Biomedical subjects

David W Hoskin

Publications and source records attributed to David W Hoskin.

30 records · Page 2Linked to original sources

PubMed2004

Reduced tumorigenicity of B16-F10 mouse melanoma cells transfected with mycobacterial antigen 85A.

Immunotherapy based on the administration of the mycobacterium bacillus Calmette-Guerin has been successfully used in the treatment of in situ transitional cell bladder cancer, and may be applicable to the treatment of cutaneous malignant melanoma. Antigen 85A (Ag85A) and heat shock protein 65 kDa (hsp65) are major secreted proteins of Mycobacterium species and potent stimulators of cell-mediated immunity. This study evaluated the ability of Ag85A and hsp65 gene transfection to limit tumor growth by B16-F10 mouse melanoma cells. Immunoblotting confirmed protein expression and secretion by B16-F10 cells that were transiently transfected with plasmid DNA containing the Ag85A or hsp65 gene. Groups of syngeneic C57BL/6 mice were injected subcutaneously with 1x10(5) untransfected B16-F10 cells or B16-F10 cells transiently transfected with either empty vector or vector containing the Ag85A or hsp65 gene. Ag85A-expressing B16-F10 cells exhibited a dramatic 76% reduction (p<0.05, Mann-Whitney U test) in tumor weight in comparison to empty vector controls at 14 days post-inoculation. In contrast, hsp65-transfected B16-F10 cells did not show any change in tumorigenicity. Decreased tumorigenicity by Ag85A-transfected B16-F10 cells was not due to a reduced ability of Ag85A-transfected B16-F10 cells to proliferate since both mock- and Ag85A-transfected B16-F10 cells showed increased in vitro proliferation in comparison to untransfected cells. Hematoxylin and eosin staining revealed that Ag85A-transfected B16-F10 tumors contained an inflammatory leukocyte infiltrate that was not present in hsp65-transfected tumors. Reduced tumor progression by Ag85A-transfected B16-F10 melanoma cells suggests that immunotherapy based on the transient induction of Ag85A expression may be an effective approach for the treatment of cutaneous malignant melanoma.

Animals↗

PubMed2003

Cutting edge: dendritic cell actin cytoskeletal polarization during immunological synapse formation is highly antigen-dependent.

Dendritic cells (DC) actively rearrange their actin cytoskeleton to participate in formation of the immunological synapse (IS). In this study, we evaluated the requirements for DC participation in the IS. DC rearrange their actin cytoskeleton toward naive CD4(+) T cells only in the presence of specific MHC-peptide complexes. In contrast, naive CD4(+) T cells polarized their cytoskeletal proteins in the absence of Ag. DC cytoskeletal rearrangement occurred at the same threshold of peptide-MHC complexes as that required for T cell activation. Furthermore, T cell activation was inhibited by specific blockade of DC cytoskeletal rearrangement. When TCR-MHC interaction was bypassed by using Con A-activated T cells, DC polarization was abrogated. In addition, directional ligation of MHC class II resulted in DC cytoskeletal polarization. Our findings suggest that a high Ag specificity is required for DC IS formation and that MHC class II signaling plays a central role in this process.

Actins↗

PubMed2003

Adenosine inhibits activation-induced T cell expression of CD2 and CD28 co-stimulatory molecules: role of interleukin-2 and cyclic AMP signaling pathways.

Adenosine is an immunosuppressive molecule that is associated with the microenvironment of solid tumors. Mouse T cells activated with anti-CD3 antibody in the presence of adenosine with or without coformycin (to prevent adenosine breakdown by adenosine deaminase) exhibited decreased tyrosine phosphorylation of some intracellular proteins and were inhibited in their ability to proliferate and synthesize interleukin (IL)-2. In addition, adenosine interfered with activation-induced expression of the co-stimulatory molecules CD2 and CD28. Activation-induced CD2 and CD28 expression was also diminished when T cells were activated in the presence of anti-IL-2 and anti-CD25 antibodies to neutralize IL-2 bioactivity. Collectively, these data suggest that CD2 and CD28 up-regulation following T cell activation is IL-2-dependent; and that adenosine inhibits activation-induced T cell expression of CD2 and CD28 by interfering with IL-2-dependent signaling. The inhibitory effect of adenosine on activation-induced CD2 and CD28 expression could not be attributed to cyclic AMP (cAMP) accumulation resulting from the stimulation of adenylyl cyclase-coupled adenosine receptors, even though cAMP at concentrations much higher than those generated following adenosine stimulation was inhibitory for both CD2 and CD28 expression. We conclude that adenosine interferes with IL-2-dependent T cell expression of co-stimulatory molecules via a mechanism that does not involve the accumulation of intracellular cAMP.

