Biomedical subjects

R H Goldfarb

Publications and source records attributed to R H Goldfarb.

72 records · Page 4Linked to original sources

PubMedDate not supplied

Matrix metalloproteinases of human NK cells.

We have previously reported that MMP-2 and MMP-9 are present in rat A-NK cells, and have recently documented that additional MMPs are present in rodent A-NK cells. To our knowledge only proMMP-9 has previously been reported for human NK and A-NK cells. Herein, we report for the first time the presence of MMP-2 and MT1-MMP in human NK cells. The importance of these enzymes for the migration of A-NK cells into tumor metastases is of great potential relevance. MMPs may be rate limiting in A-NK cells, following their adoptive transfer, to traverse basement membrane and accumulate within established cancer metastases, a likely pre-requisite to their cytolytic function. Human NK cells express and produce MMP-2, MMP-9, MT1-MMP and the inhibitor TIMP-1. Moreover, human A-NK cells degrade the extracellular matrix equivalent (Matrigel) in a seemingly IL-2 dependent manner. It is therefore likely that A-NK cell MMPs play crucial roles in contributing to A-NK cell localisation and positioning the cells in vivo to allow for triggering their cytolytic potential.

Blotting, Western↗

PubMedDate not supplied

Expression of matrix metalloproteinases and their inhibitors by rat NK cells: inhibition of their expression by genistein.

In this study, we describe rat NK cell-derived MMPs including membrane-type MMPs (MT-MMPs) and tissue inhibitors of MMP (TIMPs). RT-PCR analysis from cDNA of rat A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-7, MMP-10, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. The RNK-16 cells expressed mRNA for MMP-7, MMP-10, MMP-11, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2, in addition to MMP-3 and MMP-13. Western blot analysis confirmed proteins for MT1-MMP and MT2-MMP in RNK-16 cells. TIMP-1 in rat A-NK cells was present at molecular mass of 34-kDa protein which may represent a highly glycosylated form. Genistein, a natural isoflavone found in soybeans, inhibited proliferation of RNK-16 cells in dosage dependent manner. In addition, it down-regulated the expression of MMP-13, MT1-MMP, TIMP-1 and TIMP-2. Moreover, genistein greatly impaired the ability of RNK-16 cells to invade through a model basement membrane. This effect might be mediated by the observed down-regulation of MMP-13 and MT1-MMP.

Animals↗

PubMedDate not supplied

Cooperation of urokinase plasminogen activator and matrix metalloproteinases in NK cell invasion.

We have previously investigated the role of the urokinase plasminogen activator (uPA) system in NK cell invasion. We have also studied NK cell-derived matrix metalloproteinases (MMPs) in extracellular matrix (ECM) degradation. We now report that both enzyme systems cooperate in NK cell invasion. Zymographic analyses detected uPA in RNK-16 cell conditioned media (CM) with the same molecular weights as the uPA we have previously shown to be associated with the rat NK cell urokinase plasminogen activator receptor. The combination of aprotinin, an inhibitor of plasmin, and Batimastat (BB94), an inhibitor of MMPs, in Matrigel invasion assays showed a more potent inhibitory effect on NK cell invasion than either inhibitor alone. Finally, a down regulation of uPA mRNA was noted following RNK-16 stimulation with collagen IV, fibronectin, and laminin.

Animals↗

PubMedDate not supplied

Regulation of IFN-gamma production following 2B4 activation in human NK cells.

IFN-gamma is a cytokine that regulates various functions of the immune system. The major producers of IFN-gamma are T cells and NK cells. 2B4 is a novel activating receptor expressed on all human NK cells, a subset of CD8+ T cells, monocytes and basophils. Activation of human NK cells through surface 2B4 enhances NK cell cytolytic function and secretion of IFN-gamma. We have examined the regulation of IFN-gamma production by the human NK cell line YT upon activation through surface 2B4. Our data indicate that ligation of surface 2B4 by mAb C1.7, that specifically recognizes 2B4, induces transcriptional activation of IFN-gamma. Partial inhibition of transcription did not prevent the transcriptional upregulation of IFN-gamma. S1 nuclease protection analysis indicated that transcriptional activation as well as mRNA stability may account for the increased production of IFN-gamma by human NK cells following 2B4 stimulation.

