that a spleen cell population which expressed high levels of HSA also bound the iron transport protein transferrin. Photofrin increases the splenic demand for iron by promoting erythropoietic activity within the tissue.

Animals↗
PubMed1998

Photodynamic treatment with benzoporphyrin derivative monoacid ring A produces protein tyrosine phosphorylation events and DNA fragmentation in murine P815 cells.

Treatment with benozopophyrin derivative monoacid ring A (BPD-MA, verteporfin) and broad-spectrum fluorescent light rapidly produced apoptosis in murine P815 mastocytoma cells. Fragmentation of DNA, a fundamental characteristic of cells undergoing apoptosis, was evident within 3 h following the photodynamic treatment. Western immunoblot analysis using the specific antiphosphotyrosine monoclonal antibody 4G10 indicated that molecular species of > 200 kDa were phosphorylated on tyrosine residues during or immediately following the irradiation of cells loaded with BPD-MA. Increased tyrosine phosphorylation of a 15 kDa protein was evident by 15 min postirradiation. In the absence of light, BPD-MA did not affect the status of cellular protein tyrosine phosphorylation or cause DNA fragmentation. The protein kinase inhibitor staurosporine prevented tyrosine phosphorylation of the > 200 kDa species but did not affect tyrosine phosphorylation of the 15 kDa protein or the level of DNA fragmentation produced by the photodynamic treatment. The protein tyrosine phosphorylation events observed for P815 cells treated with cytotoxic levels of BPD-MA and light may not be directly related to the induction of the apoptotic cell death pathway.

Animals↗
PubMed1997

Inhibition of contact hypersensitivity with different analogs of benzoporphyrin derivative.

Four structural analogs of benzoporphyrin derivative (BPD), a potent anti-tumor photosensitizer, were evaluated for their capacity to influence the immunologically-mediated contact hypersensitivity (CHS) response against the hapten 2,4-dinitrofluorobenzene (DNFB). Immunocompetent hairless strain mice received BPD monoacid ring A (BPD-MA, verteporfin) and returned to normal housing conditions or treated with 690 nm red light (transcutaneous photodynamic therapy, PDT). Unexpectedly, we found that mice given BPD-MA exhibited significantly reduced CHS ear swelling responses to DNFB upon antigenic challenge, whether or not they had been treated with PDT. A significant reduction in the CHS response to DNFB was observed when BPD-MA or PDT was given 48 or 24 h prior to, on the same day, or 24 or 72 h after DNFB sensitization. However, the magnitude of the CHS response was unaffected if these treatments were given 96 h after DNFB sensitization, 24 h before challenge with DNFB. Significantly reduced CHS responses also occurred in Balb/c mice given BPD-MA with or without PDT. Mice given BPD-MA but retained in total darkness throughout the experimental period generated full-fledged ear swelling responses to DNFB indicating that CHS suppression with BPD-MA was light dependent. BPD monoacid ring B (BPD-MB) strongly reduced the CHS response of Balb/c mice kept under ambient light while BPD diacid ring A (BPD-DA) and BPD diacid ring B (BPD-DB) also lowered the CHS response but were less effective than the monoacid forms. Other photosensitizers including Photofrin, tin etiopurpurin, and zinc phthalocyanine did not alter the CHS response of Balb/c mice maintained under ambient light. The ability of different BPD analogs to inhibit the CHS response in mice held under ambient light conditions appears related to the potent photosensitizing activity of these compounds.

Adjuvants, Immunologic↗
PubMed1997

Photodynamic therapy induces caspase-3 activation in HL-60 cells.

Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. In this study, we show for the first time that photodynamic therapy (PDT), using benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) as the photosensitizer, induces the complete cleavage and subsequent activation of caspase-3 (CPP32/Yama/Apopain) but not caspase-1 (ICE) in human promyelocytic leukemia HL-60 cells. Poly(ADP-ribose) polymerase (PARP) and the catalytic subunit of DNA dependent protein kinase (DNA PK(CS)) were cleaved within 60 min of light activation of BPD-MA. The general caspase inhibitor Z-Asp-2,6 dichlorobenzoyloxymethylketone (Z-Asp-DCB) blocked PARP cleavage while the serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and N-tosyl-lysyl chloromethyl ketone (TLCK) blocked the cleavage of caspase-3 suggesting that they act upstream of caspase-3 activation. All three inhibitors were able to block DNA fragmentation that was induced by treatment with BPD-MA followed by light application. These studies demonstrate that protease activity, particularly that of caspase-3, is triggered in HL-60 cells treated with lethal levels of BPD-MA and visible light.

Journal Article↗
PubMed1997

Influence of Photofrin on the hematopoietic accessory function of murine peritoneal cells.

The porphyrin photochemotherapeutic agent Photofrin stimulates hematopoietic activity within the bone marrow (BM) and spleens of normal mice. We found that the intraperitoneal (i.p.) administration of Photofrin also caused a 3-4 fold increase in peritoneal cell (PC) numbers, particularly cells bearing granulocyte surface antigens. Little granulocyte-macrophage progenitor activity was detectable within the PC population of control and Photofrin-injected mice suggesting that Photofrin had elicited an influx of inflammatory cells into the region. In contrast to cells from control animals, PC obtained 6 h to 168 h after the i.p. injection of Photofrin exhibited a consistently inferior capacity to support the growth of BM colony forming units granulocyte-macrophage (CFU-GM). When PC from control or Photofrin-treated mice were added to the BM culture system in the presence of defined growth factors there was no effect on the number of colonies formed. This finding indicated that negative regulatory elements were not responsible for the reduced hematopoietic accessory activity exhibited by PC from Photofrin-treated mice. Supernatants conditioned by PC from Photofrin-injected mice poorly supported BM CFU-GM growth in vitro and in contrast to PC supernatants prepared from control mice did not contain detectable amounts of granulocyte-macrophage colony stimulating factor (GM-CSF). The cellular changes which occur within the peritoneal cavity represent bystander events not directly related to the hematostimulatory action of Photofrin.

Animals↗
PubMed1996

Transcutaneous photodynamic therapy alters the development of an adoptively transferred form of murine experimental autoimmune encephalomyelitis.

Transcutaneous photodynamic therapy (PDT), utilizing benzoporphyrin derivative monoacid ring A (BPD, verteporfin) and whole-body light exposure, was assessed for its capacity to modify the course of adoptively transferred experimental autoimmune encephalomyelitis (EAE) in PL mice. Using a novel cell culture technique to facilitate the induction of this neurodegenerative condition, disease signs commenced 3-4 weeks after the transfer of myelin basic protein (MBP)-reactive lymph node or spleen cells to naive syngeneic recipients. Mice administered MBP-sensitized lymph node cells preincubated with BPD followed by whole-body 690 nm light irradiation (15 J/cm2) did not display symptoms of EAE. Although almost all animals given MBP-sensitized spleen cells developed EAE, mice given BPD (1 mg/kg) and the light treatment 24, 48 or 120 h after spleen cell transfer exhibited significantly less severe disease symptoms than control animals. Mice given the photodynamic treatment 24 h after spleen cell transfer also exhibited a significantly later disease onset than the control animals. Treatment of mice with PDT 24 h prior to spleen cell transfer did not influence subsequent disease severity but modestly delayed its onset. In the absence of directed light, BPD did not influence the development of EAE. Spinal cord tissues were evaluated for the presence of T cell receptor (TCR) V alpha 4 mRNA transcripts that specifically encode for the TCR alpha-chain of MBP-reactive T cells of PL mice. Using the polymerase chain reaction, V alpha 4 TCR mRNA transcripts were present in spinal cord samples prepared from almost all control mice but in only about one-half of spinal cord samples prepared from mice treated with PDT 24 h after spleen cell transfer. These observations indicated that PDT had limited the expansion of MBP-specific V alpha 4+ T cells within the central nervous system. Transcutaneous PDT represents a new technique with which to approach the treatment of autoimmune disease.