Adenosine↗

PubMed2003

Thy-1 signaling in the context of costimulation provided by dendritic cells provides signal 1 for T cell proliferation and cytotoxic effector molecule expression, but fails to trigger delivery of the lethal hit.

Cross-linking of the GPI-anchored protein Thy-1 results in T cell proliferation and IL-2 synthesis. However, the exact function of Thy-1 in the process of T cell activation remains unknown, as does the effect of costimulation on Thy-1-driven T cell responses. In this study, we have investigated the ability of Thy-1 to substitute for traditional signal 1 in the context of costimulation provided by dendritic cells. Dendritic cells dramatically enhanced T cell proliferation and IL-2 synthesis in response to Thy-1 triggering by anti-Thy-1 mAb. This effect was not dependent on dendritic cell Fcgamma receptors, but was a result of B7-mediated costimulation (signal 2). T cells were also activated when microbeads coated with a combination of anti-Thy-1 and anti-CD28 mAbs were used to supply signals 1 and 2, respectively. Thy-1-stimulated T cells adhere to target cells and express perforin, granzyme B, and Fas ligand, but fail to kill target cells due to an inability to reorganize their secretion machinery. Moreover, in contrast to TCR signaling, Thy-1 triggering failed to induce cytotoxicity in redirected lysis assays. We conclude that Thy-1 triggering can partially substitute for signal 1, which, in combination with a strong signal 2, leads to robust T cell proliferation, IL-2 synthesis, and cytotoxic effector molecule expression, but does not induce cytolytic function. The block at the level of cytotoxic effector function that results when T cells are activated in the absence of a classical, Ag-specific signal 1 may constitute a mechanism to ensure the specificity of CTL responses and prevent potentially harmful promiscuous cytotoxicity.

Amino Acid Sequence↗

PubMed2003

Exposure to paclitaxel or vinblastine down-regulates CD11a and CD54 expression by P815 mastocytoma cells and renders the tumor cells resistant to killing by nonspecific cytotoxic T lymphocytes induced with anti-CD3 antibody.

Paclitaxel and vinblastine are drugs with anti-microtubule activity that are commonly used in the treatment of numerous types of cancer. In this study, we investigated the effect of prior exposure to submaximal cytotoxic concentrations (EC(25) and EC(50)) of paclitaxel or vinblastine on the subsequent susceptibility of surviving P815 murine mastocytoma cells to cytolysis by major histocompatibility complex (MHC)-unrestricted mouse cytotoxic T lymphocytes that had been induced with anti-CD3 antibody. P815 cells that had survived culture for 24 h in the presence of paclitaxel (5 or 50 micro g/ml) or vinblastine (1.5 or 15 micro g/ml) were rendered resistant to anti-CD3-activated killer-T (AK-T) cell-mediated cytolysis in a standard (51)Cr-release assay. Resistance to killing was associated with a reduced ability of AK-T cells to form conjugates with drug-treated P815 target cells, suggesting a possible effect on adhesion molecules. Flow cytometric analysis of paclitaxel- or vinblastine-treated P815 cells revealed reduced cell-surface expression of the adhesion molecules LFA-1 (CD11a /CD18) and ICAM-1 (CD54). Similar results were obtained following paclitaxel or vinblastine treatment of Yac-1 lymphoma cells. RT-PCR analysis revealed reduced levels of mRNAs coding for CD11a and CD54 in paclitaxel- or vinblastine-pretreated P815 cells. Collectively, these data lead us to conclude that paclitaxel and vinblastine render P815 mastocytoma cells resistant to T cell-mediated cytotoxicity by interfering with CD11a and CD54 expression by the tumor cells. A similar effect by these drugs on tumor cells and/or leukocytes in cancer patients might compromise tumor-specific cell-mediated immune responses.

Animals↗

PubMed2003

CD2-CD48 interactions promote cytotoxic T lymphocyte induction and function: anti-CD2 and anti-CD48 antibodies impair cytokine synthesis, proliferation, target recognition/adhesion, and cytotoxicity.

The role of CD2 signaling in cytotoxic T lymphocyte (CTL) development was examined by stimulating mouse T cells with anti-CD3 monoclonal antibody (mAb) in the absence or presence of anti-CD2 mAb or anti-CD48 mAb or both. Induction of nonspecific CTL and interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) synthesis were impaired in the absence of CD2-CD48 interactions. Anti-CD2 mAb also inhibited activation-induced expression of the high-affinity IL-2 receptor (IL-2R). In contrast, IFN-gamma receptor (IFNGR) expression was increased in the presence of anti-CD2 mAb. Reduced cytotoxicity by CTL induced in the absence of CD2-CD48 interactions was associated with a diminished ability of CTL to conjugate with target cells and reduced expression of granzyme B and perforin. Anti-CD2 mAb did not affect expression of Fas ligand and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by anti-CD3-activated T cells. Cytotoxic effector function and granzyme B and perforin expression were rescued when exogenous IL-2 and IFN-gamma were added in combination with anti-CD2 mAb to anti-CD3-activated T cells at initiation of culture. We conclude that CD2-CD48 interactions during T cell activation are critical for the synthesis of sufficient IL-2 and IFN-gamma to drive CD8(+) T cells to differentiate into functional cytotoxic effector cells.