Actins↗

PubMedDate not supplied

Tumor blood supply and tumor localization by adoptively transferred IL-2 activated natural killer cells.

The circulatory pattern of IL-2 activated natural killer (A-NK) cells was studied in C57BL/6 mice bearing 10 day-old pulmonary and subcutaneous (s.c.) metastases of the B16 melanoma in order to evaluate the roles of the concentration of A-NK cells in the blood and of tumor blood flow on accumulation of A-NK cells in tumors. Kinetic studies of the presence of A-NK cells in peripheral blood after adoptive transfer revealed that these cells rapidly disappear from the blood. Via intravital microscopy of animals with exposed lung tissue, we have shown that the vast majority of transferred A-NK cells become efficiently arrested within the lung microcirculation at their first encounter with this organ, thereby explaining the fast disappearance of the cells from the bloodstream. Despite the low number of A-NK cells circulating in the blood, systemically injected A-NK cells (20 million per mouse) localized significantly (70-80 million cells/g) into most pulmonary metastases within 8-16 hours. In contrast, very few A-NK cells (< 0.2 million cells/g) were found in the s.c. metastases. Based on measurements of tumor blood flow (showing a classic inverse relationship between tumor size and tumor blood flow) and the blood concentration of A-NK cells, we estimated the highest intratumoral density of A-NK cells that theoretically can be generated by A-NK cells transported to the tumor by way of the blood. In s.c. tumors, the observed density of A-NK cells was at all times lower (10-50 fold) than the estimated density, indicating that only a few percent of the A-NK cells arriving at these tumors become retained in them. In contrast, the observed density of A-NK cells in pulmonary metastases was at all times higher (2-3 fold) than the estimated density. This finding indicates that A-NK cells might not reach the pulmonary metastases solely by way of the blood stream. In conclusion, i.v. injected A-NK cells become immediately entrapped in the lungs and, consequently, circulate poorly. While lung metastases become significantly infiltrated by i.v. injected A-NK cells, metastases in organs down-stream from the lungs become poorly infiltrated. We hypothesize that only a part of the A-NK cells found in lung metastases 8-16 hours following injection reach these metastases by way of the blood-vascular system. They might also migrate into the metastases from the surrounding normal lung tissue.

Animals↗

PubMedDate not supplied

Expression of neutrophil collagenase (MMP-8) in Jurkat T leukemia cells and its role in invasion.

BACKGROUND: Previous studies have shown that MMP-8, the neutrophil collagenase, was expressed in neutrophils, chondrocytes and rheumatoid synovial fibroblasts. MATERIALS AND METHODS: We used semi-quantitative RT-PCR analysis, Western blotting, and immunofluorescence assays to determine the expression of MMP-8 in Jurkat T cells. RESULTS: We have determined the expression of MMP-8 from Jurkat cells and the down-regulation of its expression by genistein, a principal soy isoflavone. Genistein inhibited the invasion of Jurkat cells through a model basement membrane by about 75%, similar to the inhibition by BB-94, a synthetic MMP inhibitor. Genistein also down-regulated the expression of MMP-13, but slightly up-regulated the expression of TIMP-1 and TIMP-2. CONCLUSIONS: Our findings documented for the first time the expression of the neutrophil collagenase by a T-cell line. We also determined the inhibition of Jurkat cell invasion by genistein, which was in part mediated through the regulation of the expression of MMPs and TIMPs.

Antineoplastic Agents↗

PubMedDate not supplied

uPA and uPAR contribute to NK cell invasion through the extracellular matrix.