Animals↗
PubMed1996

Monoclonal antibody LR-1 recognizes murine heat-stable antigen, a marker of antigen-presenting cells and developing hematopoietic cells.

The rat monoclonal antibody LR-1 was initially described to be reactive with an antigen present on murine splenic B lymphocytes. However, flow-cytometric analyses of cells obtained from thymus, bone marrow, spleen, and lymph nodes showed that LR-1 stained approximately 95, 95, 60-70 and 20% of cells present within these tissues in normal DBA/2 mice. The marker recognized by LR-1 was present on peripheral erythrocytes and splenic dendritic cells, and activation with lipopolysaccharide A further increased expression of this antigen by splenic B cells. This particular tissue and cellular distribution was similar to that delineated with monoclonal antibodies reactive with heat-stable antigen (HSA). Duallabelling studies were conducted to compare the reactivity patterns of LR-1 and the HSA-reactive monoclonal antibody J11d and indicated that both antibodies recognized splenocytes bearing B cell (IgM) or erythroid (TER-119, CD71) but not T cell (CD4, CD8) markers. Splenocytes exposed to phosphoinostol-specific phospholipase C showed marked reduction in LR-1 binding, indicating that this antibody recognized a glycosylphosphatidylinositol-anchored cell surface protein, consistent with the known structure of HSA. Mixing of LR-1 with the HSA-specific antibodies J11d or M1/69 provided flow-cytometric profiles indistinguishable from those obtained with either antibody alone. However, LR-1 inhibited M1/69 binding to splenocytes by 83%, while J11d reduced M1/69 binding to these cells by only 18%. This finding suggested that LR-1 and M1/69 recognize identical splenic HSA epitopes, while LR-1 and J11d bind distinct antigenic determinants of spleen HSA. Western blot analysis of splenocyte, thymocyte, bone marrow cell and erythrocyte detergent extracts revealed that LR-1 reacted with glycoforms of HSA of known molecular weights (30-55 kD). Thus, LR-1 recognizes HSA, the murine analogue of human CD24, and will be a useful reagent with which to investigate the role of HSA in the immune response and hematopoiesis.

Animals↗
PubMed1995

Accelerated myelopoietic recovery in irradiated mice treated with Photofrin.

The porphyrin photosensitizer, Photofrin porfimer sodium (Photofrin), has been widely studied for its capacity to evoke destruction of malignant tissues. In addition to its photosensitizing properties, Photofrin may exert myelostimulatory effects in normal and immunosuppressed mice in the absence of activating light. In the present set of experiments, we examined the effect of Photofrin upon the immunohematopoietic axis of sublethally irradiated DBA/2 mice. Administration of Photofrin (10 mg/kg) 1 and 4 days following irradiation (4 Gy) significantly enhanced the recovery of spleen cellularity, spleen and bone granulocyte/macrophage progenitors (colony-forming units, CFU-GM) and peripheral blood leukocytes levels. Proliferative responses to the T-cell mitogen concanavalin A by spleen cells prepared from Photofrin-treated mice were significantly less than those of cells from irradiated control mice 8 and 15 days post-irradiation. Photofrin given 1, 4 and 7 days following irradiation elevated splenic CFU-GM 3 to 4-fold 10 days post-irradiation relative to the irradiated controls and mice given only two injections of Photofrin. In contrast to the effect of two injections, multiple (three or four) injections of Photofrin did not elevate bone marrow CFU-GM above control levels beyond 8 days post-irradiation. In addition, splenic CFU-GM levels in animals receiving three or four injections of Photofrin were no different than those of the irradiated controls later than 10 days post-irradiation. These findings indicate that prolonged exposure to Photofrin in sublethally irradiated mice may induce regulatory factors which dampen the enhanced myelopoietic recovery stimulated by only two injections of the drug.

Animals↗
PubMed1995

Sensitivity of activated murine peritoneal macrophages to photodynamic killing with benzoporphyrin derivative.