Animals↗

PubMed2002

Adenosine acts through an A3 receptor to prevent the induction of murine anti-CD3-activated killer T cells.

Adenosine, a purine nucleoside found at high levels in solid tumors, is able to suppress the recognition/adhesion and effector phases of killer lymphocyte-mediated tumor cell destruction. Here, we demonstrate that adenosine, at concentrations that are typically present in the extracellular fluid of solid tumors, exerts a profound inhibitory effect on the induction of mouse cytotoxic T cells, without substantially affecting T-cell viability. T-cell proliferation in response to mitogenic anti-CD3 antibody was impaired in the presence of 10 microM adenosine (plus coformycin to inhibit endogenous adenosine deaminase). Antigen-specific T-cell proliferation was similarly inhibited by adenosine. Anti-CD3-activated killer T (AK-T) cells induced in the presence of adenosine exhibited reduced major histocompatibility complex-unrestricted cytotoxicity against P815 mastocytoma cells in JAM and (51)Cr-release assays. Diminished tumoricidal activity correlated with reduced expression of mRNAs coding for granzyme B, perforin, Fas ligand and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), as well as with diminished Nalpha-CBZ-L-lysine thiobenzylester (BLT) esterase activity. Interleukin-2 and interferon-gamma synthesis by AK-T cells was also inhibited by adenosine. AK-T cells express mRNA coding for A(2A), A(2B) and A(3) receptors, but little or no mRNA coding for A(1) receptors. The inhibitory effect of adenosine on AK-T cell proliferation was blocked by an A(3) receptor antagonist (MRS1191) but not by an A(2) receptor antagonist (3,7-dimethyl-1-propargylxanthine [DMPX]). The A(3) receptor agonists (N(6)-2-(4-aminophenyl)ethyladenosine [APNEA] and N(6)-benzyl-5'-N-ethylcarboxamidoadenosine [N(6)-benzyl-NECA]) also inhibited AK-T cell proliferation. Adenosine, therefore, acts through an A(3) receptor to prevent AK-T cell induction. Tumor-associated adenosine may act through the same mechanism to impair the development of tumor-reactive T cells in cancer patients.

Adenosine↗

PubMed2002

Adenosine suppresses alpha(4)beta(7) integrin-mediated adhesion of T lymphocytes to colon adenocarcinoma cells.

The interaction of T lymphocytes with tumor cells, a key step in the antitumor immune response, is suppressed by adenosine, a nucleoside produced at increased levels within the hypoxic tumor environment. We have explored the mechanism by which adenosine interferes with the lymphocyte:tumor cell interaction. The adhesion of anti-CD3-stimulated T cells to syngeneic MCA-38 mouse colon adenocarcinoma cells did not involve LFA-1 (alpha(L)beta(2)) or VLA-5 (alpha(5)beta(1)). However, antibodies against either lymphocyte alpha(4) or beta(7) (but not beta(1)) integrin subunits, or against VCAM-1 on the tumor cells, significantly suppressed adhesion, showing that the recognition of MCA-38 cells by T cells is strongly dependent upon the association of alpha(4)beta(7) on the effector cells with VCAM-1 on the tumor targets. This association is modulated by adenosine: The ability of adenosine to suppress T cell adhesion to MCA-38 cells was lost if alpha(4)beta(7) was functionally blocked with anti-alpha(4) antibodies (i) prior to or (ii) during the adhesion assay or if (iii) alpha(+)(4) cells were depleted from the T lymphocyte population. The binding of T cells to fibronectin through alpha(4)beta(1) was not suppressed by adenosine. We conclude that adenosine partially inhibits the interaction of T lymphocytes with tumor cells by blocking the function of integrin alpha(4)beta(7).

Adenocarcinoma↗

PubMed2002

Activation-induced expression of cell surface CD28 on mouse T lymphocytes is inhibited by cyclosporine A.