BACKGROUND: The urokinase plasminogen activator (uPA) system has been implicated in cellular invasiveness of tumor cells and immune cells. Herein we provide evidence for the production by natural killer (NK) cells of both uPA and its receptor (uPAR). MATERIALS AND METHODS: Western blot analysis, RTPCR, casein/plasminogen zymography, and fluorescence microscopy were employed to detect uPA and uPAR on NK cells. NK cell invasiveness was examined using Matrigel invasion assays. RESULTS: NK cell uPA appeared at its characteristic molecular weights, is enzymatically active in casein/plasminogen zymography, and is recognized by monoclonal antibodies. uPAR was detected by RTPCR and fluorescence microscopy. Matrigel invasion assays demonstrated an active role of uPA in NK cell invasion. CONCLUSION: The uPA system contributes to extracellular matrix (ECM) degradation by NK cells, which may be essential for NK cell accumulation into metastases, and may be prerequisite for their killing of tumor cells following NK cell adoptive transfer.

3T3 Cells↗

PubMedDate not supplied

Tissue distribution of adoptively transferred adherent LAK cells: role of the route of administration.

The ability of LAK and adherent LAK (A-LAK) cells to migrate to and localize into tumors might be a limiting factor for the efficacy of adoptive immunotherapy. Employing different routes of inoculation of A-LAK cells labeled with 125IUdR, we have investigated how murine A-LAK cells circulate in the bloodstream and localize into various tissues. After intravenous injection, most of the A-LAK cells migrated to the lungs with less than 15% redistributing to other organs. Following left ventricular inoculation, after which the injected cells do not have to pass the lung capillaries before reaching other organs, higher numbers of A-LAK cells were found in liver, carcass, kidney and gut compared to intravenous injection. However, most of the A-LAK cells were cleared from these organs within 24 h, and surprisingly at this time the overall survival of the injected A-LAK cells was not more than 10-20% regardless of the route of injection. We found that a substantial accumulation of A-LAK cells in the liver could be achieved only when the cells were injected directly into the portal vein. Following this route of administration, more than 40% of the injected A-LAK cells were still in the liver at 24 h, whereas only 3-5% had redistributed to other organs. Our data suggest that A-LAK cells circulate inefficiently and survive poorly following systemic administration. However, enhanced accumulation of these cells in a particular organ might be achieved by direct administration of the A-LAK cells into the vessels feeding the organ.

Animals↗

PubMedDate not supplied

Neuroendocrine modulation of tumor metastases. I. Effect of adrenalectomy on B16 melanoma metastases.

There is increasing evidence to suggest a link between the neuroendocrine system and the immune system. Since it is well known that the immune response influences the establishment, progression or elimination of malignancy, we have examined the effects of adrenalectomy on B16 melanoma pulmonary tumor metastases and immune function in C57BL/6 mice to investigate the role of adrenal corticosteroids. Adrenalectomized mice were injected with 10(5) B16 melanoma cells on day 0. On day 9 the mice were sacrificed and the number of lung colonies counted. Adrenalectomized animals had a greater than 3 to 4-fold increase in the number of metastases as compared with sham operated animals. Steroid replacement therapy using dexamethasone delivered at 1 microgram/hr did not lead to any reduction in tumor metastases in adrenalectomized animals. These studies indicate that normal levels of adrenal steroids may influence the ability of tumor cells to colonize target organs and/or the ability of the immune system to mount an effective response.

Adrenalectomy↗

PubMedDate not supplied

Accumulation of adoptively transferred A-NK cells in murine metastases: kinetics and role of interleukin-2.