This study compared the ability of highly purified resting and activated DBA/2 mouse peritoneal macrophages to survive treatment with the photosensitizer benzoporphyrin derivative (BPD, verteporfin) and light. Culture of macrophages with recombinant murine interferon-gamma (rIFN-gamma, 100 U/mL) for 72 h imparted a phenotypic and functional activation by dramatically increasing cell surface expression of major histocompatibility complex Class II (Ia) molecules and the formation of nitric oxide. The rIFN-gamma-activated macrophages were significantly (P < 0.05) more sensitive (lethal dose to cause a 50% reduction in cell survival, LD50 = 14.4 +/- 1.1 ng/mL) to photodynamic killing with BPD and light (10 J/cm2) than cells (LD50 = 18.2 +/- 2.0 ng/mL) cultured in medium alone. In contrast, macrophages treated with different concentrations of bacterial lipopolysaccharide (LPS) were as resistant or more resistant to photodynamic killing than cells cultured in medium alone. No cytotoxic effect of BPD was detected in cultures containing the drug but protected from light. Comparable amounts of BPD were taken up in vitro by unactivated and rIFN-gamma-activated macrophages, as detected by flow cytometric analysis. However, cells cultured with LPS (10 micrograms/mL) took up more BPD than macrophages cultured in medium alone or with rIFN-gamma. The DBA/2 P815 mastocytoma cells took up greater amounts of the drug and were subsequently more vulnerable to treatment with BPD and light (LD50 = 6.9 ng/mL) than macrophages cultured under any condition. The explanation for the increased vulnerability of rIFN-gamma-activated macrophages and the greater resistance of LPS-activated macrophages, relative to medium-cultured macrophages, to photodynamic killing with BPD is uncertain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Rude awakenings.

A new nurse recently complained in a national nursing publication that her manager, a 22-year veteran, refers to her staff as"dodos," "dipsticks" and "resident idiots." The vexed grad wanted to know whether she had to put up with this verbal abuse. It's amazing that she even had to ask. The Los Angeles Times reports that rudeness is epidemic in America, as "embedded in the workplace culture as time cards, fluorescent lighting and computers." EMTs and paramedics are prime targets. You endure it from patients, their families, bystanders, coworkers, managers, ED staffers and cops. You no doubt take a deep breath, hold your tongue and prepare to move on to the next call. But what if you're mad as hell and don't want to take it anymore? On the following pages, experts share their experiences and offer communication strategies. So, hurry up! Turn the page! (Oops. We apologize for being rude...)

Communication↗
PubMed1994

Studies of human cord blood dendritic cells: evidence for functional immaturity.

We have isolated low-density, nonadherent, nonphagocytic, HLA-DR+ve cells with the morphology of dendritic cells (DCs) from the cord blood of full-term newborn infants. Relative to adult DCs, cord blood DCs were poor stimulators of the mixed leukocyte reaction when either adult or cord blood mononuclear cells (MNCs) or T lymphocytes were used as responder cells. In contrast, cord blood T cells and MNCs responded normally to allogeneic adult DCs. Cord blood DCs performed poorly as accessory cells for T-lymphocyte mitogenic responses at suboptimal concentrations of concanavalin A (Con A) and phytohemagglutinin A or at optimal concentrations of mitogen and low numbers of DCs. Addition of recombinant interleukin-2 (rIL-2) or recombinant interferon-gamma (rIFN-gamma) to cord blood DC-T-cell cultures containing a suboptimal concentration of Con A potentiated the proliferative response. In contrast, rIL-2 and rIFN-gamma exerted little effect on the proliferative response of adult T cells cultured with Con A and DCs. Flow cytometric studies showed that levels of intercellular adhesion molecule-1 (ICAM-1; CD54) and major histocompatibility complex (MHC) class I HLA-ABC and class II HLA-DR antigens on cord blood DCs were significantly lower than those on adult blood DCs. These findings suggest that the relative inefficiency of cord blood DCs in the activation of T cells may be related to their low cell surface expression of MHC and cell adhesion molecules. The demonstrated impairment of cord blood DC function could be of importance in understanding the immunologic relationship between the fetus and mother and could contribute to the susceptibility of newborns to infection.

Adult↗