T-cell activation requires both T-cell receptor signaling and a costimulatory signal provided by CD28 which enhances and prolongs interleukin-2 (IL-2) production. To determine the effect of cyclosporine A (CsA) on constitutive and activation-induced CD28 expression, mouse T cells were exposed to CsA (0.1 microM) in the absence or presence of anti-CD3 monoclonal antibody (mAb). CD28 expression was then determined by flow cytometry. CsA treatment prevented activation-induced CD28 expression but did not affect constitutive CD28 expression. Inhibition of inducible CD28 expression by CsA was not rapidly reversible, requiring 48h of restimulation in the absence of CsA for CD28 expression to return to control levels. T cells activated in the presence of combined anti-IL-2 and anti-CD25 mAb (both 10 microg/mL) also exhibited reduced CD28 expression, suggesting that activation-induced CD28 expression is, at least in part, an IL-2-dependent process. However, the inhibitory effect of CsA on activation-induced CD28 expression was maintained in the presence of exogenous IL-2 (250 U/mL). We conclude that CsA, by inhibiting activation-induced expression of costimulatory CD28 molecules by T lymphocytes, may interfere with the ability of CD28 to provide an optimal costimulatory signal for sustained IL-2 production following T-cell activation.

Animals↗

PubMedDate not supplied

Chemosensitization of T-47D breast carcinoma cells to TRAIL and Fas receptor-induced killing.

BACKGROUND: Although chemotherapeutic agents are known to sensitize some tumour types to killing through cell surface death receptors for Fas ligand and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), chemosensitization of breast carcinoma cells has not yet been explored. MATERIALS AND METHODS: Mitochondrial thiazole tetrazolium assays were used to measure changes in MCF-7 and T-47D breast carcinoma cell viability. Semiquantitative RT-PCR was used to determine Fas and TRAIL receptor mRNA expression. RESULTS: Treatment with suboptimal concentrations of etoposide or doxorubicin rendered T-47D cells sensitive to anti-Fas antibody or TRAIL, consistent with Fas and TRAIL-R1 mRNA expression by T-47D cells following drug treatment. In contrast, neither drug sensitized MCF-7 cells to TRAIL- or anti-Fas antibody, most likely due to diminished Fas and TRAIL-R1 expression following drug treatment. CONCLUSION: These findings suggest that some breast carcinomas may respond favorably to a combination of chemotherapy and immunotherapy.

Antibodies, Monoclonal↗

PubMedDate not supplied

Human mast cells induce caspase-independent DNA fragmentation in leukemic T cells.

Mast cells are known to play an important role in inflammation and host defense. Recent evidence suggests that mast cells may also participate in immune surveillance against cancer cells. In this study, we demonstrate that human mast cells are able to trigger apoptosis in Jurkat T leukemia cells. Fragmentation of Jurkat cell DNA was detectable by JAM assay within 4 h of exposure to HMC-1 mast cells or cultured human cord blood-derived mast cells. HMC-1 mast cells that were fixed with paraformaldehyde retained the ability to induce apoptosis in Jurkat cells while cell-free conditioned supernatants from HMC-1 cell cultures failed to elicit DNA fragmentation, suggesting that mast cells mediate anti-tumor activity via a cell-surface molecule. Surprisingly, the apoptosis-inducing activity of HMC-1 mast cells was not mediated by known tumor necrosis factor (TNF) superfamily members (TNF-alpha, Fas ligand, TRAIL, TWEAK), nor did the fragmentation of Jurkat cell DNA by mast cells require the activity of caspase-3, -8, or -9. Collectively, these data indicate that mast cells trigger caspase-independent apoptosis in leukemic cells via a novel cell-surface molecule, and are consistent with a role for mast cells as anti-tumor effector cells.

Apoptosis Regulatory Proteins↗

PubMedDate not supplied

Paclitaxel inhibits natural killer cell binding to target cells by down-regulating adhesion molecule expression.

BACKGROUND: Chemotherapy with paclitaxel is associated with impaired natural killer (NK) cell function. The purpose of this study was to determine the effect of paclitaxel treatment on NK cell adherence to target cells. MATERIALS AND METHODS: Human NK-like YT cells or NK-sensitive K562 cells were exposed to submaximal cytotoxic concentrations (EC10 and EC30) of paclitaxel. The ability of surviving YT or K562 cells to adhere to untreated K562 cells or YT cells, respectively, was assessed in a conjugation assay. The effect of paclitaxel on adhesion molecule expression was determined by flow cytometry. RESULTS: Paclitaxel treatment resulted in decreased conjugate formation, as well as decreased alpha4, alphaL, and beta7 integrin expression by YT cells and decreased ICAM-1 expression by K562 cells. CONCLUSIONS: Paclitaxel inhibition of adhesion molecule expression resulted in impaired NK cell binding to target cells, which may have a negative impact on immune surveillance.

Antineoplastic Agents, Phytogenic↗