Infiltration of lung metastases by adoptively transferred A-NK cells was time- and dose-dependent. Infiltration became significant 6 hours after injection and reached a maximum after 16-24 hours. When no exogenous IL-2 was administered to support the injected cells, hardly any A-NK cells infiltrated the tumors. More A-NK cells accumulated in the tumors when high doses of IL-2 were given compared to lower doses. Significantly more A-NK cells accumulated in the tumours when the total dose of IL-2 was given as many small doses at short intervals compared to larger doses given less frequently. Use of IL-2 complexed to Polyethylene-glycol (Peg-IL-2) resulted in a significantly better tumor infiltration by A-NK cells than use of regular IL-2. IL-2 administered in mini-osmotic pumps did not lead to a better infiltration than ip injections of the same amount of IL-2. The route of administration of IL-2 did not seem to influence the degree of infiltration.

Animals↗

PubMedDate not supplied

Zymographic analysis of extracellular, released and cell-associated proteases of rat interleukin 2-activated natural killer (A-NK) cells.

The biochemical properties of heretofore uncharacterized extracellular proteases of rat interleukin-2-activated natural killer (A-NK) cells were investigated. Following p-aminobenzamidine- and D-phenylalanine-agarose affinity chromatography both trypsin- and chymotrypsin-like protease activities were found. Zymography revealed "trypsin-like" activities, with apparent molecular weights of approximately 42 kDa to 20.5 kDa, and "chymotrypsin-like" activities of approximately 27 kDa, to 22 kDa. These proteases might contribute to diverse NK cell functions, and the characteristics of these proteolytic activities are compared with those of granzymes of lytic granules and the proteasome of A-NK cells.

Animals↗

PubMedDate not supplied

Interleukin-2 (IL-2) activated natural killer (A-NK) cells: binding to microvascular endothelial cells and BRM enhancement of cytolytic activity.

A syngeneic in vitro system was developed for investigation of the binding of purified murine interleukin-2 (IL-2) activated natural killer (A-NK) cells to murine microvascular endothelial cells. This system is potentially useful as a model to investigate biochemical and molecular events underlying the binding of A-NK cells to microvascular endothelial cells within metastases in vivo that we have previously noted using electron microscopy. A-NK/endothelial cell binding in the syngeneic system was compared with the binding of allogeneic A-NK cells to the endothelial cells. Among test agents used to pretreat the A-NK cells for modulation of binding, lipopolysaccharide (LPS) most consistently enhanced binding. Additionally, test agents were also used in concurrent assays of A-NK cytolytic activity versus YAC-1 and P815 tumor cell targets. Polyinosinic: polycytidylic acid (poly IC), and gamma interferon (gamma IFN) were among the test agents which enhanced A-NK cell cytolytic activity.

Animals↗

PubMedDate not supplied

Natural killer cells and gene therapy: potential of gene transfection for optimizing effector cell functions and for targeting gene products into tumor metastases.

Fluorescently labeled, adoptively transferred interleukin (IL)-2 activated natural killer (A-NK) cells have the ability to selectively accumulate within established pulmonary or hepatic metastases, binding to tumor cells and/or to microvascular endothelial cells. A-NK cells have also been shown to exert antimetastatic therapy in animal models and in the clinic. Transfection of genes for cytokines or possibly other molecules has the potential to improve the therapeutic potency and efficacy of the effector cells. Gene transfection to induce autocrine production of IL-2 and/or other cytokines is expected to augment their antimetastatic activities, while avoiding toxicity from systemic administration of high doses of cytokines. An alternative or complementary strategy for gene therapy is to transfect A-NK cells with genes for cytotoxic molecules, to selectively target them to metastatic sites.

Animals↗

PubMedDate not supplied

Immunophenotypically varied cell subpopulations in primary and metastatic human melanomas. Monoclonal antibodies for diagnosis, detection of neoplastic progression and receptor directed immunotherapy.

During a systematic, immunocytochemical screening of 40 human cutaneous melanomas (30 primary and 10 metastatic) for immunophenotype (IP) heterogeneity, we employed a library of 20 well characterized, commercially available mono- and polyclonal antibodies. The use of the sensitive, indirect, four to six step immunoperoxidase or alkaline phosphatase conjugated streptavidin-biotin antigen detection technique provided excellent results. The immunocytochemically most characteristic IP for primary cutaneous melanoma, as detected by us was: HMB45+, S-100+, CEA+, vimentin+, cytokeratin 19+, p53+, Rbgene+, nm23+, HLA-DR+, HL.A-DP+, c-erbB3/HER-3+/-, cytokeratin 10/13+/-, HLA-DQ-, cytokeratin 5/8-, EMA-, c-myc-, and actin-. During melanoma progression, a tendency toward poor differentiation (dedifferentiation) and an increase in c-myc expression have both been observed, the latter downregulating HLA-A,B,C expression and consequently diminishing the possibility of melanoma cell Iysis by powerful CD8+, cytotoxic T lymphocytes (CTL) or other cytotoxic cells which requires HLA class I antigens. The development of the metastatic potential in melanomas caused an increase in CEA expression, eliminated the presence of nm23, and prompted the appearance of actin among the intermediate filaments, composing the cytoskeleton of these malignant tumor cells. The most characteristic IP for MMs, identified by this study was HMB45+, S-100+, CEA+, EMA+, vimentin+, HLA-DR+, HLA-DP+, cytokeratin 19+, actin-, c-erbB3/HER-3+, p53+, cytokeratin 10/13+/-, c-myc+/-, c-erbB2/HER-2+/-, HLA-DQ-, cytokeratin 5/8-, Rb gene-, nm23-. It has been observed that adhesion molecules and integrins play a significant role in the complex process of melanoma metastasis and thus we propose a blocking of these de novo expressed molecules with the appropriate antibodies as a form of immunotherapy of PMs and early stages of MMs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMedDate not supplied

Retention of adoptively transferred interleukin-2-activated natural killer cells in tumor tissue.

BACKGROUND: Adoptively transferred interleukin-2 activated natural killer (A-NK) cells are capable of selectively infiltrating tumor, however, only at low efficiency. The aim of this study was to investigate the intratumoral A-NK cell retention using an ex-vivo tissue-isolated tumor preparation. METHODS: R3230AC mammary adenocarcinoma and CSE fibrosarcoma were implanted in the ovarian fat pad of Fisher 344 rats. The tumors were perfused ex vivo 14 to 15 days post-implant with a known number of fluorescent labelled A-NK cells, and the effluent collected serially over time. Non stimulated splenocytes (N-SS) were used as controls. RESULTS: In group 1, tumors were perfused with either A-NK (n = 16) or N-SS (n = 7) cells. The mean number of the cells which remained intratumorally at the completion of the perfusion was 48.37% +/- 14.94 for A-NK cells and 34.68% +/- 13.20 of N-SS (p = 0.048). In group 2, tumors were perfused with a suspension containing both A-NK and N-SS cell (n = 11). The difference in tumor retention between A-NK cells and N-SS was 22.5% (p = 0.0053) for R3230AC tumors (retention of intratumoral A-NK cells was 45.1% +/- 6.47 vs. 22.6% +/- 19.09 for N-SS) and 15.88% (p = 0.028) for the fibrosarcomas (34.01% +/- 15.96 vs. 18.12 +/- 17.78 for A-NK and N-SS, respectively). No difference with respect to retention of A-NK cells or N-SS cells was observed between tumor types (p = 0.23 and p = 0.71, respectively). CONCLUSIONS: The retention of A-NK cells in tumor tissues was significantly better than the retention of N-SS when administered directly. Since the retention of A-NK cells in tumor tissue was high (35-50%), this factor does not explain the low efficiency of adoptively transferred A-NK cells accumulating in tumors when administered systemically.

Adoptive Transfer↗

PubMedDate not supplied

Augmentation of IL-2 activated natural killer cell adoptive immunotherapy with cyclophosphamide.

UNLABELLED: We have previously documented that adoptively transferred, IL-2 activated natural killer (A-NK) cells can accumulate within established pulmonary metastases. Since we have observed that increases in the accumulation of A-NK cells do not always lead to increases in therapeutic efficacy, we examined the ability of cyclophosphamide to enhance the therapeutic efficacy of A-NK cells. Animals with established B16 melanoma or Lewis lung carcinoma pulmonary metastases were treated with A-NK cell adoptive immunotherapy, either alone or following treatment with chemotherapeutic doses of cyclophosphamide. Adoptive immunotherapy studies with A-NK cells yielded at most a 30% reduction in the number of pulmonary metastases; however, cyclophosphamide (300 mg/kg) consistently reduced the size of metastatic colonies. In contrast, the combination therapy of A-NK cells plus cyclophosphamide was more effective than adoptive immunotherapy alone. In addition, polyethylene glycol IL-2 is superior to IL-2 in these studies. CONCLUSIONS: Our studies suggest that chemoimmunotherapy with A-NK cells plus cyclophosphamide may be more effective than adoptive immunotherapy alone since it results in the reduction in both the size and number of pulmonary metastases.

Adoptive Transfer↗

PubMedDate not supplied

Cytolytic activities of IL-2 activated NK cells from MMTV/v-Ha-ras transgenic oncomice during tumor progression.

IL-2 activated natural killer (A-NK) cells have the capacity to infiltrate metastatic tumors and lyse tumor cells. Nevertheless, adoptive immunotherapy with lymphokine-activated killer cells has been only modestly effective in the clinic and has not routinely provided long-term survival in patients with established cancer metastases. This may indicate the need for more carefully investigating the role of effector cells of the immune response, including A-NK cells, in models of tumor progression. Herein we describe the use of the MMTV/v-Ha-ras transgenic mouse model as a system for exploring the role of NK cells during tumor progression. We have examined the lytic capacity of A-NK cells generated from tumor-free and tumor-bearing transgenic oncomice against standard A-NK cell targets (YAC-1 and P815) in addition to tumor cells isolated from these animals. A-NK cells generated from mice without obvious tumor burden show higher lytic activity than A-NK cells generated from mice with evident tumors, i.e., those at a more advanced stage of tumor progression. Only long term (8-day) cultures of late passage A-NK cells generated from tumor-bearing mice showed significant increases in lytic activity over those generated from tumor-free mice. These results suggest that experimental protocols using transgenic oncomice at various stages of tumor growth may constitute a novel model for testing the role of A-NK cells for their capacity to interfere with cancer progression.

Animals↗

PubMedDate not supplied

Flavone acetic acid enhances accumulation of IL-2 activated NK cells within established metastases.

Flavone acetic acid, an agent which has been implicated in both tumor vasculature collapse and NK cell activations, has been tested recently as a potential anti-cancer chemotherapeutic agent. We have tested this agent in combination with adoptive immunotherapy using IL-2 activated natural killer (A-NK) cells in a metastatic B16 melanoma model in C57BL/6 mice. By using rhodamine-labeled A-NK cells we have been able to quantitate both the number of A-NK cells that localize within each tumor section and the percentage of the tumor area occupied by A-NK cells. This has been accomplished using an image analysis system. Flavone acetic acid (200 mg/kg, i.p.) given one day prior to the injection of A-NK cells increased the area of the tumor occupied by A-NK cells and the area of individual A-NK cells approximately 2-fold; however, it did not appear to increase the number of A-NK cells per tumor cross-section. Nevertheless, this increase did not lead to any significant change in the therapeutic efficacy of A-NK cell adoptive immunotherapy. Our studies therefore suggest that mere enhancement of A-NK cell recruitment into tumor metastases does not necessarily translate into enhanced metastatic therapeutic efficacy. Moreover, this method may be a useful tool for pre-screening of compounds which enhance the accumulation of adoptively transferred cells into tumor metastases prior to in vivo screening for therapeutic efficacy.

Adjuvants, Immunologic↗